**Phenomenal RNA Interference: From Mechanism to Application**

Pallavi Mittal1,\*, Rashmi Yadav2, Ruma Devi3,

Shubhangini Sharma4 and Aakash Goyal5 *1ITS Paramedical College, Ghaziabad, 2All India Institute of Medical Science, Delhi, 3PAU Regional Station, Gurdaspur, 4Aptara (Techbook International), Delhi 5Bayer Crop Science Saskatoon, 1,2,3,4India 5Canada* 

#### **1. Introduction**

60 Crop Plant

Mills, G., Buse, A., Gimeno, B., et al. 2007. A synthesis of AOT40-based response functions

Pandey, J., Agrawal, M. 1992. Ozone: concentration variabilities in a seasonally dry tropical

Pang, J., Kobayashi, K., Zhu, J. 2009. Yield and photosynthetic characteristics of flag leaves

Rai, R., Agrawal, M., Agrawal, S.B. 2007. Assessment of yield losses in tropical wheat using

Rai, R., Agrawal, M. 2008. Evaluation of physiological and biochemical responses of two rice

Sarkar, A., Agrawal, S.B. 2010. Identification of ozone stress in Indian rice through foliar injury and differential protein profile. Environmental Monitoring Assessment 161: 283-302. Sarkar, A., Rakwal, R., Agrawal, S.B, et al. 2010. Investigating the impact of elevated levels

and proteomics approaches. Journal of Proteome Research 9: 4565-4584. Singh, A., Sarin, S.M, Shanmugam, P., et al. 1997. Ozone distribution in the urban environment of Delhi during winter months. Atmospheric Environment 31: 3421-3427. Sioshansi, R., Oren, S. 2007. How good are supply function equilibrium models: An empirical analysis of the ERCOT balancing market. Journal of Regulatory Economics 31: 1-35. Tiwari, S., Rai, R. Agrawal, M., 2008. Annual and seasonal variations in tropospheric ozone

Cambridge, UK, NY, USA.

Environment 41: 2630-2643.

climate. Environment International 18: 515-520.

sensitivity. Plant Physiology and Biochemistry 40: 599-603.

open top chambers. Atmospheric Environment 41: 9543-9554.

rural site in India. Science of the Total Environment 407: 679-691.

fourth assessment report of IPCC on climate change. Cambridge University Press,

and critical levels of ozone for agricultural and horticultural crops. Atmospheric

in chinese rice (Oryza sativa L.) varieties subjected to free- air release of ozone. Agriculture, Ecosystems and Environment (doi. 10.1016/j.agee.2009.03. 012). Pasqualini, S., Antonielli, M., Ederli, L., et al. 2002. Ozone uptake and its effect on

photosynthetic parameters of two tobacco cultivars with contrasting ozone

(Oryza sativa L.) cultivars to ambient air pollution using open top chambers at a

of O3 on tropical wheat using integrated phenotypical, physiological, biochemical

concentrations around Varanasi. International Journal of Remote Sensing 29, 4499- 4514.

2007. Gel based proteomics reveals potential novel protein markers of ozone stress in leaves of cultivated bean and maize species of Panama. Electrophoresis 28: 4369-4381. Tripathi, R., Sarkar, A., Pandey Rai, S., Agrawal, S.B., 2011. Supplemental ultraviolet-B and

ozone: Impact on antioxidants, proteome and genome of linseed (*Linum* 

related photochemical oxidants (Final). U.S. Environmental Protection Agency,

background station in eastern China 1991–2006: enhanced variability. Atmospheric

Torres, N.L, Cho, K., Shibato, J., Kubo, A., Iwahashi, H., Jwa, N.S., Agrawal, G.K., Rakwal, R.

US Environmental Protection Agency U.S. EPA. 2006. Air quality criteria for ozone and

Varshney, C.K, Aggarwal, M. 1992. Ozone pollution in the urban atmosphere of Delhi.

Vingarzan, R. 2004. A review of surface ozone background levels and trends. Atmospheric

Woodward, F.I. 2002. Potential impacts of global elevated CO2 concentrations on plants.

Xu, X., Lin, W., Wang, T., et al. 2008. Long term trend of surface ozone at a regional

*usitatissimum* L. cv Padmini). Plant Biology 13: 93-105.

Washington, DC, EPA/600/R-05/004aF-cF.

Atmospheric Environment 26: 291-294.

Current Opinion in Plant Biol. 5: 207-211.

Chemistry Physics Discussion 8: 215-243

Environment 38: 3431-3442.

The phenomenon of dsRNA-mediated interference (RNAi), was first demonstrated in nematodes in 1998 by *Professor* Andrew Z. Fire *at Stanford University, California, USA* and *Professor* Craig C. Mello at University of Massachusetts *Medical School in Worcester, USA.* It is thought to have evolved as a type of "genetic immune system" to protect organisms from the presence of foreign or unwanted genetic material. To be more specific, RNAi probably evolved as a mechanism to block the replication of viruses and/or to suppress the movements of transposons within the genome, because both of these potentially dangerous processes typically involve the formation of dsRNA intermediates. Cells can recognize dsRNAs as "undesirable" because such molecules are not produced by the cell's normal genetic activities.

The RNAi was introduced to the public in mid-2001 and in just about few years it became one of the most widely used technologies in both academic and industrial research environments. In recognition of overwhelming importance of RNAi is an biological process and universally applicable tool, the leading Journal Science proclaimed it "The breakthrough of the year, 2002". During the last decade, our knowledge repertoire of RNAmediated functions has hugely increased, with the discovery of small non-coding RNAs which play a central part in a process called RNA silencing. Ironically, the very important phenomenon of co-suppression has recently been recognized as a manifestation of RNA interference (RNAi), an endogenous pathway for negative post-transcriptional regulation. RNAi has revolutionized the possibilities for creating custom "knock-downs" of gene activity. RNAi operates in both plants and animals, and uses double stranded (dsRNA) as a trigger that targets homologous mRNAs for degradation or inhibiting its transcription or

<sup>\*</sup> Corresponding author

Phenomenal RNA Interference: From Mechanism to Application 63

Similar PTGS like effect "Quelling" (Cogoni and Macino, 1999) was also seen in fungi *Neurospora crassa*. Another RNAi related phenomenon, coat protein mediated protection (CPMP) in plants gave insight into the mechanism of PTGS. In 1995 Guo and Kempheus first studied RNA silencing in animals. They used antisense RNA technique to silence par1 mRNA expression in *C. elegans* but found that par1 mRNA itself repressed *par1* gene and concluded that both sense and antisense RNA could cause silencing. Their observation inspired the experiment of Fire, Mello and colleagues. Thus began the journey of the newly dubbed technology RNA Interference. The mystery of molecular mechanism responsible for gene silencing now known as RNA interference (RNAi) exploded in 1998. It was discovered that PTGS was triggered by double-stranded RNA (dsRNA), provided most unexpected

Andrew Fire and Craig Mello published their break-through study on the mechanism of RNA interference in 1998, *Nature*. It was earlier known that antisense RNA, but remarkably also sense RNA could silence genes, but the results were inconsistent and the effects usually modest. However, due to the fact that both sense and antisense RNA could cause silencing Mello argued that the mechanism could not just be a pairing of antisense RNA to mRNA, and he coined the term RNA interference for the unknown mechanism. In their *Nature* paper, Fire and Mello tested the phenotypic effect of RNA injected into the worm *C. elegans.* They established that annealed sense/antisense RNA, but neither antisense nor sense RNA alone, caused the predicted phenotype. Furthermore, only injection of double-stranded RNA (dsRNA) led to an efficient loss of the target mRNA. Fire and Mello made the remark that RNAi could provide an explanation for a phenomenon studied in plants for several years: posttranscriptional gene silencing (PTGS). Finally, they ended their paper by speculating about the possibility that "dsRNA could be used by the organism for physiological gene silencing". This discovery later won Fire and Mello the 2006 Nobel Prize in Physiology or Medicine. Thus it was clear that co-suppression in plants, quelling in fungi and RNAi in nematodes all shared a common mechanism. Further work showed that this

effect was even more widespread, occurring in fruitflies and mammals too.

**2. Proteins involved in RNAi/ PTGS/Quelling** 

The biochemistry of RNAi was further elucidated in an *in vitro* system, built on *Drosophila* cultured cells, Elbashir et al., 2011 demonstrated that Double-stranded RNA is cut into short interfering RNA (siRNA) by the endonuclease Dicer (Lee et al., 2002). The antisense strand is loaded into the RISC (RNA-induced silencing complex) and links the complex to the endogenous mRNA by base-pairing (Martinez et al., 2002). The RISC complex cuts the mRNA strand, and the mRNA is subsequently degraded. In certain systems, in particular plants, worms and fungi, an RNA dependent RNA polymerase (RdRP) plays an important role in generating and/or amplifying siRNA. Thus, within few years a vast amount of information accumulated on the specific proteins and protein complexes involved in RNAi and molecular machinery involved in RNAi was subsequently revealed (Thakur, 2003).

To understand the basis of RNA silencing both genetic and biochemical approaches have been undertaken. Genetic screens were carried out to search for mutants defective in quelling, RNAi or PTGS and a large number of genes whose products are somehow implicated in RNA silencing have been identified in *C. elegans*, *D. melanogaster*, *Homo sapiens*,

explanation with many profound consequences.

translation (Almedia and Allshire 2005; Cotta-Ramusion et al., 2011) whereby susceptible genes can be silenced. Hence, RNA interference is the newest kid on the genetic block, allows the scientists to selectively turn off genes and finally promises to set the scientific world alight with its therapeutic potential and it has provided new platforms for developing eco-friendly molecular tools for crop improvement by suppressing the genes responsible for various stresses and improving novel traits in plants including disease resistance.

#### **1.1 Landmarks in RNAi discovery**

The discovery of RNAi was preceded first by observations of transcriptional inhibition by antisense RNA expressed in transgenic plants and more directly by reports of unexpected outcomes in experiments performed *in 1990s*. In an attempt to produce more intense purple coloured *Petunias*, researchers introduced additional copies of a transgene encoding chalcone synthase (a key enzyme for flower pigmentation). However, they were surprised at the result that instead of a darker flower, the *Petunias* were either variegated or completely white (Figure 1). They called this phenomenon co-suppression of gene expression (Napoli et al., 1990)*,* since both the expression of the existing gene (the initial purple colour) and the introduced gene /transgene (to deepen the purple) were suppressed. It was subsequently reported by Christine, 2008 that suppression of gene activity could take place at the transcriptional level (transcriptional gene silencing, TGS) or at the posttranscriptional level (posttranscriptional gene silencing, PTGS).

Fig. 1. Upon injection of the transgene responsible for purple colouring in *Petunias,* the flowers became variegated or white rather than deeper purple as was expected**.** 

translation (Almedia and Allshire 2005; Cotta-Ramusion et al., 2011) whereby susceptible genes can be silenced. Hence, RNA interference is the newest kid on the genetic block, allows the scientists to selectively turn off genes and finally promises to set the scientific world alight with its therapeutic potential and it has provided new platforms for developing eco-friendly molecular tools for crop improvement by suppressing the genes responsible for

The discovery of RNAi was preceded first by observations of transcriptional inhibition by antisense RNA expressed in transgenic plants and more directly by reports of unexpected outcomes in experiments performed *in 1990s*. In an attempt to produce more intense purple coloured *Petunias*, researchers introduced additional copies of a transgene encoding chalcone synthase (a key enzyme for flower pigmentation). However, they were surprised at the result that instead of a darker flower, the *Petunias* were either variegated or completely white (Figure 1). They called this phenomenon co-suppression of gene expression (Napoli et al., 1990)*,* since both the expression of the existing gene (the initial purple colour) and the introduced gene /transgene (to deepen the purple) were suppressed. It was subsequently reported by Christine, 2008 that suppression of gene activity could take place at the transcriptional level (transcriptional gene silencing, TGS) or at the posttranscriptional level

Fig. 1. Upon injection of the transgene responsible for purple colouring in *Petunias,* the flowers became variegated or white rather than deeper purple as was expected**.** 

various stresses and improving novel traits in plants including disease resistance.

**1.1 Landmarks in RNAi discovery** 

(posttranscriptional gene silencing, PTGS).

Similar PTGS like effect "Quelling" (Cogoni and Macino, 1999) was also seen in fungi *Neurospora crassa*. Another RNAi related phenomenon, coat protein mediated protection (CPMP) in plants gave insight into the mechanism of PTGS. In 1995 Guo and Kempheus first studied RNA silencing in animals. They used antisense RNA technique to silence par1 mRNA expression in *C. elegans* but found that par1 mRNA itself repressed *par1* gene and concluded that both sense and antisense RNA could cause silencing. Their observation inspired the experiment of Fire, Mello and colleagues. Thus began the journey of the newly dubbed technology RNA Interference. The mystery of molecular mechanism responsible for gene silencing now known as RNA interference (RNAi) exploded in 1998. It was discovered that PTGS was triggered by double-stranded RNA (dsRNA), provided most unexpected explanation with many profound consequences.

Andrew Fire and Craig Mello published their break-through study on the mechanism of RNA interference in 1998, *Nature*. It was earlier known that antisense RNA, but remarkably also sense RNA could silence genes, but the results were inconsistent and the effects usually modest. However, due to the fact that both sense and antisense RNA could cause silencing Mello argued that the mechanism could not just be a pairing of antisense RNA to mRNA, and he coined the term RNA interference for the unknown mechanism. In their *Nature* paper, Fire and Mello tested the phenotypic effect of RNA injected into the worm *C. elegans.* They established that annealed sense/antisense RNA, but neither antisense nor sense RNA alone, caused the predicted phenotype. Furthermore, only injection of double-stranded RNA (dsRNA) led to an efficient loss of the target mRNA. Fire and Mello made the remark that RNAi could provide an explanation for a phenomenon studied in plants for several years: posttranscriptional gene silencing (PTGS). Finally, they ended their paper by speculating about the possibility that "dsRNA could be used by the organism for physiological gene silencing". This discovery later won Fire and Mello the 2006 Nobel Prize in Physiology or Medicine. Thus it was clear that co-suppression in plants, quelling in fungi and RNAi in nematodes all shared a common mechanism. Further work showed that this effect was even more widespread, occurring in fruitflies and mammals too.

The biochemistry of RNAi was further elucidated in an *in vitro* system, built on *Drosophila* cultured cells, Elbashir et al., 2011 demonstrated that Double-stranded RNA is cut into short interfering RNA (siRNA) by the endonuclease Dicer (Lee et al., 2002). The antisense strand is loaded into the RISC (RNA-induced silencing complex) and links the complex to the endogenous mRNA by base-pairing (Martinez et al., 2002). The RISC complex cuts the mRNA strand, and the mRNA is subsequently degraded. In certain systems, in particular plants, worms and fungi, an RNA dependent RNA polymerase (RdRP) plays an important role in generating and/or amplifying siRNA. Thus, within few years a vast amount of information accumulated on the specific proteins and protein complexes involved in RNAi and molecular machinery involved in RNAi was subsequently revealed (Thakur, 2003).

#### **2. Proteins involved in RNAi/ PTGS/Quelling**

To understand the basis of RNA silencing both genetic and biochemical approaches have been undertaken. Genetic screens were carried out to search for mutants defective in quelling, RNAi or PTGS and a large number of genes whose products are somehow implicated in RNA silencing have been identified in *C. elegans*, *D. melanogaster*, *Homo sapiens*,

Phenomenal RNA Interference: From Mechanism to Application 65

The identification of the quelling-defective gene *qde-1* in *Neurospora* was the first experimental evidence of the involvement of an RdRP in PTGS. The *C. elegans* nuclear genome also contains four members of this gene family: *ego-1*, *rrf-1*, *rrf-2* and *rrf-3*. Among all three of these *rrf-1* was found as the gene coding for RdRp involved in RNAi (Correa et al., 2010). Therefore, RdRP may also be responsible for the amplification and maintenance of the silencing signal by synthesis of secondary dsRNA trigger molecules, which in turn would be processed into secondary siRNAs. However, no RdRp has been identified by

RNA interference is a classical mechanism of gene regulation found in eukaryotes as diverse as in yeast and mammals and, probably plays a central role in controlling gene expression, by inhibiting gene expression at the stage of translation or by hindering the transcription of specific genes. The RNAi pathway is initiated by the enzyme dicer, which trims long double stranded RNA, to form small interfering RNA (si RNA) or microRNA (miRNA). These processed RNAs are incorporated into the RNA-induced silencing complex (RISC), which targets messenger RNA to prevent translation. Using a recently developed *Drosophila in vitro*  system, molecular mechanism underlying RNAi was examined. It was found that RNAi is ATP dependent yet uncoupled from mRNA translation. RNAi pathway can be divided into

This is the first step in which, dsRNA is converted into 21-23bp small fragments by the enzyme Dicer. Dicer is the enzyme involved in the initiation of RNAi. It is a member of Rnase III family of dsRNA specific endonuclease that cleaves dsRNA in ATP dependent, processive manner to generate siRNA duplexes of length 21-23 bp with characteristic 2

The siRNAs generated in the initiator step now join a multinuclease effector complex RISC that mediates unwinding of the siRNA duplex. RISC is a ribonucleoprotien complex and its two signature components are the single-stranded siRNA and Argonaute family protien. The active components of an RISC are endonucleases called argonaute proteins, which cleave the target mRNA strand complementary to their bound siRNA, therefore argonaute contributes "Slicer" activity to RISC. As the fragments produced by dicer are double-stranded, they could each in theory produce a functional siRNA. However, only one of the two strands, which is known as the guide strand, binds the argonaute protein and directs gene silencing. The other anti-guide strand or passenger strand is degraded during RISC activation. The process is actually ATPindependent and performed directly by the protein components of RISC. Although it was first believed that an ATP-dependent helicase separated these two strands, the process is actually ATP-independent and performed directly by the protein components

homology in the genomes of either flies or humans.

**3. Mechanism of action** 

three major steps:

**3.1 Initiator step: dsRNA cleavage** 

of RISC (Senapedis et al., 2011).

nucleotide overhang at 3'- OH termini and 5' PO4.

**3.2 Effector step: Entry of si RNA into RISC** 

*Dictyostelium discoideum*, *N. crassa*, *Chlamydomonas reinhardtii* and *A. thaliana*. The identified genes encode various components some of which identified as initiators while others serve as effectors, amplifiers and transmitters of the gene silencing process. In the years to come, many other components as well as their interrelations will be revealed. Here, we outline what is known so far.

#### **2.1 Dicer**

The endonuclease enzyme called Dicer was first discovered in *Drosophila* by Bernstein et al., 2001. It belongs to the RNase III-family that shows specificity for dsRNAs and cleaves them with 3′ overhangs of 2 to 3 nucleotides and 5′-phosphate and 3′-hydroxyl termini (Elbashier et al., 2001; Nicholson, 1999). Dicer is involved in the first step of RNA silencing -the production of siRNAs. Owing to its ability to digest dsRNA into uniformly sized small interfering RNAs (siRNA), this enzyme was named Dicer (DCR). Dicer is ATP-dependent and contains four distinct domains an N-terminal helicase domain, a PAZ domain, a 110 amino-acid domain conservative throughout evolution found in Piwi/Argonaute/Zwille proteins in *Drosophila, Arabidopsis* and involved in developmental control (Catalanotto et al., 2000; Tabara et al., 1999) dual RNase III domains and a double stranded RNA-binding domain, ruler helix. Evolutionarily conserved Dicer homologues from many different sources were also identified and tested in plants, fungi and mammals (Bernstein et al., 2001; Ketting et al., 2000). Furthermore, some recombinant Dicers have also been examined *in vitro*, and phylogenetic analysis of the known Dicer-like proteins indicates a common ancestry of these proteins (Golden et al., 2002).

#### **2.2 RNA-induced silencing complex (RISC)**

During studies on the biochemistry of RNAi several proteins engaged in RISC formation were characterised. After partial purification of crude extracts from *Drosophila* embryolysate and human HeLa cells through differential centrifugation and anion exchange chromatography, the nuclease cofractionated with a discrete ≈25-nucleotide RNA species (siRNAs) are part of an effector nuclease which targets homologous RNAs for degradation (Hammond et al., 2000). This complex is referred to as the RNA-induced silencing complex (RISC). It is made up of a group of proteins which use the siRNA as a guide, presumably identifying the substrate through Watson and Crick base-pairing. The proteins in this complex are members of the Argonaute protein family, which are defined as having a PAZ and PIWI domains. The Argonaute PAZ domain most likely holds the 3' end of siRNA, providing the proper orientation for recognition and cleavage of mRNA. PIWI contains the active site for cleaving the mRNA, shown by the scissors in the schematic (Nykanen et al., 2001). The Argonaute family members have been linked both to the gene-silencing phenomenon and to the control of development in diverse species. The first link between Argonaute protein and RNAi was shown by isolation of *rde1* mutants of *C. elegans* in a screen for RNAi-deficient mutants. Argonaute family members have been shown to be involved in RNAi in *Neurospora crassa* (QDE3) as well as in *A. thaliana* AGO1 (Fagard et al., 2000).

#### **2.3 RNA-dependent RNA polymerase**

As a result of screening for genes involved in RNAi a family of proteins that exhibit the activity of RNA-dependent RNA polymerase (RdRP) was also identified (Birchler, 2009). The identification of the quelling-defective gene *qde-1* in *Neurospora* was the first experimental evidence of the involvement of an RdRP in PTGS. The *C. elegans* nuclear genome also contains four members of this gene family: *ego-1*, *rrf-1*, *rrf-2* and *rrf-3*. Among all three of these *rrf-1* was found as the gene coding for RdRp involved in RNAi (Correa et al., 2010). Therefore, RdRP may also be responsible for the amplification and maintenance of the silencing signal by synthesis of secondary dsRNA trigger molecules, which in turn would be processed into secondary siRNAs. However, no RdRp has been identified by homology in the genomes of either flies or humans.

#### **3. Mechanism of action**

64 Crop Plant

*Dictyostelium discoideum*, *N. crassa*, *Chlamydomonas reinhardtii* and *A. thaliana*. The identified genes encode various components some of which identified as initiators while others serve as effectors, amplifiers and transmitters of the gene silencing process. In the years to come, many other components as well as their interrelations will be revealed. Here, we outline

The endonuclease enzyme called Dicer was first discovered in *Drosophila* by Bernstein et al., 2001. It belongs to the RNase III-family that shows specificity for dsRNAs and cleaves them with 3′ overhangs of 2 to 3 nucleotides and 5′-phosphate and 3′-hydroxyl termini (Elbashier et al., 2001; Nicholson, 1999). Dicer is involved in the first step of RNA silencing -the production of siRNAs. Owing to its ability to digest dsRNA into uniformly sized small interfering RNAs (siRNA), this enzyme was named Dicer (DCR). Dicer is ATP-dependent and contains four distinct domains an N-terminal helicase domain, a PAZ domain, a 110 amino-acid domain conservative throughout evolution found in Piwi/Argonaute/Zwille proteins in *Drosophila, Arabidopsis* and involved in developmental control (Catalanotto et al., 2000; Tabara et al., 1999) dual RNase III domains and a double stranded RNA-binding domain, ruler helix. Evolutionarily conserved Dicer homologues from many different sources were also identified and tested in plants, fungi and mammals (Bernstein et al., 2001; Ketting et al., 2000). Furthermore, some recombinant Dicers have also been examined *in vitro*, and phylogenetic analysis of the known Dicer-like proteins indicates a common

During studies on the biochemistry of RNAi several proteins engaged in RISC formation were characterised. After partial purification of crude extracts from *Drosophila* embryolysate and human HeLa cells through differential centrifugation and anion exchange chromatography, the nuclease cofractionated with a discrete ≈25-nucleotide RNA species (siRNAs) are part of an effector nuclease which targets homologous RNAs for degradation (Hammond et al., 2000). This complex is referred to as the RNA-induced silencing complex (RISC). It is made up of a group of proteins which use the siRNA as a guide, presumably identifying the substrate through Watson and Crick base-pairing. The proteins in this complex are members of the Argonaute protein family, which are defined as having a PAZ and PIWI domains. The Argonaute PAZ domain most likely holds the 3' end of siRNA, providing the proper orientation for recognition and cleavage of mRNA. PIWI contains the active site for cleaving the mRNA, shown by the scissors in the schematic (Nykanen et al., 2001). The Argonaute family members have been linked both to the gene-silencing phenomenon and to the control of development in diverse species. The first link between Argonaute protein and RNAi was shown by isolation of *rde1* mutants of *C. elegans* in a screen for RNAi-deficient mutants. Argonaute family members have been shown to be involved in RNAi in *Neurospora* 

As a result of screening for genes involved in RNAi a family of proteins that exhibit the activity of RNA-dependent RNA polymerase (RdRP) was also identified (Birchler, 2009).

what is known so far.

ancestry of these proteins (Golden et al., 2002).

**2.2 RNA-induced silencing complex (RISC)** 

*crassa* (QDE3) as well as in *A. thaliana* AGO1 (Fagard et al., 2000).

**2.3 RNA-dependent RNA polymerase** 

**2.1 Dicer** 

RNA interference is a classical mechanism of gene regulation found in eukaryotes as diverse as in yeast and mammals and, probably plays a central role in controlling gene expression, by inhibiting gene expression at the stage of translation or by hindering the transcription of specific genes. The RNAi pathway is initiated by the enzyme dicer, which trims long double stranded RNA, to form small interfering RNA (si RNA) or microRNA (miRNA). These processed RNAs are incorporated into the RNA-induced silencing complex (RISC), which targets messenger RNA to prevent translation. Using a recently developed *Drosophila in vitro*  system, molecular mechanism underlying RNAi was examined. It was found that RNAi is ATP dependent yet uncoupled from mRNA translation. RNAi pathway can be divided into three major steps:

#### **3.1 Initiator step: dsRNA cleavage**

This is the first step in which, dsRNA is converted into 21-23bp small fragments by the enzyme Dicer. Dicer is the enzyme involved in the initiation of RNAi. It is a member of Rnase III family of dsRNA specific endonuclease that cleaves dsRNA in ATP dependent, processive manner to generate siRNA duplexes of length 21-23 bp with characteristic 2 nucleotide overhang at 3'- OH termini and 5' PO4.

#### **3.2 Effector step: Entry of si RNA into RISC**

The siRNAs generated in the initiator step now join a multinuclease effector complex RISC that mediates unwinding of the siRNA duplex. RISC is a ribonucleoprotien complex and its two signature components are the single-stranded siRNA and Argonaute family protien. The active components of an RISC are endonucleases called argonaute proteins, which cleave the target mRNA strand complementary to their bound siRNA, therefore argonaute contributes "Slicer" activity to RISC. As the fragments produced by dicer are double-stranded, they could each in theory produce a functional siRNA. However, only one of the two strands, which is known as the guide strand, binds the argonaute protein and directs gene silencing. The other anti-guide strand or passenger strand is degraded during RISC activation. The process is actually ATPindependent and performed directly by the protein components of RISC. Although it was first believed that an ATP-dependent helicase separated these two strands, the process is actually ATP-independent and performed directly by the protein components of RISC (Senapedis et al., 2011).

Phenomenal RNA Interference: From Mechanism to Application 67

In addition to naturally occurring and manufactured siRNAs (Sigoillot and King, 2011),

These are an abundant class of short (19–25 nt) single-stranded RNAs that are expressed in all higher eukaryotes. They are encoded in the host genome and are processed by Rnase III nuclease Dicer from 70nt hairpin precursors). They can silence gene activity through destruction of homologous mRNA in plants or blocking its translation in plants and animals (Cullen, 2004; Novina and Sharp 2004). Recent work has identified their specific roles in the regulation of early haematopoiesis and lineage commitment). They are sometimes referred to as small temporal RNAs as a reflection of their importance in the regulation of

These are single-stranded 25–31 nt RNAs which have recently been detected in mouse, rat, and human testes. They have been shown to associate with Piwi protein (a subclass of Argonaute proteins) and the human RecQ1 protein to form a Piwi-interacting RNA complex (piRC). These complexes are thought to regulate the genome within developing sperm cells

Short hairpin RNAor shRNA or is a synthetically manufactured RNA molecule of 19–29 nucleotides that contains a sense strand, antisense strand, and a short loop sequence between the sense and antisense fragments. Due to the complementarity of the sense and antisense fragments in their sequence, such RNA molecules tend to form hairpin-shaped double-stranded RNA (dsRNA). shRNA is cloned into a vector, allowing for expression by a pol III type promoter. The expressed shRNA is then exported into the cytoplasm where it is processed by dicer into siRNA which then get incorporated into the siRNA induced

These are short, double-stranded RNAs which are found in the nucleus of neural stem cells of mice. They play a critical role in mediating neuronal differentiation through

RNA interference occurs in plants, animals, and humans. It is of great importance for the regulation of gene expression, participates in defense against viral infections, and keeps jumping genes under control. Presently RNA interference has become attractive tool for various researchers and widely used in basic science as a method to study the function of

there have been recent publications of alternative forms ofRNA, these are

developmental timing (Chen et al., 2004; Medema et al., 2004).

**4. Other forms of RNA interference** 

**4.1 Micro (mi)–RNAs** 

**4.2 Piwi-interacting (pi) RNAs** 

**4.3 Short-Hairpin (sh) RNAs** 

silencing complex (RISC)(Medema, 2004).

dsRNA/protein interaction (Kuwabara etal., 2004).

genes and it may lead to novel therapies in the future.

**4.4 Small modulatory (sm)RNAs** 

**5. RNAi application** 

(Carthew, 2006).

#### **3.3 Step 3: Sequence specific cleavage to targeted mRNA**

The active RISC further promotes unwinding of siRNA through an ATP dependent process and the unwound antisense strand guides active RISC to the complementary mRNA. The targeted mRNA is cleaved by RISC at a single site that is defined with regard to where the 5' end of the antisense strand is bound to mRNA target sequence. The RISC cleaves the complimentary mRNA in the middle, ten nucleotides upstream of the nucleotide paired with the 5' end of the guide siRNA. This cleavage reaction is independent of ATP. The target RNA hydrolysis reaction requires Mg2+ ions. Cleavage is catalyzed by the PIWI Domain of a subclass of Argonaute proteins. This domain is a structural homolog of RNase H, a Mg2+ dependant endoribonuclease that cleaves the RNA strand of RNA- DNA hybrids. But each cleavage- competent RISC can break only one phosphodiester bond in its RNA target. The siRNA guide delivers RISC to the target region, the target is cleaved, and then siRNA departs intact with the RISC.

Thus the two important conditions to be fulfilled for the success of silencing by RNAi are established as: 5' phoshorylation of the antisense strand and the double helix of the antisense target mRNA duplex to be in the A form. The A-form helix is required for the stabilization of the heteroduplex formation between the siRNA antisense strand and its target mRNA.

Fig. 2. Mechanism of action of RNAi. Double stranded RNA is introduced into a cell is chopped bydicer to form siRNA, which binds to the RISC complex and is unwound. The anitsense RNA complexed with RISC binds to its corresponding mRNA which is the cleaved by the enzyme slicer rendering it inactive (Christine, 2008).

### **4. Other forms of RNA interference**

In addition to naturally occurring and manufactured siRNAs (Sigoillot and King, 2011), there have been recent publications of alternative forms ofRNA, these are

#### **4.1 Micro (mi)–RNAs**

66 Crop Plant

The active RISC further promotes unwinding of siRNA through an ATP dependent process and the unwound antisense strand guides active RISC to the complementary mRNA. The targeted mRNA is cleaved by RISC at a single site that is defined with regard to where the 5' end of the antisense strand is bound to mRNA target sequence. The RISC cleaves the complimentary mRNA in the middle, ten nucleotides upstream of the nucleotide paired with the 5' end of the guide siRNA. This cleavage reaction is independent of ATP. The target RNA hydrolysis reaction requires Mg2+ ions. Cleavage is catalyzed by the PIWI Domain of a subclass of Argonaute proteins. This domain is a structural homolog of RNase H, a Mg2+ dependant endoribonuclease that cleaves the RNA strand of RNA- DNA hybrids. But each cleavage- competent RISC can break only one phosphodiester bond in its RNA target. The siRNA guide delivers RISC to the target region, the target is cleaved, and then siRNA

Thus the two important conditions to be fulfilled for the success of silencing by RNAi are established as: 5' phoshorylation of the antisense strand and the double helix of the antisense target mRNA duplex to be in the A form. The A-form helix is required for the stabilization of

the heteroduplex formation between the siRNA antisense strand and its target mRNA.

Fig. 2. Mechanism of action of RNAi. Double stranded RNA is introduced into a cell is chopped bydicer to form siRNA, which binds to the RISC complex and is unwound. The anitsense RNA complexed with RISC binds to its corresponding mRNA which is the cleaved

by the enzyme slicer rendering it inactive (Christine, 2008).

**3.3 Step 3: Sequence specific cleavage to targeted mRNA** 

departs intact with the RISC.

These are an abundant class of short (19–25 nt) single-stranded RNAs that are expressed in all higher eukaryotes. They are encoded in the host genome and are processed by Rnase III nuclease Dicer from 70nt hairpin precursors). They can silence gene activity through destruction of homologous mRNA in plants or blocking its translation in plants and animals (Cullen, 2004; Novina and Sharp 2004). Recent work has identified their specific roles in the regulation of early haematopoiesis and lineage commitment). They are sometimes referred to as small temporal RNAs as a reflection of their importance in the regulation of developmental timing (Chen et al., 2004; Medema et al., 2004).

#### **4.2 Piwi-interacting (pi) RNAs**

These are single-stranded 25–31 nt RNAs which have recently been detected in mouse, rat, and human testes. They have been shown to associate with Piwi protein (a subclass of Argonaute proteins) and the human RecQ1 protein to form a Piwi-interacting RNA complex (piRC). These complexes are thought to regulate the genome within developing sperm cells (Carthew, 2006).

#### **4.3 Short-Hairpin (sh) RNAs**

Short hairpin RNAor shRNA or is a synthetically manufactured RNA molecule of 19–29 nucleotides that contains a sense strand, antisense strand, and a short loop sequence between the sense and antisense fragments. Due to the complementarity of the sense and antisense fragments in their sequence, such RNA molecules tend to form hairpin-shaped double-stranded RNA (dsRNA). shRNA is cloned into a vector, allowing for expression by a pol III type promoter. The expressed shRNA is then exported into the cytoplasm where it is processed by dicer into siRNA which then get incorporated into the siRNA induced silencing complex (RISC)(Medema, 2004).

#### **4.4 Small modulatory (sm)RNAs**

These are short, double-stranded RNAs which are found in the nucleus of neural stem cells of mice. They play a critical role in mediating neuronal differentiation through dsRNA/protein interaction (Kuwabara etal., 2004).

#### **5. RNAi application**

RNA interference occurs in plants, animals, and humans. It is of great importance for the regulation of gene expression, participates in defense against viral infections, and keeps jumping genes under control. Presently RNA interference has become attractive tool for various researchers and widely used in basic science as a method to study the function of genes and it may lead to novel therapies in the future.

Phenomenal RNA Interference: From Mechanism to Application 69

studies have been undertaken to elucidate the role of specific genes in basic cellular processes like DNA damage response and cell cycle control (Brummelkamp et al., 2002), general cell metabolism, signaling, the cytoskeleton and its rearrangement during mitosis,

Heterochromatin is composed of highly repetitive sequences interspersed with transposons and is non-coding. The condensation pattern is determined by both DNA and histone modification. Recently it has been found to produce RNAi which appear to be the key factor in epigenetic regulation of gene expression, chromosome behaviour and evolution. It may be the mechanism underlying genome imprinting whereby chromosomal condensation pattern is determined by parent-of-origin. Even the phenomenon of hybrid dysgenesis may be explained if siRNA pools that are largely maternal, do not match polymorphic repeats from the paternal chromosome it may result in mobilisation of transposons and consequent

RNAi has been adapted with high-throughput screening formats in *C. elegans*, for which the recombination-based gene knockout technique was not established. Recently, a large-scale functional analysis of 19,427 predicted genes of *C. elegans*, was carried out with RNA interference. This study identified mutant phenotypes for 1,722genes. RNAi technology has been similarly used in the identification of several genes in *D. melanogaster* involved in

In plants, gene knockdown-related functional studies are being carried out efficiently with transgenes present in the form of hairpin (or RNAi) constructs. Plant endotoxins could be removed if the toxin biosynthesis genes are knocked out. SiRNA results in partial knockout, which is an advantage over complete knockout in that it helps in investigating the effect of various phenotypes. Thus the method holds a great potential to become the most commonly

Prior to the discovery of RNAi, scientists applied various methods such as insertion of T-DNA elements, transposons, treatment with mutagens or irradiation. These approaches are very cumbersome and the above methods did not always work adequately. For instance, transposons and T-DNA elements were found to occasionally insert randomly in the genome resulting in highly variable gene expression. Furthermore, in many instances the particular phenotype or a trait could not be correlated with the function of a gene of interest. At the same time to improve crop plants transgenes are mainly introduced into the genomes of most model plant species using *Agrobacterium tumefaciens*, a common soil bacterium, and the mechanism of which relies on T-DNA (transfer DNA), that is carried on a resident plasmid. Single T-DNAs can integrate into the genome, but it is very common for multiple copies to integrate in variously permuted head-to-head, tail-to-tail and head-to tail arrays. As we all know till now the most effective genetic approach to pest control has been to make plants that produce a protein called Bt toxin, which causes insects to slow down, then stop eating crops, then die. More than 120,000 square miles of crops genetically engineered to produce Bt were grown last year. But Bt isn't effective against many pests, including corn rootworm, which can cause such extensive damage to corn plants' root systems that the plants blow over in the wind and researchers are concerned that insect pests are becoming

membrane trafficking, transcription and, DNA methylation.

biochemical signaling cascade as well as embryonic development.

used technique for gene annotation in the near future.

**5.2 Genetic improvement of crop plants** 

chromosomal disruption.

Over the past decade "RNA interference" has emerged as a natural antiviral mechanism for protecting organisms from viruses. It blocks infection by RNA viruses especially in plants and lower animals. For instance, replication of plant viruses, many of which produce dsRNA replication intermediates, very effectively cause a type of RNA silencing called VIGS (Virus induced gene silencing). When viruses or transgenes are introduced into plants, they trigger a post transcriptional gene silencing response in which double stranded RNA molecules, which may be generated by replicative intermediates of viral RNAs or by aberrant transgene coded RNAs. The dsRNAs are then digested into 21-25 nt small interfering RNAs or siRNAs. The siRNAs subsequently assemble into a nuclease complex called RISC, guiding the complex to bind and destroy homologous transcripts. PTGS is believed to be an anti-viral response. Viral RNAs not only trigger PTGS, but they also serve as targets. Cleavages of viral RNA results in reduce virus titers in local and distant leaves and a plant recovery phenotype. In response numerous plant viruses have evolved proteins to suppress PTGS (Elbashir et al., 2001). The results by Silhavy and his colleagues suggest that tombavirus p19 protein suppresses local PTGS by binding to 21-25nt siRNAs. Therefore, siRNAs and not the longer dsRNAs, act as mobile silencing element since p19 can in hibit systemic silencing and p19 can only bind to siRNAs (Sui et al., 2002).

RNA interference besides being working as a genetic immune system against virus**,** it holds a promising key for maintaining the genome stability by suppressing the movement of mobile genetic elements such as transposons. Jumping genes, also known as transposons, are DNA sequences that can move around in the genome. They are present in all organisms and can cause deleterious effects in the genome if they end up in the wrong place. Many transposons operate by copying their DNA to RNA, which is then reverse-transcribed back to DNA and inserted at another site in the genome. Part of this RNA molecule is often double-stranded and can be targeted by RNA interference. **.** Thus in this way, foreign elements in the genome (viruses and transposons) can be kept silent.

Moreover, RNAi can be used greatly in both the mammer both "forward genetic" experiments (identifying the gene responsible for a given phenotype) and "reverse genetic" experiments (identifying the function of a known gene). From an application point of view, RNAi may also be useful as a therapy for diseases arising from aberrant gene expression. Typical reverse genetic experiments involve designing siRNAs (chemically or enzymatically synthesized) expressing constructs targeting a gene of interest. Following transient transfection of siRNAs, the phenotype of the cells is assessed using appropriate functional assays.

#### **5.1 Functional genomics**

The technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes. Genome sequencing projects have generated wealth of information regarding gene sequences but still clarity on functional role of all genes is missing. The use of small interfering RNA (siRNA) to knock down/ knockout expression of specific gene have opened up exciting possibilities in the study of functional genomics. The ability to easily and economically silence genes promises to elucidate numerous signaling, developmental, metabolic, and related disease pathways. Various

Over the past decade "RNA interference" has emerged as a natural antiviral mechanism for protecting organisms from viruses. It blocks infection by RNA viruses especially in plants and lower animals. For instance, replication of plant viruses, many of which produce dsRNA replication intermediates, very effectively cause a type of RNA silencing called VIGS (Virus induced gene silencing). When viruses or transgenes are introduced into plants, they trigger a post transcriptional gene silencing response in which double stranded RNA molecules, which may be generated by replicative intermediates of viral RNAs or by aberrant transgene coded RNAs. The dsRNAs are then digested into 21-25 nt small interfering RNAs or siRNAs. The siRNAs subsequently assemble into a nuclease complex called RISC, guiding the complex to bind and destroy homologous transcripts. PTGS is believed to be an anti-viral response. Viral RNAs not only trigger PTGS, but they also serve as targets. Cleavages of viral RNA results in reduce virus titers in local and distant leaves and a plant recovery phenotype. In response numerous plant viruses have evolved proteins to suppress PTGS (Elbashir et al., 2001). The results by Silhavy and his colleagues suggest that tombavirus p19 protein suppresses local PTGS by binding to 21-25nt siRNAs. Therefore, siRNAs and not the longer dsRNAs, act as mobile silencing element since p19 can

in hibit systemic silencing and p19 can only bind to siRNAs (Sui et al., 2002).

elements in the genome (viruses and transposons) can be kept silent.

assays.

**5.1 Functional genomics** 

RNA interference besides being working as a genetic immune system against virus**,** it holds a promising key for maintaining the genome stability by suppressing the movement of mobile genetic elements such as transposons. Jumping genes, also known as transposons, are DNA sequences that can move around in the genome. They are present in all organisms and can cause deleterious effects in the genome if they end up in the wrong place. Many transposons operate by copying their DNA to RNA, which is then reverse-transcribed back to DNA and inserted at another site in the genome. Part of this RNA molecule is often double-stranded and can be targeted by RNA interference. **.** Thus in this way, foreign

Moreover, RNAi can be used greatly in both the mammer both "forward genetic" experiments (identifying the gene responsible for a given phenotype) and "reverse genetic" experiments (identifying the function of a known gene). From an application point of view, RNAi may also be useful as a therapy for diseases arising from aberrant gene expression. Typical reverse genetic experiments involve designing siRNAs (chemically or enzymatically synthesized) expressing constructs targeting a gene of interest. Following transient transfection of siRNAs, the phenotype of the cells is assessed using appropriate functional

The technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes. Genome sequencing projects have generated wealth of information regarding gene sequences but still clarity on functional role of all genes is missing. The use of small interfering RNA (siRNA) to knock down/ knockout expression of specific gene have opened up exciting possibilities in the study of functional genomics. The ability to easily and economically silence genes promises to elucidate numerous signaling, developmental, metabolic, and related disease pathways. Various studies have been undertaken to elucidate the role of specific genes in basic cellular processes like DNA damage response and cell cycle control (Brummelkamp et al., 2002), general cell metabolism, signaling, the cytoskeleton and its rearrangement during mitosis, membrane trafficking, transcription and, DNA methylation.

Heterochromatin is composed of highly repetitive sequences interspersed with transposons and is non-coding. The condensation pattern is determined by both DNA and histone modification. Recently it has been found to produce RNAi which appear to be the key factor in epigenetic regulation of gene expression, chromosome behaviour and evolution. It may be the mechanism underlying genome imprinting whereby chromosomal condensation pattern is determined by parent-of-origin. Even the phenomenon of hybrid dysgenesis may be explained if siRNA pools that are largely maternal, do not match polymorphic repeats from the paternal chromosome it may result in mobilisation of transposons and consequent chromosomal disruption.

RNAi has been adapted with high-throughput screening formats in *C. elegans*, for which the recombination-based gene knockout technique was not established. Recently, a large-scale functional analysis of 19,427 predicted genes of *C. elegans*, was carried out with RNA interference. This study identified mutant phenotypes for 1,722genes. RNAi technology has been similarly used in the identification of several genes in *D. melanogaster* involved in biochemical signaling cascade as well as embryonic development.

In plants, gene knockdown-related functional studies are being carried out efficiently with transgenes present in the form of hairpin (or RNAi) constructs. Plant endotoxins could be removed if the toxin biosynthesis genes are knocked out. SiRNA results in partial knockout, which is an advantage over complete knockout in that it helps in investigating the effect of various phenotypes. Thus the method holds a great potential to become the most commonly used technique for gene annotation in the near future.

#### **5.2 Genetic improvement of crop plants**

Prior to the discovery of RNAi, scientists applied various methods such as insertion of T-DNA elements, transposons, treatment with mutagens or irradiation. These approaches are very cumbersome and the above methods did not always work adequately. For instance, transposons and T-DNA elements were found to occasionally insert randomly in the genome resulting in highly variable gene expression. Furthermore, in many instances the particular phenotype or a trait could not be correlated with the function of a gene of interest. At the same time to improve crop plants transgenes are mainly introduced into the genomes of most model plant species using *Agrobacterium tumefaciens*, a common soil bacterium, and the mechanism of which relies on T-DNA (transfer DNA), that is carried on a resident plasmid. Single T-DNAs can integrate into the genome, but it is very common for multiple copies to integrate in variously permuted head-to-head, tail-to-tail and head-to tail arrays. As we all know till now the most effective genetic approach to pest control has been to make plants that produce a protein called Bt toxin, which causes insects to slow down, then stop eating crops, then die. More than 120,000 square miles of crops genetically engineered to produce Bt were grown last year. But Bt isn't effective against many pests, including corn rootworm, which can cause such extensive damage to corn plants' root systems that the plants blow over in the wind and researchers are concerned that insect pests are becoming

Phenomenal RNA Interference: From Mechanism to Application 71

The ability to tap this native RNAi pathway has been recognized as one the most exciting biotechnology advances in the last decade. Given the gene-specific features of RNAi, it is conceivable that this method will play an important role in therapeutic applications and possibly of most commercial interest in the use of RNAi as a therapeutic agent. Indeed, RNAi has revolutionized biology research, including drug target discovery, by allowing for rapid identification and validation of gene function. There are three main time points at which a disease can be stopped. These are transcriptional, post-transcriptional, and posttranslational intervention. Before the discovery of antisense RNA and RNAi, most of the drug targets have been proteins, and therefore, post- translational intervention. RNAi is a way to control the development of a disease earlier on in the process. Furthermore, the genespecific features and potential of RNAi for knocking out a protein without harming a cell has established its most believable role into therapeutic applications. The inhibitory action of siRNAs has been documented for numerous diseases. Some of the examples are

Gregory Hannon and colleagues have used RNAi to silence expression of p53 — the 'guardian of the genome', which protects against any tumour-associated DNA damage — by introducing several p53-targeting shRNAs into stem cells and looking at the effect in mice (Hemann et al., 2003). The shRNAs produced a wide range of clinical effects, ranging from benign to malignant tumours, the severity and type of which correlated with the extent to which the shRNA had silenced p53. As tumour suppressors such as p53 usually work as part of a complex and finely regulated network, the ability to dampen these networks to varying degrees in these libraries- which the authors term an epi-allelic series of hypomorphic mutations -will be of enormous value when it comes to investigating the early stages of disease. The success of these modified stem cells also gives hope that this could treat diseases in which stem cells can be modified *ex vivo* and then re-introduced into the

Researchers at the charity Cancer Research UK and the Netherlands Cancer Institute have recently announced that they intend to generate a large library of human cells, each containing a silenced gene. They initially want to silence 300–8,000 cancer genes, and hope to eventually cover the entire human genome. Their aim is to uncover all the genes that become overexpressed in human cancers and to find out precisely what needs to be taken away from a cancerous cell in order to make it normal again (Hoffman et al., 2010).

HIV infection can be blocked by targeting either viral genes (for example, *gag*, *rev*, *tat* and *env*) or human genes (for example, *CD4*, the principal receptor for HIV) that are involved in the HIV life cycle.The strategy used was to silence the main structural protein in the virus, p24, and the human protein *CD4*, which the virus needs to enter the cells. This impairs the virus in infected cells and limits its spread into healthy cells (Paddison et al., 2002). Hence the production of virus is inhibited either by blocking new infections or blocking the production of new viral particles in infected cells (Lohman et

**5.3 RNA interference as a novel therapeutic agent** 

highlighted below:

affected individual.

**5.3.2 HIV protection** 

**5.3.1 Cancer protection** 

resistant to Bt. Here RNAi play a vital role. Now a day, researchers are trying to create plants that kill insects by disrupting their gene expression. The crops, which initiate a genesilencing response called RNA interference, are a step beyond existing genetically modified crops that produce toxic proteins. Because the new crops target particular genes in particular insects, some researchers suggest that they will be safer and less likely to have unintended effects than other genetically modified plants. Moreover, the quality of crop plants can be improved by RNAi for example Kusaba M 2004 have made significant contribution by applying RNAi to improve rice plants. They were able to reduce the level of glutenin and produced a rice variety called *LGC-1* (low glutenin content 1).The rice mutant line LGC-1 (Low Glutenin Content-1) was the first commercially useful cultivar produced by RNAi. It is low-protein rice and is useful for patients with kidney disease whose protein intake is restricted. This dominant mutation produces hairpin RNA (hpRNA) from an inverted repeat for glutenin, the gene for the major storage protein glutenin, leading to lower glutenin content in the rice through RNAi. Rice down regulation can also be achieved through mutation-based reverse genetics and a gene targeting system (Terada et al., 2002; Shinozuka et al., 2003). However, RNAi has some advantages over these systems. One of these is its applicability to multigene families and polyploidy (Lawrence and Pikaard 2003), as it is not straightforward to knockout a multigene family by the accumulation of mutations for each member of the family by conventional breeding, particularly if members of the family are tightly linked. Another advantage of RNAi lies in the ability to regulate the degree of suppression. Agronomic traits are often quantitative, and a particular degree of suppression of target genes may be required. Control of the level of expression of dsRNA through the choice of promoters with various strengths is thought to be useful in regulating the degree of suppression. However, for wider application of transgene- based RNAi to the genetic improvement of crop plants further feasibility studies are needed.

Engineering of food plants that produce lower levels of natural plant toxins also possible through RNAi. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. For example, cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol, making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of deltacadinene synthase, a key enzyme in gossypol production, without affecting the enzyme's production in other parts of the plant, where gossypol is important in preventing damage from plant pest (Kumar et al., 2006). Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plant (Siitunga and Sayre 2003). Although no plant products that use RNAi-based genetic engineering have yet passed the experimental stage, development efforts have successfully reduced the levels of allergens in tomato plants and decreased the precursors of likely carcinogens in tobacco plant (Gavilano et al., 2006). Other plant traits that have been engineered in the laboratory include the production of non-narcotic natural products by the opium poppy (Allen et al., 2004) resistance to common plant viruses (Zadeh and Foster 2004) and fortification of plants such as tomatoes with dietary antioxidants (Niggeweg et al., 2004). In plants, gene knockdownrelated functional studies are being carried out efficiently when transgenes are present in the form of hairpin (or RNAi) constructs. Plant endotoxins could also be removed if the toxin biosynthesis genes are targeted with the RNAi constructs. Therefore, RNAi soon caught the world-wide attention and became a powerful and useful tool for molecular breeders to produce improved crop varieties.

#### **5.3 RNA interference as a novel therapeutic agent**

The ability to tap this native RNAi pathway has been recognized as one the most exciting biotechnology advances in the last decade. Given the gene-specific features of RNAi, it is conceivable that this method will play an important role in therapeutic applications and possibly of most commercial interest in the use of RNAi as a therapeutic agent. Indeed, RNAi has revolutionized biology research, including drug target discovery, by allowing for rapid identification and validation of gene function. There are three main time points at which a disease can be stopped. These are transcriptional, post-transcriptional, and posttranslational intervention. Before the discovery of antisense RNA and RNAi, most of the drug targets have been proteins, and therefore, post- translational intervention. RNAi is a way to control the development of a disease earlier on in the process. Furthermore, the genespecific features and potential of RNAi for knocking out a protein without harming a cell has established its most believable role into therapeutic applications. The inhibitory action of siRNAs has been documented for numerous diseases. Some of the examples are highlighted below:

#### **5.3.1 Cancer protection**

70 Crop Plant

resistant to Bt. Here RNAi play a vital role. Now a day, researchers are trying to create plants that kill insects by disrupting their gene expression. The crops, which initiate a genesilencing response called RNA interference, are a step beyond existing genetically modified crops that produce toxic proteins. Because the new crops target particular genes in particular insects, some researchers suggest that they will be safer and less likely to have unintended effects than other genetically modified plants. Moreover, the quality of crop plants can be improved by RNAi for example Kusaba M 2004 have made significant contribution by applying RNAi to improve rice plants. They were able to reduce the level of glutenin and produced a rice variety called *LGC-1* (low glutenin content 1).The rice mutant line LGC-1 (Low Glutenin Content-1) was the first commercially useful cultivar produced by RNAi. It is low-protein rice and is useful for patients with kidney disease whose protein intake is restricted. This dominant mutation produces hairpin RNA (hpRNA) from an inverted repeat for glutenin, the gene for the major storage protein glutenin, leading to lower glutenin content in the rice through RNAi. Rice down regulation can also be achieved through mutation-based reverse genetics and a gene targeting system (Terada et al., 2002; Shinozuka et al., 2003). However, RNAi has some advantages over these systems. One of these is its applicability to multigene families and polyploidy (Lawrence and Pikaard 2003), as it is not straightforward to knockout a multigene family by the accumulation of mutations for each member of the family by conventional breeding, particularly if members of the family are tightly linked. Another advantage of RNAi lies in the ability to regulate the degree of suppression. Agronomic traits are often quantitative, and a particular degree of suppression of target genes may be required. Control of the level of expression of dsRNA through the choice of promoters with various strengths is thought to be useful in regulating the degree of suppression. However, for wider application of transgene- based RNAi to the

genetic improvement of crop plants further feasibility studies are needed.

breeders to produce improved crop varieties.

Engineering of food plants that produce lower levels of natural plant toxins also possible through RNAi. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. For example, cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol, making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of deltacadinene synthase, a key enzyme in gossypol production, without affecting the enzyme's production in other parts of the plant, where gossypol is important in preventing damage from plant pest (Kumar et al., 2006). Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plant (Siitunga and Sayre 2003). Although no plant products that use RNAi-based genetic engineering have yet passed the experimental stage, development efforts have successfully reduced the levels of allergens in tomato plants and decreased the precursors of likely carcinogens in tobacco plant (Gavilano et al., 2006). Other plant traits that have been engineered in the laboratory include the production of non-narcotic natural products by the opium poppy (Allen et al., 2004) resistance to common plant viruses (Zadeh and Foster 2004) and fortification of plants such as tomatoes with dietary antioxidants (Niggeweg et al., 2004). In plants, gene knockdownrelated functional studies are being carried out efficiently when transgenes are present in the form of hairpin (or RNAi) constructs. Plant endotoxins could also be removed if the toxin biosynthesis genes are targeted with the RNAi constructs. Therefore, RNAi soon caught the world-wide attention and became a powerful and useful tool for molecular Gregory Hannon and colleagues have used RNAi to silence expression of p53 — the 'guardian of the genome', which protects against any tumour-associated DNA damage — by introducing several p53-targeting shRNAs into stem cells and looking at the effect in mice (Hemann et al., 2003). The shRNAs produced a wide range of clinical effects, ranging from benign to malignant tumours, the severity and type of which correlated with the extent to which the shRNA had silenced p53. As tumour suppressors such as p53 usually work as part of a complex and finely regulated network, the ability to dampen these networks to varying degrees in these libraries- which the authors term an epi-allelic series of hypomorphic mutations -will be of enormous value when it comes to investigating the early stages of disease. The success of these modified stem cells also gives hope that this could treat diseases in which stem cells can be modified *ex vivo* and then re-introduced into the affected individual.

Researchers at the charity Cancer Research UK and the Netherlands Cancer Institute have recently announced that they intend to generate a large library of human cells, each containing a silenced gene. They initially want to silence 300–8,000 cancer genes, and hope to eventually cover the entire human genome. Their aim is to uncover all the genes that become overexpressed in human cancers and to find out precisely what needs to be taken away from a cancerous cell in order to make it normal again (Hoffman et al., 2010).

#### **5.3.2 HIV protection**

HIV infection can be blocked by targeting either viral genes (for example, *gag*, *rev*, *tat* and *env*) or human genes (for example, *CD4*, the principal receptor for HIV) that are involved in the HIV life cycle.The strategy used was to silence the main structural protein in the virus, p24, and the human protein *CD4*, which the virus needs to enter the cells. This impairs the virus in infected cells and limits its spread into healthy cells (Paddison et al., 2002). Hence the production of virus is inhibited either by blocking new infections or blocking the production of new viral particles in infected cells (Lohman et

Phenomenal RNA Interference: From Mechanism to Application 73

dimensions in the various field of science. However, a better and comprehensive understanding of RNAi should allow future researchers to work effectively and efficiently

Allen, R., Millgate, A., Chitty, J., Thisleton, J., Miller, J., Fist, A., Gerlach, W., & Larkin, P.

Almeida, R. & Allshire, R. C. (2005) RNA silencing and genome regulation. *Trends Cell* 

Bernstein, E. A., Caudy, A., Hammond, S. M. & Hannon, G. J. (2001) Role for a

Brichler, J. A. (2009) Ubiquitous RNA-dependent RNA polymerase and gene silencing.

Brummelkamp, T. R., Bernards, R. & Agami, R. (2002) A system for stable expression of

Carrington, J. C. & Ambros, V. (2003) Role of Micro RNAs in plant and animal development.

Chen, C., Li, L., Lodish, H. F. & Bartel, D. P. (2004) MicroRNAs modulate haematopoeitic

Cogoni, C. & Macino, G. (1999) Homology dependent gene silencing in plants and

Corrêa, R. L., Steiner, F. A., Berezikov, E. & Ketting, R. F. (2010) MicroRNA–Directed siRNA

Cotta-Ramusino, C., McDonald, E. R. 3rd, Hurov, K., Sowa, M. E., Harper, J. W. &

Cullen, B. R. (2004) Derivation and function of small interfering RNAs and microRNAs.

Elbashir, S. M., Lendeckel, W. & Tuschl, T. (2001) RNA interference is mediated by 21- and

Fagard, M., Boutet, S., Morel, J. B., Bellini, C. & Vaucheret, H. (2000) AGO1, QDE-2, and

fungi : a number of variations on the same theme. *Curr. Opin. Microbiol.* 2(6):

Elledge, S. J. (2011) A DNA damage response screen identifies RHINO, a 9-1-1 and TopBP1 interacting protein required for ATR signaling. *Science.* 332:1313-

RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals. *Proc. Natl. Acad. Sci.*

Carthew, R. W. (2006) A new RNA dimension to genome control. *Science.* 313: 305-306. Catalanotto, C., Azzalin, G., Macino, G. & Cogoni, C. (2000) Gene silencing in worms and

Christine, A. (2008) Antisense RNA *The Science Creative Quarterly*, Issue Three 25-30.

biogenesis in *Caenorhabditis elegans*. *PLoS Genet.* 6(4): e1000903.

short interfering RNAs in mammalian cells. *Science.* 296:550-553.

reticuline in opium poppy". *Nat. Biotechnol.* 22: 1559-1566.

(2004) RNAi-mediated replacement of morphine with the nonnarcotic alkaloid

bidentate ribonuclease in the initiation step of RNA interference. *Nature.* 409:363-

in order to manage the phenomenon.

*Biol.* 15(5):251-258.

*Genome Biol.* 10:243.1-243.3.

*Science* 301: 336-338.

fungi. *Nature.* 404:245.

657-662.

1317.

*Virus Res*. 102:3-9.

97:11650-11654.

lineage differentiation. *Science.* 303: 83-86.

22-nucleotide RNAs. *Genes Dev.* 15:188-200.

**7. References** 

366.

al., 1999). The concept of silencing genes in HIV is straightforward: Hit the virus where it counts by eliminating a protein it needs to reproduce or cause infection. siRNA molecules (shorter than 30 base pairs) are added to the cells, where the cell recognizes and degrades mRNA corresponding to the target sequence. As a result little or no protein is produced.

What makes RNAi so exciting to the researchers is its potential for knocking out a protein without harming a cell. By comparison chemotherapy kills tumors by destroying cancerous as well as healthy cells. RNAi strategy includes multiple targets to kill HIV. These could be the targets that block entry into the cells and disrupts the virus life cycle inside the cells. This technology will help researchers dissect the biology of HIV infection and design drugs based on the information. Researchers at City of Hope Cancer Centre in Duarte have developed a DNA-based delivery system in which human cells are generated that produce siRNA against REV protein, which is important in causing human disease (Yu et al., 2002).

#### **5.3.3 Hepatitis protection**

This has provided the first tangible evidence for RNAi as a therapy for diseases in live animals. Early RNAi studies noted that RNA silencing was prominent in the liver, which made this organ an attractive target for therapeutic approaches. Many immune-related liver diseases are characterized by apoptosis, which is mediated by a protein called Fas. So Judy Lieberman's group injected siRNA targeting Fas intravenously into two models of autoimmune hepatitis in mice. This decreased Fas mRNA and protein levels in hepatocytes and protected the cells against liver injury from apoptosis, even when siRNA was administered after the induction of injury. Extending these findings to other liver diseases looks hopeful, but the authors concluded that other strategies, such as viral vectors, might be required to target organs in which RNA silencing is less effective than in the liver.

#### **6. Conclusion and future outlook of RNAi**

The field of RNAi is moving at an impressive pace, generating exciting results and has established a novel archetype with far-reaching consequences in the field of transcription regulation. The RNA silencing has practical use because of the ability to reduce gene expression in a manner that is highly sequence specific as well as technologically facile, economical and having potential in finding out the function of genes at a faster speed and in agriculture specifically for nutritional improvement of plants and the management of mascotous plant diseases. In addition it has kindled hope for the treatment of several diseases, which have bothered mankind as untreatable by providing an innovative technology for development of therapeutics. However, the major obstacles hindering its immediate applications include selection of targeting sequences and in the delivery of siRNA. The key issues are 1) how to select silencing targets for a particular disease and 2) how to efficiently deliver siRNAs into specific cell types *in vivo*? Besides, RNAi technology can be considered an eco-friendly, biosafe ever green technology as it eliminates even certain risks associated with development of transgenic and it has already added new dimensions in the various field of science. However, a better and comprehensive understanding of RNAi should allow future researchers to work effectively and efficiently in order to manage the phenomenon.

#### **7. References**

72 Crop Plant

al., 1999). The concept of silencing genes in HIV is straightforward: Hit the virus where it counts by eliminating a protein it needs to reproduce or cause infection. siRNA molecules (shorter than 30 base pairs) are added to the cells, where the cell recognizes and degrades mRNA corresponding to the target sequence. As a result little or no

What makes RNAi so exciting to the researchers is its potential for knocking out a protein without harming a cell. By comparison chemotherapy kills tumors by destroying cancerous as well as healthy cells. RNAi strategy includes multiple targets to kill HIV. These could be the targets that block entry into the cells and disrupts the virus life cycle inside the cells. This technology will help researchers dissect the biology of HIV infection and design drugs based on the information. Researchers at City of Hope Cancer Centre in Duarte have developed a DNA-based delivery system in which human cells are generated that produce siRNA against REV protein, which is important in causing human disease

This has provided the first tangible evidence for RNAi as a therapy for diseases in live animals. Early RNAi studies noted that RNA silencing was prominent in the liver, which made this organ an attractive target for therapeutic approaches. Many immune-related liver diseases are characterized by apoptosis, which is mediated by a protein called Fas. So Judy Lieberman's group injected siRNA targeting Fas intravenously into two models of autoimmune hepatitis in mice. This decreased Fas mRNA and protein levels in hepatocytes and protected the cells against liver injury from apoptosis, even when siRNA was administered after the induction of injury. Extending these findings to other liver diseases looks hopeful, but the authors concluded that other strategies, such as viral vectors, might be required to target organs in which RNA silencing is less effective than in

The field of RNAi is moving at an impressive pace, generating exciting results and has established a novel archetype with far-reaching consequences in the field of transcription regulation. The RNA silencing has practical use because of the ability to reduce gene expression in a manner that is highly sequence specific as well as technologically facile, economical and having potential in finding out the function of genes at a faster speed and in agriculture specifically for nutritional improvement of plants and the management of mascotous plant diseases. In addition it has kindled hope for the treatment of several diseases, which have bothered mankind as untreatable by providing an innovative technology for development of therapeutics. However, the major obstacles hindering its immediate applications include selection of targeting sequences and in the delivery of siRNA. The key issues are 1) how to select silencing targets for a particular disease and 2) how to efficiently deliver siRNAs into specific cell types *in vivo*? Besides, RNAi technology can be considered an eco-friendly, biosafe ever green technology as it eliminates even certain risks associated with development of transgenic and it has already added new

protein is produced.

(Yu et al., 2002).

the liver.

**5.3.3 Hepatitis protection** 

**6. Conclusion and future outlook of RNAi** 


Phenomenal RNA Interference: From Mechanism to Application 75

Napoli, C., Lemiex, C. & Jorgenson, R. A. (1990) Introduction of a chimeric chalcone

Nicholson, A. W. (1999) Function, mechanism and regulation of bacterial ribonucleases.

Niggeweg, R., Michael, A. & Martin, C. (2004) Engineering plants with increased levels of

Nykanen, A., Haley, B. & Zamore, P. D. (2001) ATP requirements and small interfering RNA

Paddison, P. J., Claudy, A. A., Bernstein, E., Hannon, G. J. & Conklin, D. S. (2002) Short

Senapedis, W. T., Kennedy, C. J, Boyle, P. M. & Silver, P. A. (2011) Whole genome siRNA

Shinozuka, Y., Onosato, K. & Hirochika, H. (2003) Target site specificity of the Tos 17

Sigoillot, F. D., King RW (2011) Vigilance and Validation: Keys to Success in RNAi

Sijen, T., Fleenor, J., Simmer, F., Thijssen, K. L., Parish, S., Timmons, L., Plasterk, R. H. A. &

Siritunga, D. & Sayre, R. (2003) Generation of cyanogen-free transgenic cassava. *Planta.* 217:

Sui, G., Sohoo, C., Affar, E. B., Gav, F., Shi, Y., Forrester, W. C. & Shi, Y. A. (2002) DNA

Kumar, S. G., Campbell, L., Puckhaber, L., Stipanovic, R. & Rathore, K. (2006) Engineering

Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A., Timmons, L., Fire, A. &

Terada, R., Urawa, H., Inagaki, Y., Tsugane, K. & Iida, S. (2002) Efficient gene

Yu, J. Y., Deruiter, S. L. & Turner, D. L. (2002) RNA interference by expression of short

Thakur, A. (2003) RNA interference revolution. *Electronic J Biotechnol.* 6: 39-49.

hairpin RNAs (shRNAs) induce sequence specific silencing in mammalian cells.

cell-based screen links mitochondria to Akt signaling network through uncoupling of electron transport chain. *Mol. Biol. of the Cell* 22:1791-1805.

retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. *Plant Cell*. 15:1771-

Fire, A. (2001) On the role of RNA amplification in dsRNA-triggered gene

vector based RNAi technology to suppress gene expression in mammalian cells.

cottonseed for use in human nutrition by tissue-specific reduction of toxic

Mello, C. C. (1999) The rde-1 gene, RNA interference and transposon silencing in *C.* 

targeting by homologous recombination in rice. *Nat Biotechnol*. 20:

interfering RNAs and hairpin RNAs in mammalian cells *Proc. Natl. Acad. Sci.*

the antioxidant chlorogenic acid. *Nat Biotechnol.* 22:746-754. Novina, C. D. & Sharp, P. A. (2004) The RNAi revolution. *Nature*. 430:161-165.

structure in the RNA interference pathway. *Cell.* 107: 309-321.

genes in trans. *Plant Cell.* 2:279-289.

Screening. *ACS Chem. Biol.* 21:47-60.

*Proc. Natl. Acad. Sci.* 99: 5515-5520.

gossypol. *Proc. Natl. Acad. Sci.* 103: 18054–18059.

silencing. *Cell.* 107: 465-476.

*elegans*. *Cell*. 99:123-132.

1030-1034.

99:6047-6052.

*FEMS Microbiol. Rev,* 23:371-390.

*Genes Dev.* 16: 948-958.

1780.

367-373.

synthase gene into Petunia results in reversible co-suppression of homologous


Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. & Mello, C. C. (1998) Potent

Gavilano, L., Coleman, N., Burnley, L., Bowman, M., Kalengamaliro, N., Hayes, A., Bush, L.

Golden, T. A., Schauer, S. E., Lang, J. D., Pien, S., Mushegian, A. R., Grossniklaus, U,.

Guo, S. & Kemphues, K. J. (1995) Par-1, a gene required for establishing polarity in C.

Hammond, S., Bernstein, E., Beach, D. & Hannon, G. J. (2000) An RNA-directed nuclease

Hemann, M. T., Fridman, J. S., Zilfou, J. T., Hernando, E., Paddison, P. J., Cordon-Cardo, C.,

Hoffman, G. R., Moerke, N. J., Hsia, M., Shamu, C. E. & Blenis, J. (2010) A high-

Ketting, R. F., Fischer, S. E. J., Bernstein, E., Sijen, T., Hannon, G. J. & Plasterk, R. H. A.

Kuwabara, T., Hsieh, J., Nakashima, K., Taira, K. & Gage, F. H. (2004) A small modulatory dsRNA specifies the fate of adult neural stem cells . *Cell*. 116: 779-93. Lawrence, R. J. & Pikaard, C. S. (2003) Transgene-induced RNA interference: a strategy for

Lee, N. S., Dohjima, T., Bauer, G., Li, H., Li, M. J., Ehsani, A., Salvaterra, P. & Rossi, J. (2002)

Lohmann, J. U., Endl, I. & Bosch, T. C . (1999) Silencing of developmental genes in

Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R. & Tuschl, T. (2002)

Medema, R. H. (2004) Optimizing RNA interference for application in mammalian cells.

in developmental timing in *C. Elegans*. *Genes Dev.* 15: 2654-2659.

nornicotine content. *J. Agric. Food Chem.* 54: 9071-9078.

elegans. *Nature.* 391:806-811.

distributed. *Cell* 81:611-620

*Dev. Technol.* 8(2):186-99.

*Plant J.* 36:114-121.

574.

human cells. *Nat. Biotechnol*. 20:500-505.

Hydra*. Dev. Biol.* 214:211-214.

*Biochem J.* 380:593-603.

296.

400.

*Arabidopsis*. *Plant Physiol.* 130:808-822.

and specific genetic interference by double-stranded RNA in Caenorhabditis

& Siminszky, B. (2006) Genetic engineering of Nicotiana tabacum for reduced

Meinke, D. W. & Ray, A. (2002) Short Integuments/ Suspensor1/ Carpel Factory, a Dicer homolog, is a maternal effect gene required for embryo development in

elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically

mediates post-transcriptional gene silencing in Drosophila cells. *Nature.* 404: 293-

Hannon, G. J. & Lowe, S.W. (2003) An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. *Nature Genet.* 33: 396-

throughput, cell-based screening method for siRNA and small molecule inhibitors of mTORC1 signaling using the in cell western technique. *Assay Drug* 

(2000) Dicer functions in RNA interference and in synthesis of small RNA involved

overcoming gene redundancy in polyploids to generate loss-of-function mutations.

Expression of mall interfering RNAs targeted against HIV-1 rev transcripts in

Singlestranded antisense siRNAs guide target RNA cleavage in RNAi. *Cell*. 110:563-


*1Faculty of Agriculture, Food, and Natural Resources, The University of Sydney, 2Research & Development Section, Fauji Fertilizer Company Ltd, Rawalpindi,* 

*4Soil Chemistry Section, Institute of Soil Chemistry & Environmental Sciences,* 

*5Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, 6School of Earth and Environment, Faculty of Natural and Agricultural Sciences,* 

Boron (B) is a unique non-metal micronutrient required for normal growth and development of plants. In 1923, it was first time reported that B is essential for cell structure of plants (Warington, 1923). The possible roles of B include sugar transport, cell wall synthesis, lignification, cell wall structure integrity, carbohydrate metabolism, ribose nucleic acid (RNA) metabolism, respiration, indole acetic acid (IAA) metabolism, phenol metabolism, and as part of the cell membranes (Parr & Loughman, 1983; Welch, 1995; Ahmad et al., 2009). In soils, concentration of total B is reported to be in the range of 20 to 200 mg B kg-1 (Mengel & Kirkby, 1987), and its available concentrations also vary greatly

Boron is absorbed by roots as undissociated boric acid [B (OH)3 or H3BO3] (Mengel & Kirkby, 1982; Marschner, 1995) which has a strong ability to form complexes with diols and polyols, particularly with cis-diols inside the plant system (Loomis & Durst, 1992). Among the elements required by plants that are taken up from the soil, B is the only element that is taken up by plants not as an ion, but as an uncharged molecule (Marschner, 1995; Miwa & Fujiwara, 2010). The factors affecting B uptake include soil type (texture, alkalinity/calcareousness, pH, organic matter content), B concentration, moisture, and plant species (Welch et al., 1991). Boron absorption by plant roots is closely related to pH and B concentration in the soil solution; and is probably a non-metabolic process (Brown & Hu, 1998). The supplying mechanism of B to plant roots is primarily through mass flow, while its distribution in plants is governed by the transpiration stream through the xylem (Raven, 1980). Boron is relatively immobile in plant, and thus its availability is essential at all stages of growth, especially during fruit/seed development. However, recent physiological studies

**1. Introduction** 

from soil to soil.

Waqar Ahmad1, Munir H. Zia2,

*1,6Australia 2,4,5Pakistan 3Canada* 

Sukhdev S. Malhi3, Abid Niaz4 and Saifullah5,6

*Ayub Agricultural Research Institute, Faisalabad,* 

*The University of Western Australia, Crawley, Perth,* 

*3Agriculture and Agri-Food Canada, Melfort, Saskatchewan,* 

Zadeh, A. & Foster, G. (2004) Transgenic resistance to tobacco ringspot virus. *Acta Viro.,* 48: 145-152. **5** 

Waqar Ahmad1, Munir H. Zia2,

Sukhdev S. Malhi3, Abid Niaz4 and Saifullah5,6 *1Faculty of Agriculture, Food, and Natural Resources, The University of Sydney, 2Research & Development Section, Fauji Fertilizer Company Ltd, Rawalpindi, 3Agriculture and Agri-Food Canada, Melfort, Saskatchewan, 4Soil Chemistry Section, Institute of Soil Chemistry & Environmental Sciences, Ayub Agricultural Research Institute, Faisalabad, 5Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, 6School of Earth and Environment, Faculty of Natural and Agricultural Sciences, The University of Western Australia, Crawley, Perth, 1,6Australia 2,4,5Pakistan 3Canada* 

#### **1. Introduction**

76 Crop Plant

Zadeh, A. & Foster, G. (2004) Transgenic resistance to tobacco ringspot virus. *Acta Viro.,* 48:

Boron (B) is a unique non-metal micronutrient required for normal growth and development of plants. In 1923, it was first time reported that B is essential for cell structure of plants (Warington, 1923). The possible roles of B include sugar transport, cell wall synthesis, lignification, cell wall structure integrity, carbohydrate metabolism, ribose nucleic acid (RNA) metabolism, respiration, indole acetic acid (IAA) metabolism, phenol metabolism, and as part of the cell membranes (Parr & Loughman, 1983; Welch, 1995; Ahmad et al., 2009). In soils, concentration of total B is reported to be in the range of 20 to 200 mg B kg-1 (Mengel & Kirkby, 1987), and its available concentrations also vary greatly from soil to soil.

Boron is absorbed by roots as undissociated boric acid [B (OH)3 or H3BO3] (Mengel & Kirkby, 1982; Marschner, 1995) which has a strong ability to form complexes with diols and polyols, particularly with cis-diols inside the plant system (Loomis & Durst, 1992). Among the elements required by plants that are taken up from the soil, B is the only element that is taken up by plants not as an ion, but as an uncharged molecule (Marschner, 1995; Miwa & Fujiwara, 2010). The factors affecting B uptake include soil type (texture, alkalinity/calcareousness, pH, organic matter content), B concentration, moisture, and plant species (Welch et al., 1991). Boron absorption by plant roots is closely related to pH and B concentration in the soil solution; and is probably a non-metabolic process (Brown & Hu, 1998). The supplying mechanism of B to plant roots is primarily through mass flow, while its distribution in plants is governed by the transpiration stream through the xylem (Raven, 1980). Boron is relatively immobile in plant, and thus its availability is essential at all stages of growth, especially during fruit/seed development. However, recent physiological studies

weather and high light intensity (Moraghan & Mascagni, 1991). Therefore, knowledge of these factors affecting B uptake is essential for the assessment of B deficiency and toxicity

Upon mineralization from organic matter or B addition to soils through irrigation or fertilization, a proportion of it remains in the soil solution while left of it is adsorbed by soil particles and other soil constituents. Tourmaline is a mineral which contains B in a very insoluble form while hydrated B minerals are the most soluble form of B minerals. These minerals do not usually determine the solubility of B in the soil solution (Goldberg, 1993), which is governed by B adsorption reactions mainly. The equilibrium exists between the soil solution and adsorbed B (Russell, 1973). Plants obtain B from the soil solution (Hatcher et al., 1959), and buffering against abrupt changes in the level of B in the soil solution is controlled by the adsorbed pool of B (Hatcher et al., 1962). Therefore, it is important to know the distribution of B between the solid and the liquid phases of the soil. Factors affecting the amount of B adsorbed by soils and the B bioavailability in soils include soil pH, texture, moisture, temperature, and management practices such as liming

Parent material is considered a dominant factor affecting supply of B from the soil. Soils are quite variable in their B and clay forming minerals contents, and therefore have a fundamental effect on the availability of B. In general, soils derived from igneous rocks, and those in tropical and temperate regions of the world, have much lower B concentrations than soils derived from sedimentary rocks, and those in arid or semi arid regions (Ho, 2000). High B concentrations are usually found in the soils that have been formed from marine shale enriched parent material. Soils derived from acid granite and other igneous rocks, fresh-water sedimentary deposits, and in coarse textured soils low in organic matter have been reported with low B concentrations (Liu et al., 1983). Boron bioavailability is also reduced in soils derived from volcanic ash (Sillanpaa & Vlek, 1985) and in soils rich in aluminum (Al) oxides (Bingham et al., 1971). Soils along the sea shore as well as those derived from mudstone are usually B enriched. Conversely, lateritic soils, and soils derived from sandstone, slate or crystalline limestone do not contain much B. The levels of total B in

**Rock type Minerals B (mg B kg-1)z B (mg B kg-1)y**  Igneous Basic: gabbro, basalt 5-20 5

rhyolite 10-30 Metamorphic Gneiss 10-30 - Sedimentary Shale 120-130 100 Sandstone 30 35

Intermediate: diorite 9-25 15

Limestone dolomite 20-30 20

under different conditions.

(Evans & Sparks, 1983).

common rocks are presented in Table 1.

Acid: granite,

zKabata-Pendias & Pendias, 1992; ySillanpaa & Vlek, 1985. Table 1. Total B concentrations in major rock types

**2.1 Parent material** 

have revealed the presence of channel-mediated facilitated diffusion and energy-dependent active transport against concentration gradients in B transport systems (Dannel et al., 2000, 2001; Stangoulis et al., 2001).

Boron deficiency is one of the major constraints to crop production (Sillanpaa, 1982), and has been reported in more than 80 countries and for 132 crops over the last 60 years (Shorrocks, 1997). Boron deficiency has been realized as the second most important micronutrient constraint in crops after that of zinc (Zn) on global scale. Boron deficiency has been reported to result considerable yield reduction in annual [fiber (cotton - *Gossypium hirsutum* L.), cereal (rice – *Oryza sativa* L., maize/corn *Zea mays* L.- , wheat – *Triticum aestivum* L.), legume/pulse (soybean – *Glycine max* L.), oilseed (groundnut – *Arachis hypogaea* L., oilseed rape/canola – *Brassica napus or B. rapa* L.)] and perennial [citrus fruit orchards, alfalfa – *Medicago sativa* L.] crops (Arora et al., 1985; Patil et al., 1987; Sakal et al., 1988; Ali & Monoranjan, 1989; Takkar et al., 1989; Dwivedi et al., 1990; Sinha et al., 1991; Borkakati & Takkar, 2000; Niaz et al., 2002, 2007; Rashid et al., 2005; Johnson, 2006; Zia et al., 2006). Rashid (2006) estimated a substantial potential net economic benefit from the use of B fertilizers in B-deficient crops.

Boron bioavailability decreases under drought condition because of reduced mobility of B from soil by mass flow to roots (Chiu & Chang, 1985; Chang et al., 1992; Chang, 1993; Barber, 1995). Boron can move relatively long distances by mass flow and diffusion to roots. Soil drying reduces B diffusion by reducing the mobility of soil solution and increasing the diffusion path length (Scott et al., 1975). The lack of moisture in soil reduces transpiration rate, thereby reducing B transport to shoots (Lovatt, 1985). Wetting and drying cycles and increasing soil temperature (25 to 45 °C) also increased B fixation by montmorillonite and kaolinite clays (Biggar & Fireman, 1960). Low temperature in spring and autumn season of temperate regions reduced availability of B to forage legumes while increased temperature enhanced B concentration for sugarcane (Gupta, 1993).

Boron deficiency has been commonly reported in soils which are highly leached and/or developed from calcareous, alluvial and loessial deposits (Takkar et al., 1989; Razzaq & Rafiq, 1996; Borkakati & Takkar, 2000). Several soil factors and conditions render soils deficient in B. For example, low soil organic matter content, coarse/sandy texture, high pH, liming, drought, intensive cultivation and more nutrient uptake than application, and the use of fertilizers poor in micronutrients are considered to be the major factors associated with the occurrence of B deficiency (Dregne & Powers, 1942; Elrashidi & O'Connor, 1982; Takkar et al., 1989; Goldberg & Forster, 1991; Rahmatullah et al., 1999; Eguchi & Yamada, 1997; Rashid et al., 1997, 2005; Mengel & Kirkby, 2001; Niaz et al., 2002, 2007; Rashid & Rayan, 2004)**.** This paper reviews the roles of B in plant nutrition and the factors affecting B availability in soils in general, while focusing on a number of case studies related to diagnosis and correction of B deficiency in soils and crops.

#### **2. Factors affecting boron availability in soils**

Boron concentrations in soil vary from 2 to 200 mg B kg-1, but generally less than 5-10% is in a form available to plants (Diana, 2006). Boron concentration and its bioavailability in soils is affected by several factors including parent material, texture, nature of clay minerals, pH, liming, organic matter content, sources of irrigation, interrelationship with other elements, and environmental conditions like moderate to heavy rainfall, dry

have revealed the presence of channel-mediated facilitated diffusion and energy-dependent active transport against concentration gradients in B transport systems (Dannel et al., 2000,

Boron deficiency is one of the major constraints to crop production (Sillanpaa, 1982), and has been reported in more than 80 countries and for 132 crops over the last 60 years (Shorrocks, 1997). Boron deficiency has been realized as the second most important micronutrient constraint in crops after that of zinc (Zn) on global scale. Boron deficiency has been reported to result considerable yield reduction in annual [fiber (cotton - *Gossypium hirsutum* L.), cereal (rice – *Oryza sativa* L., maize/corn *Zea mays* L.- , wheat – *Triticum aestivum* L.), legume/pulse (soybean – *Glycine max* L.), oilseed (groundnut – *Arachis hypogaea* L., oilseed rape/canola – *Brassica napus or B. rapa* L.)] and perennial [citrus fruit orchards, alfalfa – *Medicago sativa* L.] crops (Arora et al., 1985; Patil et al., 1987; Sakal et al., 1988; Ali & Monoranjan, 1989; Takkar et al., 1989; Dwivedi et al., 1990; Sinha et al., 1991; Borkakati & Takkar, 2000; Niaz et al., 2002, 2007; Rashid et al., 2005; Johnson, 2006; Zia et al., 2006). Rashid (2006) estimated a substantial potential net economic benefit from the use of B fertilizers in B-deficient crops. Boron bioavailability decreases under drought condition because of reduced mobility of B from soil by mass flow to roots (Chiu & Chang, 1985; Chang et al., 1992; Chang, 1993; Barber, 1995). Boron can move relatively long distances by mass flow and diffusion to roots. Soil drying reduces B diffusion by reducing the mobility of soil solution and increasing the diffusion path length (Scott et al., 1975). The lack of moisture in soil reduces transpiration rate, thereby reducing B transport to shoots (Lovatt, 1985). Wetting and drying cycles and increasing soil temperature (25 to 45 °C) also increased B fixation by montmorillonite and kaolinite clays (Biggar & Fireman, 1960). Low temperature in spring and autumn season of temperate regions reduced availability of B to forage legumes while increased temperature

Boron deficiency has been commonly reported in soils which are highly leached and/or developed from calcareous, alluvial and loessial deposits (Takkar et al., 1989; Razzaq & Rafiq, 1996; Borkakati & Takkar, 2000). Several soil factors and conditions render soils deficient in B. For example, low soil organic matter content, coarse/sandy texture, high pH, liming, drought, intensive cultivation and more nutrient uptake than application, and the use of fertilizers poor in micronutrients are considered to be the major factors associated with the occurrence of B deficiency (Dregne & Powers, 1942; Elrashidi & O'Connor, 1982; Takkar et al., 1989; Goldberg & Forster, 1991; Rahmatullah et al., 1999; Eguchi & Yamada, 1997; Rashid et al., 1997, 2005; Mengel & Kirkby, 2001; Niaz et al., 2002, 2007; Rashid & Rayan, 2004)**.** This paper reviews the roles of B in plant nutrition and the factors affecting B availability in soils in general, while focusing on a number of case studies related to

Boron concentrations in soil vary from 2 to 200 mg B kg-1, but generally less than 5-10% is in a form available to plants (Diana, 2006). Boron concentration and its bioavailability in soils is affected by several factors including parent material, texture, nature of clay minerals, pH, liming, organic matter content, sources of irrigation, interrelationship with other elements, and environmental conditions like moderate to heavy rainfall, dry

2001; Stangoulis et al., 2001).

enhanced B concentration for sugarcane (Gupta, 1993).

diagnosis and correction of B deficiency in soils and crops.

**2. Factors affecting boron availability in soils** 

weather and high light intensity (Moraghan & Mascagni, 1991). Therefore, knowledge of these factors affecting B uptake is essential for the assessment of B deficiency and toxicity under different conditions.

Upon mineralization from organic matter or B addition to soils through irrigation or fertilization, a proportion of it remains in the soil solution while left of it is adsorbed by soil particles and other soil constituents. Tourmaline is a mineral which contains B in a very insoluble form while hydrated B minerals are the most soluble form of B minerals. These minerals do not usually determine the solubility of B in the soil solution (Goldberg, 1993), which is governed by B adsorption reactions mainly. The equilibrium exists between the soil solution and adsorbed B (Russell, 1973). Plants obtain B from the soil solution (Hatcher et al., 1959), and buffering against abrupt changes in the level of B in the soil solution is controlled by the adsorbed pool of B (Hatcher et al., 1962). Therefore, it is important to know the distribution of B between the solid and the liquid phases of the soil. Factors affecting the amount of B adsorbed by soils and the B bioavailability in soils include soil pH, texture, moisture, temperature, and management practices such as liming (Evans & Sparks, 1983).

#### **2.1 Parent material**

Parent material is considered a dominant factor affecting supply of B from the soil. Soils are quite variable in their B and clay forming minerals contents, and therefore have a fundamental effect on the availability of B. In general, soils derived from igneous rocks, and those in tropical and temperate regions of the world, have much lower B concentrations than soils derived from sedimentary rocks, and those in arid or semi arid regions (Ho, 2000). High B concentrations are usually found in the soils that have been formed from marine shale enriched parent material. Soils derived from acid granite and other igneous rocks, fresh-water sedimentary deposits, and in coarse textured soils low in organic matter have been reported with low B concentrations (Liu et al., 1983). Boron bioavailability is also reduced in soils derived from volcanic ash (Sillanpaa & Vlek, 1985) and in soils rich in aluminum (Al) oxides (Bingham et al., 1971). Soils along the sea shore as well as those derived from mudstone are usually B enriched. Conversely, lateritic soils, and soils derived from sandstone, slate or crystalline limestone do not contain much B. The levels of total B in common rocks are presented in Table 1.


zKabata-Pendias & Pendias, 1992; ySillanpaa & Vlek, 1985.

Table 1. Total B concentrations in major rock types

**Sand dune Sandy loam Black clay** 

3.8 1.2 3.0 2.0 1.2 3.8 7.8 2.2 6.4 3.6 3.8 6.2 16.5 3.5 14.8 5.2 8.5 11.5 25.0 5.0 24.0 6.0 14.0 21.0 34.0 6.0 33.0 7.0 14.0 21.0 43.0 7.0 42.2 8.0 25.0 25.0

Organic matter (OM) is the storehouse for most nutrients in soil and is known to improve soil health and availability of plant nutrients. Many researchers have suggested that the level of soil organic matter (SOM) influences the nutrient bioavailability (Sarwar & Mubeen, 2009). Boron may bind with OM or with carbohydrates released during humification. Boron associated with humic colloids is the principal B pool for plant growth in most of the agricultural soils (Jones, 2003). However, there is limited information on the role of OM in B nutrition. The strongest evidence that OM affects the availability of soil B is derived from studies that show a positive correlation between levels of SOM and the amount of hotwater-soluble B (Kao & Juang, 1978; Chang et al., 1983; Takkar et al., 1989; Niaz et al., 2002;

The association between B and SOM is said to be caused by the assimilation of B by soil microbes (Gupta et al., 1985). Albeit, B present in SOM is not immediately available to plants, it seems to be a major source of available B when it is released through mineralization (Gupta et al., 1985). It is well documented, that the interaction of dissolved organic matter (DOM) with soil is affected by the presence of OM and hydroxides in the clay fraction particles (McDowell & Likens, 1988; Jardine et al., 1989; Donald et al., 1993). The role of DOM to affect B availability/adsorption has already been reported by Mackin (1986) from pore waters of marine sediments and recently by Communar & Keren (2008) for soilplant system. Further the B solution concentration assessment may be driven through interaction of effluent DOM with native soil OM, B complexation with DOM, and adsorption of B and B–DOM complexes by soil. Correlations of total dissolved boron (TDB) and ratios of B to chloride with DOM, in organic-rich sediments, predispose that the fraction of dissolved boron (DB) that is complexed by OM is a function of dissolved organic matter concentration (Mackin, 1986). It can be inferred that DB concentrations equilibrium is highly related with organic-B complexes. Further, such potentially useful approximation should also be applied for determining concentrations of organic-B complexes in marine waters and sediments. Both deep understanding of the mechanisms of these relations and parameterization according to the local conditions permit to improve the model for B transport in soil (Communar et al., 2004; Communar & Keren, 2005, 2006). But all these investigations call for more extensive research on role of DOM pertaining to B

**B adsorbed Equilibrium** 

**conc.** 

**B adsorbed** 

**B adsorbed Equilibrium** 

Table 2. Boron adsorption (mg B kg -1) in soils as affected by texturez

**conc.** 

**Equilibrium conc.** 

zSource: Gupta, 1979a.

**2.4 Organic matter** 

desorption/release.

2007; Raza et al., 2002; Shafiq et al., 2008).

#### **2.2 Soil reaction (pH)**

Soil pH is one of the most important factors affecting the availability of B in soils. Its bioavailability becomes less at the higher solution pH. Consequently, application of lime to acid soils, in excessive amounts, can sometimes render plants deficient in B. There is a close association with the pH of the soil solution and the level of soluble B in soils (Elrashidi & O'Connor, 1982; Takkar et al., 1989; Rashid et al., 1994; Niaz et al., 2002, 2007). Boron uptake by plants growing in soil, with the same water soluble B concentration, was noticed to be higher where pH of the soil solution was lower (Wear & Patterson, 1962). The adsorption of B by soils is much dependent on pH of the soil solution. Boron adsorption by soils increased when the pH rose from 3 to 9 (Bingham et al., 1971; Mezuman & Keren, 1981; Keren & Bingham, 1985; Barrow, 1989), and it decreased when the pH was increased further in the range 10 to 11.5 (Goldberg & Glaubig, 1986). In several studies, highest levels of B adsorption by soil depicted close correlation with the pH of the soil solution (Okazaki & Chao, 1968; Evans, 1987; Shafiq et al., 2008).

#### **2.3 Soil texture and clay minerals**

Coarse-textured soils often contain less available B than fine-textured soils (Takkar et al., 1989; Raza et al., 2002; Malhi et al., 2003). This might be one of the reasons that B deficiencies in crop plants have often been observed on sandy soils (Gupta, 1968; Fleming, 1980). Niaz et al. (2002) concluded from a study in Punjab, Pakistan that B concentrations of coarse- and medium-textured soils and plants grown in such soils were lower than their respective critical levels, because these soils were well drained and had good leaching. Besides aluminum and iron oxides, calcium carbonate and organic matter, clay minerals are considered to be amongst the primary B adsorbing surfaces in soils (Goldberg, 1997). The mechanism of B adsorption on these surfaces is considered to be ligand exchange with reactive surface hydroxyl groups leading to strong specific adsorption (Goldberg & Chunming, 2007). Boron adsorption in fine-textured soils is higher compared with the coarse- and medium-textured soils at the same equilibrium concentration (Table 2). The level of native B is also closely related to the clay content of the soil (Elrashidi & O'Connor, 1982; Raza et al., 2002). At the same time, water soluble B concentration and B uptake are reported to be higher in plants grown in coarse-textured soils (Wear & Patterson, 1962). The level of B adsorbed by the soil thus largely depends on soil texture in addition to pH of soil solution. It increases with an increase in clay content (Bhatnager et al., 1979; Wild & Mazaheri, 1979; Mezuman & Keren, 1981; Elrashidi & O'Connor, 1982).

More B adsorption is commonly found in illite as compared with kaolinite or montmorillonite clay types. . In fact, kaolinite adsorbs B the least (Hingston, 1964; Fleet, 1965). Frederickson & Reynolds (1959) proposed that most of the B in the clay mineral fraction of sedimentary rocks is contained in the illite fraction. Sims & Bingham (1967, 1968a, 1968b) found that B adsorption was greater for iron (Fe) and Al coated kaolinite or montmorillonite than for uncoated clays. It was concluded that hydroxyl of Fe and Al compounds present in the layer as silicates or as impurities dominate over clay mineral species per se in determining B adsorption characteristics. Bingham et al. (1971) and Schalscha et al. (1973) also inferred that B adsorption by certain soils was primarily due to their Al oxide content.


zSource: Gupta, 1979a.

80 Crop Plant

Soil pH is one of the most important factors affecting the availability of B in soils. Its bioavailability becomes less at the higher solution pH. Consequently, application of lime to acid soils, in excessive amounts, can sometimes render plants deficient in B. There is a close association with the pH of the soil solution and the level of soluble B in soils (Elrashidi & O'Connor, 1982; Takkar et al., 1989; Rashid et al., 1994; Niaz et al., 2002, 2007). Boron uptake by plants growing in soil, with the same water soluble B concentration, was noticed to be higher where pH of the soil solution was lower (Wear & Patterson, 1962). The adsorption of B by soils is much dependent on pH of the soil solution. Boron adsorption by soils increased when the pH rose from 3 to 9 (Bingham et al., 1971; Mezuman & Keren, 1981; Keren & Bingham, 1985; Barrow, 1989), and it decreased when the pH was increased further in the range 10 to 11.5 (Goldberg & Glaubig, 1986). In several studies, highest levels of B adsorption by soil depicted close correlation with the pH of the soil solution (Okazaki &

Coarse-textured soils often contain less available B than fine-textured soils (Takkar et al., 1989; Raza et al., 2002; Malhi et al., 2003). This might be one of the reasons that B deficiencies in crop plants have often been observed on sandy soils (Gupta, 1968; Fleming, 1980). Niaz et al. (2002) concluded from a study in Punjab, Pakistan that B concentrations of coarse- and medium-textured soils and plants grown in such soils were lower than their respective critical levels, because these soils were well drained and had good leaching. Besides aluminum and iron oxides, calcium carbonate and organic matter, clay minerals are considered to be amongst the primary B adsorbing surfaces in soils (Goldberg, 1997). The mechanism of B adsorption on these surfaces is considered to be ligand exchange with reactive surface hydroxyl groups leading to strong specific adsorption (Goldberg & Chunming, 2007). Boron adsorption in fine-textured soils is higher compared with the coarse- and medium-textured soils at the same equilibrium concentration (Table 2). The level of native B is also closely related to the clay content of the soil (Elrashidi & O'Connor, 1982; Raza et al., 2002). At the same time, water soluble B concentration and B uptake are reported to be higher in plants grown in coarse-textured soils (Wear & Patterson, 1962). The level of B adsorbed by the soil thus largely depends on soil texture in addition to pH of soil solution. It increases with an increase in clay content (Bhatnager et al., 1979; Wild & Mazaheri, 1979; Mezuman & Keren, 1981; Elrashidi &

More B adsorption is commonly found in illite as compared with kaolinite or montmorillonite clay types. . In fact, kaolinite adsorbs B the least (Hingston, 1964; Fleet, 1965). Frederickson & Reynolds (1959) proposed that most of the B in the clay mineral fraction of sedimentary rocks is contained in the illite fraction. Sims & Bingham (1967, 1968a, 1968b) found that B adsorption was greater for iron (Fe) and Al coated kaolinite or montmorillonite than for uncoated clays. It was concluded that hydroxyl of Fe and Al compounds present in the layer as silicates or as impurities dominate over clay mineral species per se in determining B adsorption characteristics. Bingham et al. (1971) and Schalscha et al. (1973) also inferred that B adsorption by certain soils was primarily due to

**2.2 Soil reaction (pH)** 

Chao, 1968; Evans, 1987; Shafiq et al., 2008).

**2.3 Soil texture and clay minerals** 

O'Connor, 1982).

their Al oxide content.

Table 2. Boron adsorption (mg B kg -1) in soils as affected by texturez

#### **2.4 Organic matter**

Organic matter (OM) is the storehouse for most nutrients in soil and is known to improve soil health and availability of plant nutrients. Many researchers have suggested that the level of soil organic matter (SOM) influences the nutrient bioavailability (Sarwar & Mubeen, 2009). Boron may bind with OM or with carbohydrates released during humification. Boron associated with humic colloids is the principal B pool for plant growth in most of the agricultural soils (Jones, 2003). However, there is limited information on the role of OM in B nutrition. The strongest evidence that OM affects the availability of soil B is derived from studies that show a positive correlation between levels of SOM and the amount of hotwater-soluble B (Kao & Juang, 1978; Chang et al., 1983; Takkar et al., 1989; Niaz et al., 2002; 2007; Raza et al., 2002; Shafiq et al., 2008).

The association between B and SOM is said to be caused by the assimilation of B by soil microbes (Gupta et al., 1985). Albeit, B present in SOM is not immediately available to plants, it seems to be a major source of available B when it is released through mineralization (Gupta et al., 1985). It is well documented, that the interaction of dissolved organic matter (DOM) with soil is affected by the presence of OM and hydroxides in the clay fraction particles (McDowell & Likens, 1988; Jardine et al., 1989; Donald et al., 1993). The role of DOM to affect B availability/adsorption has already been reported by Mackin (1986) from pore waters of marine sediments and recently by Communar & Keren (2008) for soilplant system. Further the B solution concentration assessment may be driven through interaction of effluent DOM with native soil OM, B complexation with DOM, and adsorption of B and B–DOM complexes by soil. Correlations of total dissolved boron (TDB) and ratios of B to chloride with DOM, in organic-rich sediments, predispose that the fraction of dissolved boron (DB) that is complexed by OM is a function of dissolved organic matter concentration (Mackin, 1986). It can be inferred that DB concentrations equilibrium is highly related with organic-B complexes. Further, such potentially useful approximation should also be applied for determining concentrations of organic-B complexes in marine waters and sediments. Both deep understanding of the mechanisms of these relations and parameterization according to the local conditions permit to improve the model for B transport in soil (Communar et al., 2004; Communar & Keren, 2005, 2006). But all these investigations call for more extensive research on role of DOM pertaining to B desorption/release.

effect of B on Al toxicity on seedlings of two soybean cultivars. The results showed that high B was found to ameliorate Al toxicity by significantly increasing the growth characters including root length under 2 mM Al stress, and epicotyl length and fresh weight under 5 mM Al stress of the two cultivars. Similar kind of study was conducted by Hossain and Hossain (2004) which confirmed the relationship of B with Al. The ratio between Ca and B in the plant is sometimes used to identify B deficiency. In a recent study, application of the both Ca and B to four cultivars of maize significantly enhanced shoot dry matter production (Kanwal et al., 2008). Nevertheless, B concentration in the shoot of maize cultivars was antagonized with Ca application. A curvilinear relation was exhibited between Ca/B ratio in shoot and relative shoot dry matter. In this regard, further work is warranted on Ca/B

utilizing association for ameliorating B deficient calcareous soils (Rashid et al., 1997).

Crop species differ in their capacity to take up B, even when they are grown in the same growth medium. These differences generally reflect different B requirements for growth. In general, dicots (cotton and leguminous plants) have 4-7 times higher B requirement (20-70 mg B kg-1) than monocots (graminae family), 5-10 mg B kg-1 (Bergmann, 1988, 1992; Marschner, 1995). As the most important functions of B in plants are thought to be its structural role in cell wall development and stimulation or inhibition of specific metabolism pathways (Gupta, 1979a, 1979b, 1993; Ahmad et al., 2009), thereby, differences in the B demand of graminaceous and dicotyledonous species are probably related to the differences in their cell wall composition, and cis-diol configuration in the cell walls, such as pectic substances. A meager amount of pectic material is constituted in the cell walls of graminaceous species (wheat and rice). Such species also have much lower Ca requirements. In fact, these two plant categories also differ in their capacity for silicon (Si) uptake, which is usually inversely related to B and Ca requirements (Loomis & Durst, 1992). All the three elements are located mainly in the cell walls. Brown & Shelp (1997) and Brown & Hu (1998) concluded that knowledge of the relative mobility of B within a particular species determines the optimum fertilization strategy and the same can also be used in partial

In summary, B deficiency is commonly induced under the following soil conditions; 1) soils which are inherently low in B, such as those derived from the parent material made from acid granite and other igneous rocks, and freshwater sedimentary deposits, 2) leaching impacted naturally acid soils from which native B has been removed, 3) light-textured sandy soils and gravelly soils, 4) alkaline and calcareous soils, 5) irrigated soils having low B concentration in irrigation water, and where salt or carbonate has been deposited, and 6)

Boron is very vulnerable to leaching, so its deficiency can temporarily be expected in countries like Pakistan and India during and after monsoon rains, especially in coarsetextured soils. However, its major source mineral (i.e., tourmaline) is highly insoluble. In Pakistan, Alfisols appear to be the soil group most likely to produce B-deficient crops (Zia et

**3. Sensitivity of crop species/cultivars to boron deficiency** 

understanding of the causes and consequences of B deficiency.

**4. Diagnosis and correction of boron deficiency** 

**4.1 Identification of boron deficiency** 

soils low in OM.

#### **2.5 Sources of irrigation water**

There are two common sources of water to irrigate crops, i.e., canal water and tube well water. The soil B status, and its availability and toxicity to plants also depend on the source of irrigation water. Underground water used for irrigation purpose has been reported to contain toxic amounts of B in many parts (Uttar Pradesh, Rajasthan, Haryana, Punjab, and Gujrat) (Chauhan & Asthana, 1981) of India. This toxicity reduces growth, particularly of shoots, and causes chlorosis starting at the leaf tip and margins of mature leaves (Nable et al., 1997; Reid et al., 2004; Reid & Fitzpatrick, 2009). Similarly, underground water for irrigation in the western desert of Egypt was also shown to be high in B (Elseewi, 1974). Boron toxicity has been reported in many crops irrigated with high-B water in Spain (Salinas et al., 1981), Arizona (Ryan et al., 1977), northern Greece (Sotiropoulos, 1997) and Philippines (Dobermann & Fairhurst, 2000). Ahmad et al. (2004) conducted a survey to determine the B concentrations in canal and ground waters used for irrigation in different villages of Faisalabad (Pakistan). The results showed that B in the tube well waters collected during February-March ranged from 0.14-0.65 mg B L-1 [standard deviation (SD) = 0.16] with a mean of 0.38 mg B L-1, and those collected in July-August ranged from 0.52 to 0.66 mg B L-1 (SD = 0.28), with a mean 0.56 mg B L-1. Boron in river water samples collected during February-March ranged from 0.11 to 0.43 mg B L-1 (SD = 0.10), with a mean of 0.21 mg B L-1. The authors concluded that tube well waters contain higher B concentrations compared to the canal waters, so farmers should get their water samples analyzed prior to irrigation and should consider these B concentrations in order to adjust B fertilizer doses to crops. This suggests that farmers using these waters for irrigation of their crops should pay attention to this potential source of B availability. This recommendation is of prime importance, because B is the unique element in the sense that there is a very narrow range between its deficient and toxic levels (< 0.5 mg B kg-1 and > 5 mg B kg-1, respectively). Farmers can calculate the amounts of B being added to their fields through irrigations of canal and tube well water. These results are also in line with those of Sillanpaa (1982) and Keren & Bingham (1985).

#### **2.6 Interactions of boron with other nutrients**

Some functions of B interrelate with those of nitrogen (N), phosphorus (P), potassium (K) and calcium (Ca) in plants (US Borax, 2009). Its interaction (synergistic, antagonistic) with most of the nutrients (N, P, K, Ca, Mg [magnesium] Al [aluminum] and Zn) may be sometimes influential in regulating B availability to plants in soil. Application of B may improve the utilization of applied N in cotton plants by increasing the translocation of N compounds into the boll (Miley et al., 1969). Smithson and Heathcote (1976) found that when B deficiency occurred in cotton, the application of 250 kg N ha-1 reduced the yield. However, when B was applied, crop biomass escalated with the same dose of N.

Graham et al. (1987) found that B uptake by barley (*Hordeum vulgare* L.) was lower when Zn was applied compared to in its absence. Further, they also showed that rate of B accumulation in plants is increased even at low levels of Zn and high levels of P. Therefore, Zn fertilization may reduce B accumulation, and lessen the risk of toxicity in plants (Ahmed et al., 2008). A significant relationship has been found between K and B fertilizers regarding their assimilation/uptake by crop plants as well as crop produce (Hill & Morrill, 1975). At heavy applications of K and other intensive production practices B may need to be applied to prevent reduction in corn yield (Woodruff et al., 1987). , Yang & Gu (2004) studied the

There are two common sources of water to irrigate crops, i.e., canal water and tube well water. The soil B status, and its availability and toxicity to plants also depend on the source of irrigation water. Underground water used for irrigation purpose has been reported to contain toxic amounts of B in many parts (Uttar Pradesh, Rajasthan, Haryana, Punjab, and Gujrat) (Chauhan & Asthana, 1981) of India. This toxicity reduces growth, particularly of shoots, and causes chlorosis starting at the leaf tip and margins of mature leaves (Nable et al., 1997; Reid et al., 2004; Reid & Fitzpatrick, 2009). Similarly, underground water for irrigation in the western desert of Egypt was also shown to be high in B (Elseewi, 1974). Boron toxicity has been reported in many crops irrigated with high-B water in Spain (Salinas et al., 1981), Arizona (Ryan et al., 1977), northern Greece (Sotiropoulos, 1997) and Philippines (Dobermann & Fairhurst, 2000). Ahmad et al. (2004) conducted a survey to determine the B concentrations in canal and ground waters used for irrigation in different villages of Faisalabad (Pakistan). The results showed that B in the tube well waters collected during February-March ranged from 0.14-0.65 mg B L-1 [standard deviation (SD) = 0.16] with a mean of 0.38 mg B L-1, and those collected in July-August ranged from 0.52 to 0.66 mg B L-1 (SD = 0.28), with a mean 0.56 mg B L-1. Boron in river water samples collected during February-March ranged from 0.11 to 0.43 mg B L-1 (SD = 0.10), with a mean of 0.21 mg B L-1. The authors concluded that tube well waters contain higher B concentrations compared to the canal waters, so farmers should get their water samples analyzed prior to irrigation and should consider these B concentrations in order to adjust B fertilizer doses to crops. This suggests that farmers using these waters for irrigation of their crops should pay attention to this potential source of B availability. This recommendation is of prime importance, because B is the unique element in the sense that there is a very narrow range between its deficient and toxic levels (< 0.5 mg B kg-1 and > 5 mg B kg-1, respectively). Farmers can calculate the amounts of B being added to their fields through irrigations of canal and tube well water. These results are also in line with those of Sillanpaa (1982) and Keren & Bingham (1985).

Some functions of B interrelate with those of nitrogen (N), phosphorus (P), potassium (K) and calcium (Ca) in plants (US Borax, 2009). Its interaction (synergistic, antagonistic) with most of the nutrients (N, P, K, Ca, Mg [magnesium] Al [aluminum] and Zn) may be sometimes influential in regulating B availability to plants in soil. Application of B may improve the utilization of applied N in cotton plants by increasing the translocation of N compounds into the boll (Miley et al., 1969). Smithson and Heathcote (1976) found that when B deficiency occurred in cotton, the application of 250 kg N ha-1 reduced the yield.

Graham et al. (1987) found that B uptake by barley (*Hordeum vulgare* L.) was lower when Zn was applied compared to in its absence. Further, they also showed that rate of B accumulation in plants is increased even at low levels of Zn and high levels of P. Therefore, Zn fertilization may reduce B accumulation, and lessen the risk of toxicity in plants (Ahmed et al., 2008). A significant relationship has been found between K and B fertilizers regarding their assimilation/uptake by crop plants as well as crop produce (Hill & Morrill, 1975). At heavy applications of K and other intensive production practices B may need to be applied to prevent reduction in corn yield (Woodruff et al., 1987). , Yang & Gu (2004) studied the

However, when B was applied, crop biomass escalated with the same dose of N.

**2.5 Sources of irrigation water** 

**2.6 Interactions of boron with other nutrients** 

effect of B on Al toxicity on seedlings of two soybean cultivars. The results showed that high B was found to ameliorate Al toxicity by significantly increasing the growth characters including root length under 2 mM Al stress, and epicotyl length and fresh weight under 5 mM Al stress of the two cultivars. Similar kind of study was conducted by Hossain and Hossain (2004) which confirmed the relationship of B with Al. The ratio between Ca and B in the plant is sometimes used to identify B deficiency. In a recent study, application of the both Ca and B to four cultivars of maize significantly enhanced shoot dry matter production (Kanwal et al., 2008). Nevertheless, B concentration in the shoot of maize cultivars was antagonized with Ca application. A curvilinear relation was exhibited between Ca/B ratio in shoot and relative shoot dry matter. In this regard, further work is warranted on Ca/B utilizing association for ameliorating B deficient calcareous soils (Rashid et al., 1997).

#### **3. Sensitivity of crop species/cultivars to boron deficiency**

Crop species differ in their capacity to take up B, even when they are grown in the same growth medium. These differences generally reflect different B requirements for growth. In general, dicots (cotton and leguminous plants) have 4-7 times higher B requirement (20-70 mg B kg-1) than monocots (graminae family), 5-10 mg B kg-1 (Bergmann, 1988, 1992; Marschner, 1995). As the most important functions of B in plants are thought to be its structural role in cell wall development and stimulation or inhibition of specific metabolism pathways (Gupta, 1979a, 1979b, 1993; Ahmad et al., 2009), thereby, differences in the B demand of graminaceous and dicotyledonous species are probably related to the differences in their cell wall composition, and cis-diol configuration in the cell walls, such as pectic substances. A meager amount of pectic material is constituted in the cell walls of graminaceous species (wheat and rice). Such species also have much lower Ca requirements. In fact, these two plant categories also differ in their capacity for silicon (Si) uptake, which is usually inversely related to B and Ca requirements (Loomis & Durst, 1992). All the three elements are located mainly in the cell walls. Brown & Shelp (1997) and Brown & Hu (1998) concluded that knowledge of the relative mobility of B within a particular species determines the optimum fertilization strategy and the same can also be used in partial understanding of the causes and consequences of B deficiency.

In summary, B deficiency is commonly induced under the following soil conditions; 1) soils which are inherently low in B, such as those derived from the parent material made from acid granite and other igneous rocks, and freshwater sedimentary deposits, 2) leaching impacted naturally acid soils from which native B has been removed, 3) light-textured sandy soils and gravelly soils, 4) alkaline and calcareous soils, 5) irrigated soils having low B concentration in irrigation water, and where salt or carbonate has been deposited, and 6) soils low in OM.

#### **4. Diagnosis and correction of boron deficiency**

#### **4.1 Identification of boron deficiency**

Boron is very vulnerable to leaching, so its deficiency can temporarily be expected in countries like Pakistan and India during and after monsoon rains, especially in coarsetextured soils. However, its major source mineral (i.e., tourmaline) is highly insoluble. In Pakistan, Alfisols appear to be the soil group most likely to produce B-deficient crops (Zia et

al. (2009).

2006).

Boron Deficiency in Soils and Crops: A Review 85

required for broadcast applications than for banded soil applications or foliar sprays. Because B is immobile in plants, B deficiency in crops growing in soils with marginal B levels can occur during peak growing periods (vegetative, flowering, and seed development stages), so a steady supply of B throughout the growing season is essential for optimum growth and seed yield. Foliar fertilization is also an effective way to supply B to plants, especially when root activity is restricted and B deficiency in crop appears under dry soil conditions in the growing season (Mortvedt, 1994). Experiments regarding the effect of B on yield, mobility and stress tolerance in different crop species revealed that B significantly enhanced yield and it was attributed to the significant increase in the panicle fertility. In extreme cases, crops on low B soils grow well until flowering when floral abortion or seed set failure can result in severe yield losses. Boron deficiency at critical stages of reproductive development has been shown to cause pod abortion with poor seed setting in wheat in Western Australia (Wong, 2003). Boron application at the onset of reproductive phase was found to be more effective, most likely due to its immobile nature in the plants depending upon the photosynthetic efficiency of the plants (Anonymous, 2007a). These findings are in agreement with the recent work of Ahmad et

Relatively small amounts of B that are normally required to make significant improvements in B status of annual crops, namely 1–2 kg B ha-1, are in broad accord with such recovery rates. For many crops, absorption of 100–200 g B ha-1 of applied B could be expected to be sufficient (Shorrocks, 1997). Application of 10 kg of boric acid ha-1 (1.7 kg B ha-1) or 18 kg of borax ha-1 (2.0 kg B ha-1) proved to be effective for 4-5 years in order to cure B deficiency in rice, wheat and cotton soils.. It was found that in case of cotton, 0.1% solution of B would be economical if used with insecticides foliar sprays. Value cost ratios (VCR) for B use in these crops have been very good , particularly in the case of cotton, where it ranged from 5:1 to 13:1 by soil application and 20:1 by foliar application of B. It was revealed that application of B significantly boosted rice yield, mainly because of increase in the panicle fertility (Anonymous, 2007b). Application of B fertilizers up to 2.5 kg B ha-1 is recommended for major crops like cotton, rice and wheat in Pakistan (Anonymous, 1998). Boron may safely be applied to orchard crops at a rate of 0.56 kg B ha-1 as a maintenance dose and at a rate of 1.12 kg B ha-1 as a deficiency dose (Zia et al., 2006) and its residual effect has generally been reported for at least two years. In the case of borax, application rates should not exceed 90 g borax per orchard tree (Zia et al.,

In India, soil application of B at 20 kg sodium tetraborate to supply 2.8 kg B ha-1 as well as two foliar sprays with 0.2% solution of this salt proved equally effective in increasing soybean grain yield and the residual effect of soil applied B on subsequent wheat crop was significantly higher as compared with direct foliar B application (Table 3). Since B undergoes less leaching in fine-textured soils, single application may produce residual effect. In view of very sharp and narrow difference between optimum and toxic levels of B, more precaution is needed in its repeat application, particularly in medium- to fine-textured soils. Boron deficiency is also invariably corrected by its soil application depending upon soil type (Arora et al., 1985; Sakal et al., 1988; Ali & Monoranjan, 1989). In calcareous soils of Bihar, the rate varying between 1.0 to 2.5 kg B ha-1 has been found to be optimum for

different crops (Sakal et al., 1988; Sinha et al., 1991).

al., 2006). Singh (2001) explored that out of 36,825 soil samples collected throughout India, 33% were deficient in B. In India, laterite and lateritic soils (Ferralsols and Dystric Nitisols) have been widely reported for the deficiency of B. Boron deficiencies are also more pronounced during drought periods when root activity is restricted. Once B has accumulated in a particular organ, it has restricted mobility in most plant species but not all. Boron is immobile in plants, so its deficiency symptoms develop firstly, and are more severe, on young leaves with marginal, dull yellow chlorosis at the tip of young leaves. Because B plays an important role in the elongation of stems and leaves, stems of B deficient plants are short and stout. If B deficiency is severe, many tillers can die before maturity, or whole plant may die before producing heads. Boron deficiency also manifests itself in poorly developed stamens, blast of pear blossoms, inadequate fruit set, bark necrosis of apple, corking in the fruit, and cracking of fruit. When leaf B levels are in the range of 20 to 25 mg B kg-1 (desired is 35 mg B kg-1) on a dry-weight basis, supplemental B is needed. Boron is taken up from the soil only at higher soil temperatures than are other elements. Most values of the critical concentration for B deficiency range from 0.15 to 0.50 mg kg-1 soil (HWE – hot water extractable). However, in highly sensitive crops and alkaline clay soils, these values can double. This is because, B sorption increases to a maximum between pH 7.5-9.5. Hence, the critical range of extractable B is generally higher in alkaline soils. For example Bell (1997) reported that for wheat grown on alkaline clay soils in northern China, a critical range of 0.32-0.38 mg B kg-1 (HWE) was proposed, whereas on loams in northern Thailand the figure was 0.12-0.15 mg B kg-1. The critical concentration of B (HWE) in soils which is considered deficient to most crops in Pakistan was 0.45-0.50 mg B kg-1 until revised recently to 0.65 mg B kg-1 (Rashid et al., 1994; Rashid, 2006). Singh (1994) concluded that depending upon groundnut genotypes and soil, the critical limits of B may vary from 0.2-0.4 mg B kg-1.

#### **4.2 Sources, rates, methods and timing of boron application**

There are eight different sources of B [borax (Na2B4O7.10H2O with 11% B), solubor - Na2B8O13.4H2O (20% B), sodium borate (Na2B4O7.5H2O with 20% B), sodium tetraborate (Na2B4O2.5H2O with 14% B), boric acid (H3BO3 with 17% B), Colemanite (Ca2B6O11.5H2O with 10% B), B frits containing 2-6% B, and boronated superphosphate being used to prevent/correct B deficiency in crops. Borax, solubor, sodium borate and sodium tetraborate have been most commonly used for soil application. Boric acid, colemanite and B frits are considered to be more promising on highly leached sandy soils as well as for long duration field crops including perennial forages and fruit plants owing to their low solubility and slow release of B. Boronated superphosphate has also been tried to correct B deficiency in crops (Patil et al., 1987).

Among these B fertilizer sources, borax is the most commonly used B fertilizer to prevent and/or correct B deficiencies in crops. Because of the narrow margin between B sufficiency and toxicity, an excess dose can easily occur and harm plant growth (Gupta, 1972; Marschner, 1995). Therefore, extreme care is needed to apply the correct dose of B fertilizer and to distribute it uniformly. Boron application rates generally range from 0.25 to 3.0 kg B ha-1, depending on crop requirement and the method of application (Arora et al., 1985; Nuttall et al., 1987; Patil et al., 1987; Sakal et al., 1988; Ali & Monoranjan, 1989; Dwivedi et al., 1990; Sinha et al., 1991; Mortvedt & Woodruff, 1993). Higher rates are

al., 2006). Singh (2001) explored that out of 36,825 soil samples collected throughout India, 33% were deficient in B. In India, laterite and lateritic soils (Ferralsols and Dystric Nitisols) have been widely reported for the deficiency of B. Boron deficiencies are also more pronounced during drought periods when root activity is restricted. Once B has accumulated in a particular organ, it has restricted mobility in most plant species but not all. Boron is immobile in plants, so its deficiency symptoms develop firstly, and are more severe, on young leaves with marginal, dull yellow chlorosis at the tip of young leaves. Because B plays an important role in the elongation of stems and leaves, stems of B deficient plants are short and stout. If B deficiency is severe, many tillers can die before maturity, or whole plant may die before producing heads. Boron deficiency also manifests itself in poorly developed stamens, blast of pear blossoms, inadequate fruit set, bark necrosis of apple, corking in the fruit, and cracking of fruit. When leaf B levels are in the range of 20 to 25 mg B kg-1 (desired is 35 mg B kg-1) on a dry-weight basis, supplemental B is needed. Boron is taken up from the soil only at higher soil temperatures than are other elements. Most values of the critical concentration for B deficiency range from 0.15 to 0.50 mg kg-1 soil (HWE – hot water extractable). However, in highly sensitive crops and alkaline clay soils, these values can double. This is because, B sorption increases to a maximum between pH 7.5-9.5. Hence, the critical range of extractable B is generally higher in alkaline soils. For example Bell (1997) reported that for wheat grown on alkaline clay soils in northern China, a critical range of 0.32-0.38 mg B kg-1 (HWE) was proposed, whereas on loams in northern Thailand the figure was 0.12-0.15 mg B kg-1. The critical concentration of B (HWE) in soils which is considered deficient to most crops in Pakistan was 0.45-0.50 mg B kg-1 until revised recently to 0.65 mg B kg-1 (Rashid et al., 1994; Rashid, 2006). Singh (1994) concluded that depending upon groundnut genotypes and soil, the critical limits of B may vary from 0.2-0.4

mg B kg-1.

crops (Patil et al., 1987).

**4.2 Sources, rates, methods and timing of boron application** 

There are eight different sources of B [borax (Na2B4O7.10H2O with 11% B), solubor - Na2B8O13.4H2O (20% B), sodium borate (Na2B4O7.5H2O with 20% B), sodium tetraborate (Na2B4O2.5H2O with 14% B), boric acid (H3BO3 with 17% B), Colemanite (Ca2B6O11.5H2O with 10% B), B frits containing 2-6% B, and boronated superphosphate being used to prevent/correct B deficiency in crops. Borax, solubor, sodium borate and sodium tetraborate have been most commonly used for soil application. Boric acid, colemanite and B frits are considered to be more promising on highly leached sandy soils as well as for long duration field crops including perennial forages and fruit plants owing to their low solubility and slow release of B. Boronated superphosphate has also been tried to correct B deficiency in

Among these B fertilizer sources, borax is the most commonly used B fertilizer to prevent and/or correct B deficiencies in crops. Because of the narrow margin between B sufficiency and toxicity, an excess dose can easily occur and harm plant growth (Gupta, 1972; Marschner, 1995). Therefore, extreme care is needed to apply the correct dose of B fertilizer and to distribute it uniformly. Boron application rates generally range from 0.25 to 3.0 kg B ha-1, depending on crop requirement and the method of application (Arora et al., 1985; Nuttall et al., 1987; Patil et al., 1987; Sakal et al., 1988; Ali & Monoranjan, 1989; Dwivedi et al., 1990; Sinha et al., 1991; Mortvedt & Woodruff, 1993). Higher rates are required for broadcast applications than for banded soil applications or foliar sprays. Because B is immobile in plants, B deficiency in crops growing in soils with marginal B levels can occur during peak growing periods (vegetative, flowering, and seed development stages), so a steady supply of B throughout the growing season is essential for optimum growth and seed yield. Foliar fertilization is also an effective way to supply B to plants, especially when root activity is restricted and B deficiency in crop appears under dry soil conditions in the growing season (Mortvedt, 1994). Experiments regarding the effect of B on yield, mobility and stress tolerance in different crop species revealed that B significantly enhanced yield and it was attributed to the significant increase in the panicle fertility. In extreme cases, crops on low B soils grow well until flowering when floral abortion or seed set failure can result in severe yield losses. Boron deficiency at critical stages of reproductive development has been shown to cause pod abortion with poor seed setting in wheat in Western Australia (Wong, 2003). Boron application at the onset of reproductive phase was found to be more effective, most likely due to its immobile nature in the plants depending upon the photosynthetic efficiency of the plants (Anonymous, 2007a). These findings are in agreement with the recent work of Ahmad et al. (2009).

Relatively small amounts of B that are normally required to make significant improvements in B status of annual crops, namely 1–2 kg B ha-1, are in broad accord with such recovery rates. For many crops, absorption of 100–200 g B ha-1 of applied B could be expected to be sufficient (Shorrocks, 1997). Application of 10 kg of boric acid ha-1 (1.7 kg B ha-1) or 18 kg of borax ha-1 (2.0 kg B ha-1) proved to be effective for 4-5 years in order to cure B deficiency in rice, wheat and cotton soils.. It was found that in case of cotton, 0.1% solution of B would be economical if used with insecticides foliar sprays. Value cost ratios (VCR) for B use in these crops have been very good , particularly in the case of cotton, where it ranged from 5:1 to 13:1 by soil application and 20:1 by foliar application of B. It was revealed that application of B significantly boosted rice yield, mainly because of increase in the panicle fertility (Anonymous, 2007b). Application of B fertilizers up to 2.5 kg B ha-1 is recommended for major crops like cotton, rice and wheat in Pakistan (Anonymous, 1998). Boron may safely be applied to orchard crops at a rate of 0.56 kg B ha-1 as a maintenance dose and at a rate of 1.12 kg B ha-1 as a deficiency dose (Zia et al., 2006) and its residual effect has generally been reported for at least two years. In the case of borax, application rates should not exceed 90 g borax per orchard tree (Zia et al., 2006).

In India, soil application of B at 20 kg sodium tetraborate to supply 2.8 kg B ha-1 as well as two foliar sprays with 0.2% solution of this salt proved equally effective in increasing soybean grain yield and the residual effect of soil applied B on subsequent wheat crop was significantly higher as compared with direct foliar B application (Table 3). Since B undergoes less leaching in fine-textured soils, single application may produce residual effect. In view of very sharp and narrow difference between optimum and toxic levels of B, more precaution is needed in its repeat application, particularly in medium- to fine-textured soils. Boron deficiency is also invariably corrected by its soil application depending upon soil type (Arora et al., 1985; Sakal et al., 1988; Ali & Monoranjan, 1989). In calcareous soils of Bihar, the rate varying between 1.0 to 2.5 kg B ha-1 has been found to be optimum for different crops (Sakal et al., 1988; Sinha et al., 1991).

**5.2 Rice** 

0.7 mg B kg-1 dry weight.

zAdapted from Rashid et al., 2005.

**5.3 Wheat** 

Boron Deficiency in Soils and Crops: A Review 87

Rice (*Oryza sativa* L.) is grown worldwide, but it is one of the most important cereal grains especially in Asia. Severe B deficiency has been reported in 10-45% rice fields in Pakistan (Tahir et al., 1990; Zia, 1993) and 1-69% (average 33%) rice fields in India (Singh, 2001). Average increase in rice paddy yield with B application in 22 field experiments was 14% over the zero-B control (Anonymous, 1998). Results of recent research have shown 15-25% increase in seed yield over N, P and Zn, coupled with appreciable improvement in grain/cooking quality (more recovery and less breakage of kernels during milling, greater grain elongation, less bursting and less stickness upon cooking) with application of B (Rashid et al., 2009). The authors also found that the B use in rice was highly profitable. Similarly, Mehmood et al. (2009) worked on three rice cultivars [viz., KS-282 (salttolerant), BG-402-4 (mixed behavior) and IR-28 (salt-sensitive)] to investigate the ameliorative nutritional aspects of B. Boron was applied at 25, 50, 100, 200, 400 and 800 ng B mL-1 in the presence (80 mol m-3) and absence (0 mol m-3) of NaCl salinity, whereas in solution culture B was applied at 1.5, 3.0 and 6.0 kg B ha-1 to saline [electrical conductivity of saturated paste extract (ECe) 9.0 dS m-1, sodium adsorption ratio (SAR) 5.46, and pH 7.8), and saline-sodic soils (ECe 9.0 dS m-1, SAR 28.2, pH 8.2]. Application of B improved all growth parameters, i.e., tillering capacity, shoot and root length, and shoot and root weight at external B application rates of 200-400 ng B mL-1 in solution culture in the presence and absence of NaCl salinity. Moreover, rice cultivars have shown differential response to B application (Table 4). In contrast, a marked increase in the paddy rice yield with the application of B was also reported in a non-saline soil (Chaudhry et al., 1976). Increasing supply of B increased the accumulation of B in roots and shoots (Nable et al., 1990; Akram et al., 2006). Vasil (1987) reported that the stigma, style and ovary often contain high concentration of B, and this B occurs in pollen at about

**B concentration (mg B kg-1) in leaves SN. Cultivar Control (no B) B applied Remarks** 

Table 4. Response of B concentration in leaves of different rice cultivars to B applicationz

Wheat (*Triticum aestivum* L.) is amongst the major cereal crops grown in almost every part of the world. Boron deficiency in wheat field was first observed almost concurrently on different sides of the world following the spread of semi-dwarf wheat in the 1960s (Rerkasem & Jamjod, 2004). Its deficiency has been reported to cause grain set failure and considerable yield losses in the

B concentration in leaves of different cultivars increased with B application

1 Super Basmati 7.58 11.24

2 Basmati-6129 9.36 16.19 3 DR-83 9.07 14.70 4 KS-282 9.86 16.67 5 Basmati-385 7.14 8.42 6 Pakhal 9.29 11.56 7 Basmati-370 7.43 9.79 8 IR-6 8.62 11.31


zSource: Dwivedi et al., 1990.

Table 3. Effect of mode of B application on grain yield of soybean and wheatz

#### **5. Yield response of selected crops to boron fertilization**

#### **5.1 Cotton**

Cotton (*Gossypium hirsutum* L.) is an important fiber crop grown in many countries of the world. There are several factors responsible for low yields of cotton, and micronutrient deficiency is one of them. Boron has been recognized as the most important micronutrient for cotton production in some countries. Its deficiency inhibits petiole and peduncle cell development and reduces growth of cotton (De Oliveira et al., 2006). In a number of studies, application of B fertilizer has been shown to increase cotton yield (Murphy & Lancaster, 1971; Rashid, 1995, 1996; Anonymous, 1998). Research has shown that as little as 1.1 kg of B ha-1 can increase cotton seed yield by more than 560 kg ha-1 (US Borax, 2002). In Pakistan, 50% cotton fields have been reported to be deficient in B (Anonymous, 1998). Cotton is very responsive to B fertilization on B deficient soils. For example, in a study in Pakistan with 30 field experiments, B application has been reported to increase cotton yield in the range of 2 to 30%, with an average value of 14%, over the zero-B control (Malik et al., 1992; Rashid, 1995, 1996; Anonymous, 1998). The value cost ratio (VCR) data indicated that by spending one rupee on B fertilizer, crop yield increase was worth Rs. 5 to 20 (average Rs. 16) in cotton (Anonymous, 1998). Use of B and Zn fertilizers proved highly profitable, benefit cost ratio being 15:1 for soil application and 30:1 for foliar spray (Rashid & Akhtar, 2006). Niaz et al. (2002) conducted field experiments on cotton at 13 different sites in Punjab, Pakistan; five were medium-textured (clay loam), two were silty clay, one was loam, and five were coarse-textured (sandy loam or loamy sand). Of the 13 soils, 12 were found deficient in B (less than 0.5 mg B kg-1 0.05M HCl extractable). Boron concentration in younger leaves, at flowering stage and harvest, ranged from 7.8 to 23.8 mg B kg-1 with an average of 11.4 mg B kg-1, whereas only one of the 13 samples had adequate B concentration (15 mg B kg-1). Similar results have also been reported from Australia (Reuter & Robinson, 1986; Shorrocks, 1997), Egypt (Ibrahim et al., 2009), Turkey (Gormus, 2005) and USA (Zhao & Oosterhuis, 2003). In Taiwan, Smithson & Heathcote (1976) found that when B deficiency occurred in cotton, the application of 250 kg N ha-1 reduced the yield. However, if B was applied, the same application of N increased the crop yield. In pot experiments, application of 0.06 g of borax to 40 kg soil, deficient in B, was sufficient to overcome B deficiency problem in cotton. In field experiments, 10 to 30 kg ha-1 of applied borax (to supply 1.1 to 3.3 kg B ha-1) was enough to prevent B deficiency. Since B is essential for the transfer and assimilation of sugars and N into complex carbohydrates (fiber) and protein, demand for this element is the greatest during lint and seed development (Lancaster et al., 1962).

#### **5.2 Rice**

86 Crop Plant

Cotton (*Gossypium hirsutum* L.) is an important fiber crop grown in many countries of the world. There are several factors responsible for low yields of cotton, and micronutrient deficiency is one of them. Boron has been recognized as the most important micronutrient for cotton production in some countries. Its deficiency inhibits petiole and peduncle cell development and reduces growth of cotton (De Oliveira et al., 2006). In a number of studies, application of B fertilizer has been shown to increase cotton yield (Murphy & Lancaster, 1971; Rashid, 1995, 1996; Anonymous, 1998). Research has shown that as little as 1.1 kg of B ha-1 can increase cotton seed yield by more than 560 kg ha-1 (US Borax, 2002). In Pakistan, 50% cotton fields have been reported to be deficient in B (Anonymous, 1998). Cotton is very responsive to B fertilization on B deficient soils. For example, in a study in Pakistan with 30 field experiments, B application has been reported to increase cotton yield in the range of 2 to 30%, with an average value of 14%, over the zero-B control (Malik et al., 1992; Rashid, 1995, 1996; Anonymous, 1998). The value cost ratio (VCR) data indicated that by spending one rupee on B fertilizer, crop yield increase was worth Rs. 5 to 20 (average Rs. 16) in cotton (Anonymous, 1998). Use of B and Zn fertilizers proved highly profitable, benefit cost ratio being 15:1 for soil application and 30:1 for foliar spray (Rashid & Akhtar, 2006). Niaz et al. (2002) conducted field experiments on cotton at 13 different sites in Punjab, Pakistan; five were medium-textured (clay loam), two were silty clay, one was loam, and five were coarse-textured (sandy loam or loamy sand). Of the 13 soils, 12 were found deficient in B (less than 0.5 mg B kg-1 0.05M HCl extractable). Boron concentration in younger leaves, at flowering stage and harvest, ranged from 7.8 to 23.8 mg B kg-1 with an average of 11.4 mg B kg-1, whereas only one of the 13 samples had adequate B concentration (15 mg B kg-1). Similar results have also been reported from Australia (Reuter & Robinson, 1986; Shorrocks, 1997), Egypt (Ibrahim et al., 2009), Turkey (Gormus, 2005) and USA (Zhao & Oosterhuis, 2003). In Taiwan, Smithson & Heathcote (1976) found that when B deficiency occurred in cotton, the application of 250 kg N ha-1 reduced the yield. However, if B was applied, the same application of N increased the crop yield. In pot experiments, application of 0.06 g of borax to 40 kg soil, deficient in B, was sufficient to overcome B deficiency problem in cotton. In field experiments, 10 to 30 kg ha-1 of applied borax (to supply 1.1 to 3.3 kg B ha-1) was enough to prevent B deficiency. Since B is essential for the transfer and assimilation of sugars and N into complex carbohydrates (fiber) and protein, demand for this element is the greatest

Soybean Wheat

Treatment Grain yield (Mg ha-1)

(20 kg sodium tetraborate ha-1) 1.41 1.46

(0.2% sodium tetraborate soil) 1.33 1.35 Control 0.89 0.66 LSD (p=0.05) 0.1 0.11

Table 3. Effect of mode of B application on grain yield of soybean and wheatz

**5. Yield response of selected crops to boron fertilization** 

during lint and seed development (Lancaster et al., 1962).

Soil Application

Foliar application

**5.1 Cotton** 

zSource: Dwivedi et al., 1990.

Rice (*Oryza sativa* L.) is grown worldwide, but it is one of the most important cereal grains especially in Asia. Severe B deficiency has been reported in 10-45% rice fields in Pakistan (Tahir et al., 1990; Zia, 1993) and 1-69% (average 33%) rice fields in India (Singh, 2001). Average increase in rice paddy yield with B application in 22 field experiments was 14% over the zero-B control (Anonymous, 1998). Results of recent research have shown 15-25% increase in seed yield over N, P and Zn, coupled with appreciable improvement in grain/cooking quality (more recovery and less breakage of kernels during milling, greater grain elongation, less bursting and less stickness upon cooking) with application of B (Rashid et al., 2009). The authors also found that the B use in rice was highly profitable. Similarly, Mehmood et al. (2009) worked on three rice cultivars [viz., KS-282 (salttolerant), BG-402-4 (mixed behavior) and IR-28 (salt-sensitive)] to investigate the ameliorative nutritional aspects of B. Boron was applied at 25, 50, 100, 200, 400 and 800 ng B mL-1 in the presence (80 mol m-3) and absence (0 mol m-3) of NaCl salinity, whereas in solution culture B was applied at 1.5, 3.0 and 6.0 kg B ha-1 to saline [electrical conductivity of saturated paste extract (ECe) 9.0 dS m-1, sodium adsorption ratio (SAR) 5.46, and pH 7.8), and saline-sodic soils (ECe 9.0 dS m-1, SAR 28.2, pH 8.2]. Application of B improved all growth parameters, i.e., tillering capacity, shoot and root length, and shoot and root weight at external B application rates of 200-400 ng B mL-1 in solution culture in the presence and absence of NaCl salinity. Moreover, rice cultivars have shown differential response to B application (Table 4). In contrast, a marked increase in the paddy rice yield with the application of B was also reported in a non-saline soil (Chaudhry et al., 1976). Increasing supply of B increased the accumulation of B in roots and shoots (Nable et al., 1990; Akram et al., 2006). Vasil (1987) reported that the stigma, style and ovary often contain high concentration of B, and this B occurs in pollen at about 0.7 mg B kg-1 dry weight.


zAdapted from Rashid et al., 2005.

Table 4. Response of B concentration in leaves of different rice cultivars to B applicationz

#### **5.3 Wheat**

Wheat (*Triticum aestivum* L.) is amongst the major cereal crops grown in almost every part of the world. Boron deficiency in wheat field was first observed almost concurrently on different sides of the world following the spread of semi-dwarf wheat in the 1960s (Rerkasem & Jamjod, 2004). Its deficiency has been reported to cause grain set failure and considerable yield losses in the

Canola or oilseed rape (*Brassica spp*. L.) is one of the major oilseed crops grown worldwide. Canola is considered to have high requirements for B. In addition, a steady supply of B during the peak vegetative, flowering, pod production and seed development stages is needed for optimum seed yield of canola. Deficiency of B at any growth stage in canola can severely affect its seed yield (US Borax, 1996). Research has shown that application of B fertilizers can be most effective if incorporated into the soil, seed and band placement may have toxic effects, and foliar application may be very effective to supply B to plants when deficiency is noted in the growing season. Foliar fertilization is an effective way to supply B to plants, especially when root activity is restricted by dry soil (Mortvedt, 1994). In a field study comparing rapeseed (*Brassica campestris* L.), barley (*Hordeum vulgare* L.) and potato (*Solanum tuberosum* L.) test crops, rapeseed showed the largest response to B fertilization (Wooding, 1985). In that study, symptoms of B deficiency on rapeseed plants did not appear until upper parts of the plants formed pods, with seed development limited to only those pods located on the lower parts of the plant. Also, B deficiency delayed maturity and kept the plants in an indeterminate stage of growth with flowers forming up to the time of the first killing frost. In other study in Alberta, Canada, B-deficient oilseed rape appeared normal in early growth stages, showed red margins and/or inter-veinal mottling at bloom stage and had reduced seed

In China, on a clayey soil with 0.7 mg B kg-1, application of B fertilizer to *Brassica napus* L. improved plant height, pod-bearing branches and pod number per plant, seed number per pod, seed yield and oil content (Hu et al., 1994). Recently, Shi & Wang (2009) reported decrease in seed yield in oilseed rape (*Brassica napus* L.) with B deficiency. In Pakistan, based on various nutrient indexing field experiments on rapeseed-mustard, Rashid (1993, 1994) reported that 65% of the tested sites were deficient in B under the agro-ecological conditions. Oilseed crops responded well to B application for the reported B-deficient sites in Pakistan (Anonymous, 1998). In other studies, canola yield was not affected by B fertilization, although B concentration in plants was significantly increased and 20-30 mg B kg-1 in plant tissue was considered adequate for optimum yield (Bullock & Sawyer, 1991). In the Parkland region of western Canada, canola grown on Gray Luvisol soils has sometimes shown failure of flower bud development and poor seed set, more often on sandy soils. Deficiency of B was suspected to be responsible for these conditions because the symptoms match B deficiency symptoms (Grant & Bailey, 1993). In an earlier study on the Parkland region soils of Saskatchewan, B fertilization was observed to enhance rapeseed yield in a greenhouse experiment but its effect was not consistent in field experiments (Nuttall et al., 1987). In another field study in Saskatchewan, Canada, application of B fertilizer did not have any consistent influence on seed yield increase of canola, grown on soils ranging between 0.11 to 0.82 mg plant-available B kg-1 (Malhi et al.,

Maize (*Zea mays* L.) belongs to Graminae family. It ranks second (after wheat) in the world cereal production. Contribution to world corn/maize production is 2% from India, while it is 10% from China, and U.S. contribution to the total maize production of the world is

**5.4 Oilseed** *Brassica spp***.** 

set (Nyborg & Hoyt, 1970).

2003).

**5.5 Maize/corn** 

wheat belt of the world's wheat growing countries (Rerkasem & Jamjod, 2004; Rerkasem et al., 2004). Since introduction of green revolution cases of severe B deficiency have been reported from several wheat growing countries of the world (Li et al., 1978; da Silva & de Andrade, 1980; Misra et al., 1992). Bangladesh, Brazil, Bulgaria, China, Finland, India, Madagascar, Nepal, Pakistan, South Africa, Sweden, Tanzania, Thailand, USA, USSR, Yugoslavia and Zambia are amongst the countries where B fertilization response based B deficiency, in wheat has been reported (Shorrocks, 1997). The B deficiency pronedregionsare believed to be in adjoining areas of eastern Nepal, northeastern India and northwestern Bangladesh, through to southwestern China (Rerkasem & Jamjod, 2004; Bhatta & Ferrara, 2005). Boron application on such fields (Bdeficient soils) can make profound contributions to grain yield in wheat (Chakraborti & Barman, 2003; Soylu & Topal, 2004). In Pakistan, Chaudhry et al. (2007) conducted a study to identify the wheat response to micronutrients (B, Fe, Zn) in rainfed areas. The authors observed an increase in the yield of wheat and other crops (rice, maize and cotton) in a number of field experiments in response to B application. Summary of 16 field experiments revealed that application of B contributed 16% increase in grain yield, and also increase in value to cost ratio (VCR) over the zero-B control (Table 5; Anonymous, 1998). Further, genotypic differences were observed among wheat cultivars for their response to B application (Table 6).


zSource: Anonymous, 1998.

Table 5. Yield responses and crop value to cost ratios (VCR) of four major crops to B fertilizer application in field experiments, Pakistanz


zAdapted from Rashid et al., 2005.

Table 6. Response of B concentration in leaves of different wheat cultivars to B applicationz

#### **5.4 Oilseed** *Brassica spp***.**

88 Crop Plant

wheat belt of the world's wheat growing countries (Rerkasem & Jamjod, 2004; Rerkasem et al., 2004). Since introduction of green revolution cases of severe B deficiency have been reported from several wheat growing countries of the world (Li et al., 1978; da Silva & de Andrade, 1980; Misra et al., 1992). Bangladesh, Brazil, Bulgaria, China, Finland, India, Madagascar, Nepal, Pakistan, South Africa, Sweden, Tanzania, Thailand, USA, USSR, Yugoslavia and Zambia are amongst the countries where B fertilization response based B deficiency, in wheat has been reported (Shorrocks, 1997). The B deficiency pronedregionsare believed to be in adjoining areas of eastern Nepal, northeastern India and northwestern Bangladesh, through to southwestern China (Rerkasem & Jamjod, 2004; Bhatta & Ferrara, 2005). Boron application on such fields (Bdeficient soils) can make profound contributions to grain yield in wheat (Chakraborti & Barman, 2003; Soylu & Topal, 2004). In Pakistan, Chaudhry et al. (2007) conducted a study to identify the wheat response to micronutrients (B, Fe, Zn) in rainfed areas. The authors observed an increase in the yield of wheat and other crops (rice, maize and cotton) in a number of field experiments in response to B application. Summary of 16 field experiments revealed that application of B contributed 16% increase in grain yield, and also increase in value to cost ratio (VCR) over the zero-B control (Table 5; Anonymous, 1998). Further, genotypic differences were

> Control yield (Mg ha-1)

Wheat 16 3.286 14 4:1 Rice 19 3.081 14 5:1 Maize 9 2.512 20 7:1

Soil 30 2.377 14 16:1 Foliar 13 2.156 12 33:1

Table 5. Yield responses and crop value to cost ratios (VCR) of four major crops to B

B concentration (mg B kg-1) in leaves SN. Cultivar Control (no B) B added Remarks

Table 6. Response of B concentration in leaves of different wheat cultivars to B applicationz

Yield increase

(%) VCR

All cultivars showed a positive response to B application as depicted from the increase in B contents of the leaves

observed among wheat cultivars for their response to B application (Table 6).

experiments

fertilizer application in field experiments, Pakistanz

1 Rohtas-90 5.2 11.0

2 Sindh-81 9.0 17.0 3 Faisalabad-85 8.0 10.3 4 Rawal-87 9.3 18.0 5 Pak-81 8.7 15.8 6 Sariab-92 10.0 19.5 7 Inqalab-91 7.2 20.7 8 Bakhtawar 11.0 21.0

Crop Field

zSource: Anonymous, 1998.

zAdapted from Rashid et al., 2005.

Cotton

Canola or oilseed rape (*Brassica spp*. L.) is one of the major oilseed crops grown worldwide. Canola is considered to have high requirements for B. In addition, a steady supply of B during the peak vegetative, flowering, pod production and seed development stages is needed for optimum seed yield of canola. Deficiency of B at any growth stage in canola can severely affect its seed yield (US Borax, 1996). Research has shown that application of B fertilizers can be most effective if incorporated into the soil, seed and band placement may have toxic effects, and foliar application may be very effective to supply B to plants when deficiency is noted in the growing season. Foliar fertilization is an effective way to supply B to plants, especially when root activity is restricted by dry soil (Mortvedt, 1994). In a field study comparing rapeseed (*Brassica campestris* L.), barley (*Hordeum vulgare* L.) and potato (*Solanum tuberosum* L.) test crops, rapeseed showed the largest response to B fertilization (Wooding, 1985). In that study, symptoms of B deficiency on rapeseed plants did not appear until upper parts of the plants formed pods, with seed development limited to only those pods located on the lower parts of the plant. Also, B deficiency delayed maturity and kept the plants in an indeterminate stage of growth with flowers forming up to the time of the first killing frost. In other study in Alberta, Canada, B-deficient oilseed rape appeared normal in early growth stages, showed red margins and/or inter-veinal mottling at bloom stage and had reduced seed set (Nyborg & Hoyt, 1970).

In China, on a clayey soil with 0.7 mg B kg-1, application of B fertilizer to *Brassica napus* L. improved plant height, pod-bearing branches and pod number per plant, seed number per pod, seed yield and oil content (Hu et al., 1994). Recently, Shi & Wang (2009) reported decrease in seed yield in oilseed rape (*Brassica napus* L.) with B deficiency. In Pakistan, based on various nutrient indexing field experiments on rapeseed-mustard, Rashid (1993, 1994) reported that 65% of the tested sites were deficient in B under the agro-ecological conditions. Oilseed crops responded well to B application for the reported B-deficient sites in Pakistan (Anonymous, 1998). In other studies, canola yield was not affected by B fertilization, although B concentration in plants was significantly increased and 20-30 mg B kg-1 in plant tissue was considered adequate for optimum yield (Bullock & Sawyer, 1991). In the Parkland region of western Canada, canola grown on Gray Luvisol soils has sometimes shown failure of flower bud development and poor seed set, more often on sandy soils. Deficiency of B was suspected to be responsible for these conditions because the symptoms match B deficiency symptoms (Grant & Bailey, 1993). In an earlier study on the Parkland region soils of Saskatchewan, B fertilization was observed to enhance rapeseed yield in a greenhouse experiment but its effect was not consistent in field experiments (Nuttall et al., 1987). In another field study in Saskatchewan, Canada, application of B fertilizer did not have any consistent influence on seed yield increase of canola, grown on soils ranging between 0.11 to 0.82 mg plant-available B kg-1 (Malhi et al., 2003).

#### **5.5 Maize/corn**

Maize (*Zea mays* L.) belongs to Graminae family. It ranks second (after wheat) in the world cereal production. Contribution to world corn/maize production is 2% from India, while it is 10% from China, and U.S. contribution to the total maize production of the world is

**5.7 Alfalfa** 

**5.8 Soybean** 

Boron Deficiency in Soils and Crops: A Review 91

Alfalfa (*Medicago sativa* L*.*; also called lucerne) is one of the most important forage crops globally. It is well adapted to a wide range of growing conditions on soils of varied fertility. Boron deficiency caused nutritional disorders are quite common (Shorrocks, 1997; Dell & Huang, 1997). Its deficiency in alfalfa is causative of leaf yellowing, reddening of the upper leaves, shorten internodes and rosette appearance of the plant. At this stage the growing point becomes dormant or dies, flowering is reduced and the flower falls before setting seed (Bell, 1997; Shorrocks, 1997). Boron and other micronutrients applications on Indian soils for alfalfa have shown positive results in the form of increase in forage and seed yield (Kormilitsyn, 1992; Hazra & Tripathi, 1998; Patel & Patel, 2003). In a study on Chinese soils, B application along with other micronutrients increased yield and crude protein content in alfalfa (Wang & Chen, 2003; Liu & Zhang, 2005). Rammah & Khedr (1984) reported positive

Alfalfa is sometimes grown on the Coastal Plain of southern United States, but poor soil fertility status is one of the production problems in these areas. Field-scale demonstrations have shown a considerable increase in alfalfa forage yields (3.9 Mg ha-1 or 159%) with B application. The sustainable economic production is possible under rainfed conditions on selected, limed Coastal Plain soils of US with improved methods of site selection, adequate

In a field study (Greece), foliar B application helped to increase the percentage of pods formed per inflorescence up to 52% as compared with the control. However, no significant difference between the different rates of B application was observed. The seed yield was increased by an average of 37% compared with the zero-B control during the second year at both locations. Moreover, foliar application of B improved seed germination and increased seed vigor which was increased by 27% in 2003 and up to 19% in 2004 as compared with the control (Dordas, 2006). Recently in a field study on calcareous soils in eastern Turkey, Turan et al. (2010) have also reported positive responses to B application. The authors concluded that lucerne production requires B addition to alleviate natural B deficiency problem in soils. This study warrants further studies with different soils and initial soil test B levels needed to conclude critical soil and tissue values for wider application across the region.

Soybean (*Glycine max* L.) belongs to Leguminosae family. China, India and Indonesia are the leading soybean growing countries after USA. The occurrence of B deficiency based on responses at farmers fields have been reported for many countries like Australasia (China, India, Korea, Thailand), Europe (USSR) and in South America (Shorrocks, 1997). Generally, B deficiency is a common problem for this crop, especially when grown on alkaline calcareous soils of the world. The alkaline, silt, and sandy loam soils in Northeast Arkansas are also known to suffer from B deficiency (Anonymous, 2007a). Soybean is known to respond positively to B application on deficient sites of the world (Wu, 1986; Kirk & Loneragan, 1988). The increase in oil content and other quality parameters in soybean with combined application of B and sulfur in India have been noticed by Dinesh & Sudkep (2009), and Kumar & Sidhu (2009). In another study, Eguchi (2000) found a depressing effect of B

deficiency on growth, yield, and protein and fat contents in the grains of soybean.

response of alfalfa to B application in some Egyptian soils.

fertility and management guidelines (Haby & Leonard, 2000, 2005).

known to be 43%. Approximately 80% of the maize production in Pakistan is concentrated in North West Frontier Province and Northern and Central Punjab. Maize in Pakistan is cultivated as a multipurpose food and forage crop, therefore the economic potential of this important crop is overwhelming (Khan et al., 2008). According to National Fertilizer Development Centre, (NFDC), forty percent (40%) maize fields in Pakistan, surveyed for fertility status, have been reported to be deficient in B (Anonymous, 1998). Boron application on nine fields exhibiting B deficiency in maize has shown to be very effective for yield increases, ranging from 12% to 35%, with an average increase of 20% over zero-B control. Maize yield increases worth of 5-15 Rs. (mean Rs. 7) have been documented after spending one rupee on B fertilizer (Anonymous, 1998). In a study in India, Mishra & Shukla (1986) reported considerable increase in plant height, metabolic rate, content of photosynthetic pigment and all dry weight fractions measured after the application of B containing amendment to maize.

#### **5.6 Groundnut/peanut**

Groundnut (*Arachis hypogaea* L.) belongs to Leguminosae family and is known as arachide in France, mani or cacahuete in Spain, pistachio di terra in Italy, erdnuss in Germany and amendoim in Portugal. Farmers in Asia and Africa grow 90% of the world's total groundnut production. The leading groundnut growing countries include India, China and USA. Groundnut is also one of the important cash crops of the Potohar plateau in the Punjab province of Pakistan. The crop is grown under rainfed conditions on relatively poorly fertile alkaline-calcareous soils with no adequate fertilization history (Rashid et al., 1997). Its average yield in Pakistan is reported to be 921 kg ha-1 (Anonymous, 2009) and is much less as compared with the average yields of some other countries, e.g., in China, 2180 kg ha-1 (Luo et al., 1990). As in alkaline and calcareous soils B is deficient (Tisdale et al., 1993), its deficiency is suspected in a highly sensitive crop species like groundnut (Katyal & Randhawa, 1983; Luo et al., 1990) when grown over such a soils. Application of B on such soils has shown positive results throughout the world.

In Pakistan, 50% B-deficient test sites have been reported in farm fields with groundnut, based on multi-locations field trials by Rashid & Qayyum (1991) and Rashid (1993, 1994). The value cost ratio (VCR) data of NFDC (Anonymous, 1998) indicated that by spending one rupee on B fertilizer, crop yield increase was worth Rs. 11 in groundnut. Seed yield increases in groundnut have been reported from 9 to 12% by borax application in B deficient Chinese soil (0.3-0.5 mg B kg-1) by Zhang et al. (1986). A 10% increase in pod yield of groundnut after B fertilization over the control) was obtained with 1 kg B ha-1 (Rashid et al., 1997) in Pakistan. Encouraging responses of groundnut to B application have also been recorded in India, with average pod yield increase of 180 kg ha-1 (Takkar & Nayyar, 1984). In China, Zhang et al. (1986) and Luo et al. (1990) indicated that 1 kg B ha-1 (borax) can be the optimal B fertilizer requirement of groundnut. Foliar application of B is also very effective and it can be used with herbicides for groundnut. Nonetheless, B use in Pakistan is not a promising practice in groundnut as it is a low-input high-risk rainfed crop. Since internal B requirements of various groundnut genotypes vary greatly; a viable and practical solution of managing B deficiency in groundnut could be the screening of the available germplasm with respect to its sensitivity to B deficiency (Rashid et al., 1997).

#### **5.7 Alfalfa**

90 Crop Plant

known to be 43%. Approximately 80% of the maize production in Pakistan is concentrated in North West Frontier Province and Northern and Central Punjab. Maize in Pakistan is cultivated as a multipurpose food and forage crop, therefore the economic potential of this important crop is overwhelming (Khan et al., 2008). According to National Fertilizer Development Centre, (NFDC), forty percent (40%) maize fields in Pakistan, surveyed for fertility status, have been reported to be deficient in B (Anonymous, 1998). Boron application on nine fields exhibiting B deficiency in maize has shown to be very effective for yield increases, ranging from 12% to 35%, with an average increase of 20% over zero-B control. Maize yield increases worth of 5-15 Rs. (mean Rs. 7) have been documented after spending one rupee on B fertilizer (Anonymous, 1998). In a study in India, Mishra & Shukla (1986) reported considerable increase in plant height, metabolic rate, content of photosynthetic pigment and all dry weight fractions measured after the application of B

Groundnut (*Arachis hypogaea* L.) belongs to Leguminosae family and is known as arachide in France, mani or cacahuete in Spain, pistachio di terra in Italy, erdnuss in Germany and amendoim in Portugal. Farmers in Asia and Africa grow 90% of the world's total groundnut production. The leading groundnut growing countries include India, China and USA. Groundnut is also one of the important cash crops of the Potohar plateau in the Punjab province of Pakistan. The crop is grown under rainfed conditions on relatively poorly fertile alkaline-calcareous soils with no adequate fertilization history (Rashid et al., 1997). Its average yield in Pakistan is reported to be 921 kg ha-1 (Anonymous, 2009) and is much less as compared with the average yields of some other countries, e.g., in China, 2180 kg ha-1 (Luo et al., 1990). As in alkaline and calcareous soils B is deficient (Tisdale et al., 1993), its deficiency is suspected in a highly sensitive crop species like groundnut (Katyal & Randhawa, 1983; Luo et al., 1990) when grown over such a soils. Application of B on such

In Pakistan, 50% B-deficient test sites have been reported in farm fields with groundnut, based on multi-locations field trials by Rashid & Qayyum (1991) and Rashid (1993, 1994). The value cost ratio (VCR) data of NFDC (Anonymous, 1998) indicated that by spending one rupee on B fertilizer, crop yield increase was worth Rs. 11 in groundnut. Seed yield increases in groundnut have been reported from 9 to 12% by borax application in B deficient Chinese soil (0.3-0.5 mg B kg-1) by Zhang et al. (1986). A 10% increase in pod yield of groundnut after B fertilization over the control) was obtained with 1 kg B ha-1 (Rashid et al., 1997) in Pakistan. Encouraging responses of groundnut to B application have also been recorded in India, with average pod yield increase of 180 kg ha-1 (Takkar & Nayyar, 1984). In China, Zhang et al. (1986) and Luo et al. (1990) indicated that 1 kg B ha-1 (borax) can be the optimal B fertilizer requirement of groundnut. Foliar application of B is also very effective and it can be used with herbicides for groundnut. Nonetheless, B use in Pakistan is not a promising practice in groundnut as it is a low-input high-risk rainfed crop. Since internal B requirements of various groundnut genotypes vary greatly; a viable and practical solution of managing B deficiency in groundnut could be the screening of the available germplasm with respect to its sensitivity to B deficiency

containing amendment to maize.

soils has shown positive results throughout the world.

**5.6 Groundnut/peanut** 

(Rashid et al., 1997).

Alfalfa (*Medicago sativa* L*.*; also called lucerne) is one of the most important forage crops globally. It is well adapted to a wide range of growing conditions on soils of varied fertility. Boron deficiency caused nutritional disorders are quite common (Shorrocks, 1997; Dell & Huang, 1997). Its deficiency in alfalfa is causative of leaf yellowing, reddening of the upper leaves, shorten internodes and rosette appearance of the plant. At this stage the growing point becomes dormant or dies, flowering is reduced and the flower falls before setting seed (Bell, 1997; Shorrocks, 1997). Boron and other micronutrients applications on Indian soils for alfalfa have shown positive results in the form of increase in forage and seed yield (Kormilitsyn, 1992; Hazra & Tripathi, 1998; Patel & Patel, 2003). In a study on Chinese soils, B application along with other micronutrients increased yield and crude protein content in alfalfa (Wang & Chen, 2003; Liu & Zhang, 2005). Rammah & Khedr (1984) reported positive response of alfalfa to B application in some Egyptian soils.

Alfalfa is sometimes grown on the Coastal Plain of southern United States, but poor soil fertility status is one of the production problems in these areas. Field-scale demonstrations have shown a considerable increase in alfalfa forage yields (3.9 Mg ha-1 or 159%) with B application. The sustainable economic production is possible under rainfed conditions on selected, limed Coastal Plain soils of US with improved methods of site selection, adequate fertility and management guidelines (Haby & Leonard, 2000, 2005).

In a field study (Greece), foliar B application helped to increase the percentage of pods formed per inflorescence up to 52% as compared with the control. However, no significant difference between the different rates of B application was observed. The seed yield was increased by an average of 37% compared with the zero-B control during the second year at both locations. Moreover, foliar application of B improved seed germination and increased seed vigor which was increased by 27% in 2003 and up to 19% in 2004 as compared with the control (Dordas, 2006). Recently in a field study on calcareous soils in eastern Turkey, Turan et al. (2010) have also reported positive responses to B application. The authors concluded that lucerne production requires B addition to alleviate natural B deficiency problem in soils. This study warrants further studies with different soils and initial soil test B levels needed to conclude critical soil and tissue values for wider application across the region.

#### **5.8 Soybean**

Soybean (*Glycine max* L.) belongs to Leguminosae family. China, India and Indonesia are the leading soybean growing countries after USA. The occurrence of B deficiency based on responses at farmers fields have been reported for many countries like Australasia (China, India, Korea, Thailand), Europe (USSR) and in South America (Shorrocks, 1997). Generally, B deficiency is a common problem for this crop, especially when grown on alkaline calcareous soils of the world. The alkaline, silt, and sandy loam soils in Northeast Arkansas are also known to suffer from B deficiency (Anonymous, 2007a). Soybean is known to respond positively to B application on deficient sites of the world (Wu, 1986; Kirk & Loneragan, 1988). The increase in oil content and other quality parameters in soybean with combined application of B and sulfur in India have been noticed by Dinesh & Sudkep (2009), and Kumar & Sidhu (2009). In another study, Eguchi (2000) found a depressing effect of B deficiency on growth, yield, and protein and fat contents in the grains of soybean.

2004; Zia et al., 2006). Its deficiency decreases growth and photosynthesis, and increases starch and hexoses in leaves of citrus seedlings (Han and Chen, 2008). Soil and plant analysis showed that > 50% of the cultivated soils of Pakistan were unable to supply sufficient B to meet the needs of many crops (Khattak, 1995) including citrus. Keeping in view the export potential for this crop, in a survey, 1250 citrus orchard growers were interviewed and soil samples were selected from their respective orchards in district Sargodha of Punjab to investigate B application trend and its suspected deficiency. The results revealed that out of the 1250 citrus growers majority (58.8%) never used the B since the establishment of their orchards, 18.4% farmers were in a practice of using recommended doses of macro and micronutrients depending upon their current financial position, while 11.6% citrus growers seasonally applied B (Table 7). Only 140 (11.2%) farmers were in a habit to use recommended doses of B. This percentage of the growers (11.2%) is the main

In Pakistan (citrus belt, Sargodha), soil samples from varied depths (0-22.5 and 22.5-45 cm) were analyzed for B concentrations by Azomethine-H method (Ponnamperuma et al., 1981). Forty eight percent (600) orchards (samples) were found to be deficient in B. Since, in the past, farmers used to fertilize their citrus orchards without any soil testing, nutrient problem were common. Other reasons for B deficiency in the orchards might be due to the effect of sampling time. As the samples were taken during the monsoon season, this sampling time could be one of the reasons to affect B concentrations. These results are in agreement with

**growers Percentage Remarks** 

of livelihood

and advisory services, good income

Never used 735 58.8 Low income, low education Seasonal users 145 11.6 Lack of interest

Conditional users 230 18.4 Low income from the other sources

Regular users 140 11.2 Education level, accessible extension

As far as B concentrations in leaves and fruits of citrus are concerned, 38% of the samples were also found to be B deficient. At the appearance of B deficiency symptoms during drought year , B concentration was below 10 mg B kg-1 in fruit peel and leaves, but at abundant precipitation B was 20 mg B kg-1 in leaves and 14 mg B kg-1 in peel, and no B deficiency symptoms were observed. In an orchard where fruit had deficiency symptoms, 0.5 M HCl-extractable-B concentrations were 0.15 mg B kg-1 in surface soil (0-22.5 cm) and 0.10 mg B kg-1 in the subsoil (22.5-45 cm). The reasons for this deficiency could be, low B status in soils of the orchards, less than recommended use of B containing fertilizers and moisture stress during the drought periods. This aspect of B deficiency induced by drought might be due to the restricted mineralization of organically bound soil B (Evans & Sparks, 1983; Flannery, 1985). Research has also shown B deficiency to be responsible for diseases and/or application of B fertilizers to correct those diseases in vegetables such as brown heart of turnips, heart rot of beets, browning of cauliflower (Hill, 1936; Greenhill, 1938), and

Table 7. Boron use matrix in Bhalwal, Sillanwali and Sahiwal tehsil of district Sargodhaz

contributor to the foreign exchange while exporting citrus.

the findings of Zia et al. (2006).

**Growers category Number of citrus** 

zSource: Ahmad et al., unpublished data.

Boron application was also found to ameliorate Al toxicity by increasing growth characters (Yang & Gu, 2004). Furthermore, the genotypic variations in responses to B and other micronutrients (Zn, Mn) deficiencies have been observed by Graham & Heavner (1993) at the cellular level. In other field experiments the susceptibility of soybean cultivars to B deficiency was examined on "Typic Tropaqualf soils" in Northern Thailand, where B deficiency depressed seed yield by 60% in different cultivars. Sometimes, B deficiency also induced a localized depression on the internal surface of cotyledons in soybean seeds resembling to the symptom of 'hollow heart' in peanut. However, addition of B either decreased or eliminated such symptoms. In a comparative study of 19 soybean cultivars, the incidence of hollow heart symptoms in seeds at control (zero-B) appeared to be 75% but by the addition of only 1% B it reduced from none to 36%. The results suggested that susceptibility to B deficiency is sufficiently important and variable among soybean genotypes to warrant its inclusion as a selection criterion when breeding cultivars for areas with low soil B (Rerkasem et al., 1993).

#### **5.9 Potato**

Potato (*Solanum tuberosum* L.) belongs to Solanaceae family. Potatoes were probably brought to the Indian sub-continent hundred years ago by the Portuguese, and its cultivation expanded under British colonial rule in the 19th century (Geddes et al., 1989). The occurrence of B deficiency in potato based on responses at farmers fields have been reported in Australasia (Australia, China, India, Pakistan), Europe (Belgium, Czechoslovakia, Finland, Germany, Hungary, Sweden, USSR), and USA (Shorrocks, 1997). Despite favorable diversity of soils, climate and agricultural practices for potato cultivation, the average yield of potato has been reported to be 20.3 Mg ha-1 in Pakistan (Anonymous, 2008). Imbalanced use of fertilizers is one of the main reasons for this low yield (Nazli, 2010). Thus, the balanced use of micro and macronutrients (B, Zn, N, P and K) can considerably increase the yield. Lora (1978) obtained B responses in potato crop on Andosols soils in Colombia. Boron has also been identified to play a key role in forming abscission layers such as scar tissues at the stem end of potato at maturity that seals the tuber and thereby preventing it from diseases and bacterial infection on its storage. In a study, the growth of potatoes in sand cultures with zero-B resulted in poor vigour, yield and quality of tuber. Application of even 1 µg mL-1 B resulted in normal growth (Hill, 1936). Potatoes have shown positive response to B application if drilled in the row with fertilizer (Midgley & Dunklee, 1947). Higher rates of B were suggested if it was broadcasted and worked into the soil. In Pakistan, application of B fertilizer on three fields exhibiting B deficiency in potato has shown to be very effective for yield increases, ranging from 15% to 30%, with an average increase of 21% over zero-B control (Anonymous, 1998). Potato yield increases worth of Rs. 9 to Rs. 40 have been documented after spending one rupee on B fertilizer.

#### **5.10 Citrus fruits**

Citrus production is one of the world's largest agricultural industries. It is sown in more than 125 countries in the belt within 35o latitude north and south of equator (Duncan and Cohn, 1990). In addition to other factors, micronutrient deficiency (including B) is also considered among constraints that are currently hampering citrus yield (Johnson, 2006). In Pakistan, B deficiencies have been exhibited in citrus and other deciduous fruits (Tariq et al.,

Boron application was also found to ameliorate Al toxicity by increasing growth characters (Yang & Gu, 2004). Furthermore, the genotypic variations in responses to B and other micronutrients (Zn, Mn) deficiencies have been observed by Graham & Heavner (1993) at the cellular level. In other field experiments the susceptibility of soybean cultivars to B deficiency was examined on "Typic Tropaqualf soils" in Northern Thailand, where B deficiency depressed seed yield by 60% in different cultivars. Sometimes, B deficiency also induced a localized depression on the internal surface of cotyledons in soybean seeds resembling to the symptom of 'hollow heart' in peanut. However, addition of B either decreased or eliminated such symptoms. In a comparative study of 19 soybean cultivars, the incidence of hollow heart symptoms in seeds at control (zero-B) appeared to be 75% but by the addition of only 1% B it reduced from none to 36%. The results suggested that susceptibility to B deficiency is sufficiently important and variable among soybean genotypes to warrant its inclusion as a selection criterion when breeding cultivars for areas

Potato (*Solanum tuberosum* L.) belongs to Solanaceae family. Potatoes were probably brought to the Indian sub-continent hundred years ago by the Portuguese, and its cultivation expanded under British colonial rule in the 19th century (Geddes et al., 1989). The occurrence of B deficiency in potato based on responses at farmers fields have been reported in Australasia (Australia, China, India, Pakistan), Europe (Belgium, Czechoslovakia, Finland, Germany, Hungary, Sweden, USSR), and USA (Shorrocks, 1997). Despite favorable diversity of soils, climate and agricultural practices for potato cultivation, the average yield of potato has been reported to be 20.3 Mg ha-1 in Pakistan (Anonymous, 2008). Imbalanced use of fertilizers is one of the main reasons for this low yield (Nazli, 2010). Thus, the balanced use of micro and macronutrients (B, Zn, N, P and K) can considerably increase the yield. Lora (1978) obtained B responses in potato crop on Andosols soils in Colombia. Boron has also been identified to play a key role in forming abscission layers such as scar tissues at the stem end of potato at maturity that seals the tuber and thereby preventing it from diseases and bacterial infection on its storage. In a study, the growth of potatoes in sand cultures with zero-B resulted in poor vigour, yield and quality of tuber. Application of even 1 µg mL-1 B resulted in normal growth (Hill, 1936). Potatoes have shown positive response to B application if drilled in the row with fertilizer (Midgley & Dunklee, 1947). Higher rates of B were suggested if it was broadcasted and worked into the soil. In Pakistan, application of B fertilizer on three fields exhibiting B deficiency in potato has shown to be very effective for yield increases, ranging from 15% to 30%, with an average increase of 21% over zero-B control (Anonymous, 1998). Potato yield increases worth of Rs. 9 to Rs. 40 have been

Citrus production is one of the world's largest agricultural industries. It is sown in more than 125 countries in the belt within 35o latitude north and south of equator (Duncan and Cohn, 1990). In addition to other factors, micronutrient deficiency (including B) is also considered among constraints that are currently hampering citrus yield (Johnson, 2006). In Pakistan, B deficiencies have been exhibited in citrus and other deciduous fruits (Tariq et al.,

with low soil B (Rerkasem et al., 1993).

documented after spending one rupee on B fertilizer.

**5.9 Potato** 

**5.10 Citrus fruits** 

2004; Zia et al., 2006). Its deficiency decreases growth and photosynthesis, and increases starch and hexoses in leaves of citrus seedlings (Han and Chen, 2008). Soil and plant analysis showed that > 50% of the cultivated soils of Pakistan were unable to supply sufficient B to meet the needs of many crops (Khattak, 1995) including citrus. Keeping in view the export potential for this crop, in a survey, 1250 citrus orchard growers were interviewed and soil samples were selected from their respective orchards in district Sargodha of Punjab to investigate B application trend and its suspected deficiency. The results revealed that out of the 1250 citrus growers majority (58.8%) never used the B since the establishment of their orchards, 18.4% farmers were in a practice of using recommended doses of macro and micronutrients depending upon their current financial position, while 11.6% citrus growers seasonally applied B (Table 7). Only 140 (11.2%) farmers were in a habit to use recommended doses of B. This percentage of the growers (11.2%) is the main contributor to the foreign exchange while exporting citrus.

In Pakistan (citrus belt, Sargodha), soil samples from varied depths (0-22.5 and 22.5-45 cm) were analyzed for B concentrations by Azomethine-H method (Ponnamperuma et al., 1981). Forty eight percent (600) orchards (samples) were found to be deficient in B. Since, in the past, farmers used to fertilize their citrus orchards without any soil testing, nutrient problem were common. Other reasons for B deficiency in the orchards might be due to the effect of sampling time. As the samples were taken during the monsoon season, this sampling time could be one of the reasons to affect B concentrations. These results are in agreement with the findings of Zia et al. (2006).


zSource: Ahmad et al., unpublished data.

Table 7. Boron use matrix in Bhalwal, Sillanwali and Sahiwal tehsil of district Sargodhaz

As far as B concentrations in leaves and fruits of citrus are concerned, 38% of the samples were also found to be B deficient. At the appearance of B deficiency symptoms during drought year , B concentration was below 10 mg B kg-1 in fruit peel and leaves, but at abundant precipitation B was 20 mg B kg-1 in leaves and 14 mg B kg-1 in peel, and no B deficiency symptoms were observed. In an orchard where fruit had deficiency symptoms, 0.5 M HCl-extractable-B concentrations were 0.15 mg B kg-1 in surface soil (0-22.5 cm) and 0.10 mg B kg-1 in the subsoil (22.5-45 cm). The reasons for this deficiency could be, low B status in soils of the orchards, less than recommended use of B containing fertilizers and moisture stress during the drought periods. This aspect of B deficiency induced by drought might be due to the restricted mineralization of organically bound soil B (Evans & Sparks, 1983; Flannery, 1985). Research has also shown B deficiency to be responsible for diseases and/or application of B fertilizers to correct those diseases in vegetables such as brown heart of turnips, heart rot of beets, browning of cauliflower (Hill, 1936; Greenhill, 1938), and

by hot water, and hot 0.01 *M* CaCl2. Tsadilas et al. (1994) in a study using diverse soils concluded that HWS-B was a valuable measure of available soil B and it correlated strongly with 0.05 *M* mannitol in 0.1 *M* CaCl2 extractable, 0.05 *M* HCl-soluble B. Another extractant, ammonium bicarbonate-diethylenetriaminepentaacetic acid (AB-DTPA) has been suggested for effective B determination in alkaline soils (Gestring & Soltanpour, 1984, 1987, Gupta, 2006). Studies involving 31 US soils (Kaplan et al., 1990) and 100 Dutch soils (Novozamsky et al., 1990) have also confirmed that B values of cold extraction using 0.01 *M* CaCl2 were highly associated with those of hot extraction (hot 0.01 *M* CaCl2). However, for hot water B extraction method, several researchers have marked some problems of significance such as problematic comparability of the basic soil parameters determined routinely, precision, time

Using irrigated rice soils (n=53, pH 3.5-8.0) Ponnamperuma et al. (1981) recommended 0.05 *M* HCl as equally good extractant as HWS-B method (r=0.96) of Berger and Truog (1939). Cartwright et al. (1983) in a study concluded that extraction of wide range of soils (pH 5.4- 10.1, CaCO3 0-85%) with 0.01 *M* CaC1, + 0.05 M mannitol was found to be a more convenient soil test for plant-available B than the standard HWS-B method, and to be as good in predicting the response in B uptake by plants. With a cold 0.01 M CaCl2 extraction (n=100, pH 3.9-6.5) equally valuable soil B values can be obtained as with the more difficult to standardize hot water extraction procedure (Novozamsky et al., 1990). Vaughan and Howe (1994) suggested sorbitol (prepared in a buffered solution of I *N* ammonium acetate and 0.1 *M* triethanolamine) as an alternate for HWS-B test in determining available soil B. The amounts of B recovered by HWS, 0.05 *M* mannitol in 0.01 *M* CaCl2 extractable B, 0.05 *M* HCl soluble B methods were strongly correlated with each other, the highest correlation obtained being between HWS-B and HC1-B. Plant B was highly correlated to the B recovered (n=50, pH 6.1-8.2, CaCO3 0-9.2%) by all the three extractants (Tsadilas et al., 1997). Amounts of extractable B with AB-DTPA and with hot water were similar (r=0.84) for ten soils (pH 5.8- 7.8, CaCO3 0-61 g/kg) studied by Matsi et al. (2000). There were highly significant positive correlations between the amounts of B extracted through hot water-soluble, 1:1 soil: distilled water and 1:2 soil:distilled water, ammonium acetate, calcium chloride - mannitol, and DTPA - sorbitol extractants (Goldberg et al., 2002). Latter, Chaudhary & Shukla (2004) also accentuated the advantageous features of sorbitol + NH4OAc + TEA and mannitol + NH4OAc + TEA extractants. Further, the simplicity of these extractants has also been compared to hot water and hot 0.01 *M* CaCl2 methods..These extractants have the tendency to demarcate the available B status of arid soils on a routine basis where a large number of samples are to analyze. DTPA-Sorbitol has been recommended as a replacement to these

cumbersome hot water extraction procedures (Shiffler et al., 2005).

The spectrophotometric technique using a colorimetric reaction with azomethine-H has been the most extensively tested B determination method for soil and plant samples (Ogner, 1980; Parker & Gardner, 1981; Porter et al., 1981; Lohse, 1982; Garcia et al., 1985; Lee et al., 1987; Chen et al., 1989; Kaplan et al., 1990; Banuelos et al., 1992; Campana et al., 1992; Nogueira et al., 1993). In this type of determination, hot water and 0.5 M HCl have commonly been used as extractants, both for acidic and alkaline soils. The use of these extractants is attributed with certain merits and demerits. For example, the HWE procedure

**8. Advances in boron analysis** 

consumption etc. (Shiffler et al., 2005).

fruits such as corky-core, blotchy cork and drought spot of apple (Hill, 1936; Greenhill, 1938; McLarty, 1940; Fritzsche, 1955), die-back of apricot (Fitzpatrick & Woodbridge, 1941) and deformed mandarin fruits of citrus (Chiu & Chang, 1985, 1986).

#### **6. Boron deficiency and crop diseases**

As discussed earlier, B deficiency is widespread in Pakistan (Rashid et al., 2009), India (Gupta, 1983, 1984; Sillanpaa & Vlek, 1985; Sakal & Singh, 1995), China (Liu Zheng et al., 1980, 1982a, 1982b, 1983, 1989) and Western Australia (Wong, 2003) and many other countries (Sillanpaa, 1982). Boron deficiency has been reported to be associated with internal tissue breakdown in root crops, groundnut/peanut, and some bean cultivars. Warncke (2005) observed the amelioration of internal black spot in cranberry bean seed with B application. Boron nutrition of cereal crops in connection to chilling tolerance has also been demonstrated by Huang & Ye (2005). Problems like stunning of cotton growth, wilting of the plants and reddening of cotton leaves have been observed in the major cotton growing areas of Sindh province in Pakistan. At present, integrated plant nutrient management including B with the best management practices seems to be the only solution of this lethal problem (Abid Niaz - personal communication). However, no clear relationships have been established between above mentioned symptoms and B nutrition. Similar relation of B application in curing the problem of rust in wheat has also been reported by Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Pakistan. Application of B also minimized the adverse effects of drought stress in crop plants during dry spell. Severe attack of rust was observed on B-deficient wheat plants exposed to drought stress, whereas no incidence of disease was fond in B treated plants (Anonymous, 2007a).

#### **7. Boron extractants and their comparative efficiency**

Several extractants used for soil B extraction have been employed over time for example, hot water for plant-available B (Berger & Truog, 1939; Parker & Gardner, 1981; Mahler et al., 1984; Rahmatullah et al., 1999), 0.05 *M* HCl for plant-available B (Ponnamperuma et al., 1981), 0.018 *M* CaCl2 for non-specifically adsorbed/readily soluble B on soil surfaces (Iyenger et al., 1981; Aitken & McCallum, 1988; Spouncer et al., 1992; Hou et al., 1996; Rahmatullah et al., 1999), 1 *M* NH4OAc for multi-element extraction (Gupta & Stewart, 1975; Chaudhary & Shukla, 2004), 0.25 *M* sorbitol-DTPA for bioavailable B (Goldberg, 1997; Miller et al., 2000; Gloldberg et al.,, 2002; Shiffler et al., 2005), 0.05 *M* mannitol prepared in 0.01 *M* CaCl2 for B in soil solution and its nonspecifically adsorbed forms to assess regenerative power of soil for B (Cartwright et al., 1983; Aitken et al., 1987; Jin et al., 1988; Rahmatullah et al., 1999;Vaughan & Howe, 1994), and 0.005 *M* AB-DTPA for multi-element extraction (Gestring & Soltanpour, 1984, 1987; Matsi et al., 2000).

There are a number of methods for extracting available B from soils. The colorimetric and other methods of determining B in the soil extract remain the same for testing on acid and alkaline soils (Bingham, 1982; Gupta, 2006). The most common extractant is hot water (Berger & Truog, 1939) because soil solution B is most important with regard to plant uptake. Li and Gupta (1991) compared hot water, 0.05 *M* HCl, and hot 0.01 *M* CaCl2 solutions as B extractants in relation to B accumulation by soybean, red clover, alfalfa, and rutabaga. The authors concluded that 0.05 *M* HCl was the best extractant (r=0.82) followed

fruits such as corky-core, blotchy cork and drought spot of apple (Hill, 1936; Greenhill, 1938; McLarty, 1940; Fritzsche, 1955), die-back of apricot (Fitzpatrick & Woodbridge, 1941) and

As discussed earlier, B deficiency is widespread in Pakistan (Rashid et al., 2009), India (Gupta, 1983, 1984; Sillanpaa & Vlek, 1985; Sakal & Singh, 1995), China (Liu Zheng et al., 1980, 1982a, 1982b, 1983, 1989) and Western Australia (Wong, 2003) and many other countries (Sillanpaa, 1982). Boron deficiency has been reported to be associated with internal tissue breakdown in root crops, groundnut/peanut, and some bean cultivars. Warncke (2005) observed the amelioration of internal black spot in cranberry bean seed with B application. Boron nutrition of cereal crops in connection to chilling tolerance has also been demonstrated by Huang & Ye (2005). Problems like stunning of cotton growth, wilting of the plants and reddening of cotton leaves have been observed in the major cotton growing areas of Sindh province in Pakistan. At present, integrated plant nutrient management including B with the best management practices seems to be the only solution of this lethal problem (Abid Niaz - personal communication). However, no clear relationships have been established between above mentioned symptoms and B nutrition. Similar relation of B application in curing the problem of rust in wheat has also been reported by Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, Pakistan. Application of B also minimized the adverse effects of drought stress in crop plants during dry spell. Severe attack of rust was observed on B-deficient wheat plants exposed to drought stress, whereas

deformed mandarin fruits of citrus (Chiu & Chang, 1985, 1986).

no incidence of disease was fond in B treated plants (Anonymous, 2007a).

Several extractants used for soil B extraction have been employed over time for example, hot water for plant-available B (Berger & Truog, 1939; Parker & Gardner, 1981; Mahler et al., 1984; Rahmatullah et al., 1999), 0.05 *M* HCl for plant-available B (Ponnamperuma et al., 1981), 0.018 *M* CaCl2 for non-specifically adsorbed/readily soluble B on soil surfaces (Iyenger et al., 1981; Aitken & McCallum, 1988; Spouncer et al., 1992; Hou et al., 1996; Rahmatullah et al., 1999), 1 *M* NH4OAc for multi-element extraction (Gupta & Stewart, 1975; Chaudhary & Shukla, 2004), 0.25 *M* sorbitol-DTPA for bioavailable B (Goldberg, 1997; Miller et al., 2000; Gloldberg et al.,, 2002; Shiffler et al., 2005), 0.05 *M* mannitol prepared in 0.01 *M* CaCl2 for B in soil solution and its nonspecifically adsorbed forms to assess regenerative power of soil for B (Cartwright et al., 1983; Aitken et al., 1987; Jin et al., 1988; Rahmatullah et al., 1999;Vaughan & Howe, 1994), and 0.005 *M* AB-DTPA for multi-element extraction

There are a number of methods for extracting available B from soils. The colorimetric and other methods of determining B in the soil extract remain the same for testing on acid and alkaline soils (Bingham, 1982; Gupta, 2006). The most common extractant is hot water (Berger & Truog, 1939) because soil solution B is most important with regard to plant uptake. Li and Gupta (1991) compared hot water, 0.05 *M* HCl, and hot 0.01 *M* CaCl2 solutions as B extractants in relation to B accumulation by soybean, red clover, alfalfa, and rutabaga. The authors concluded that 0.05 *M* HCl was the best extractant (r=0.82) followed

**7. Boron extractants and their comparative efficiency** 

(Gestring & Soltanpour, 1984, 1987; Matsi et al., 2000).

**6. Boron deficiency and crop diseases** 

by hot water, and hot 0.01 *M* CaCl2. Tsadilas et al. (1994) in a study using diverse soils concluded that HWS-B was a valuable measure of available soil B and it correlated strongly with 0.05 *M* mannitol in 0.1 *M* CaCl2 extractable, 0.05 *M* HCl-soluble B. Another extractant, ammonium bicarbonate-diethylenetriaminepentaacetic acid (AB-DTPA) has been suggested for effective B determination in alkaline soils (Gestring & Soltanpour, 1984, 1987, Gupta, 2006). Studies involving 31 US soils (Kaplan et al., 1990) and 100 Dutch soils (Novozamsky et al., 1990) have also confirmed that B values of cold extraction using 0.01 *M* CaCl2 were highly associated with those of hot extraction (hot 0.01 *M* CaCl2). However, for hot water B extraction method, several researchers have marked some problems of significance such as problematic comparability of the basic soil parameters determined routinely, precision, time consumption etc. (Shiffler et al., 2005).

Using irrigated rice soils (n=53, pH 3.5-8.0) Ponnamperuma et al. (1981) recommended 0.05 *M* HCl as equally good extractant as HWS-B method (r=0.96) of Berger and Truog (1939). Cartwright et al. (1983) in a study concluded that extraction of wide range of soils (pH 5.4- 10.1, CaCO3 0-85%) with 0.01 *M* CaC1, + 0.05 M mannitol was found to be a more convenient soil test for plant-available B than the standard HWS-B method, and to be as good in predicting the response in B uptake by plants. With a cold 0.01 M CaCl2 extraction (n=100, pH 3.9-6.5) equally valuable soil B values can be obtained as with the more difficult to standardize hot water extraction procedure (Novozamsky et al., 1990). Vaughan and Howe (1994) suggested sorbitol (prepared in a buffered solution of I *N* ammonium acetate and 0.1 *M* triethanolamine) as an alternate for HWS-B test in determining available soil B. The amounts of B recovered by HWS, 0.05 *M* mannitol in 0.01 *M* CaCl2 extractable B, 0.05 *M* HCl soluble B methods were strongly correlated with each other, the highest correlation obtained being between HWS-B and HC1-B. Plant B was highly correlated to the B recovered (n=50, pH 6.1-8.2, CaCO3 0-9.2%) by all the three extractants (Tsadilas et al., 1997). Amounts of extractable B with AB-DTPA and with hot water were similar (r=0.84) for ten soils (pH 5.8- 7.8, CaCO3 0-61 g/kg) studied by Matsi et al. (2000). There were highly significant positive correlations between the amounts of B extracted through hot water-soluble, 1:1 soil: distilled water and 1:2 soil:distilled water, ammonium acetate, calcium chloride - mannitol, and DTPA - sorbitol extractants (Goldberg et al., 2002). Latter, Chaudhary & Shukla (2004) also accentuated the advantageous features of sorbitol + NH4OAc + TEA and mannitol + NH4OAc + TEA extractants. Further, the simplicity of these extractants has also been compared to hot water and hot 0.01 *M* CaCl2 methods..These extractants have the tendency to demarcate the available B status of arid soils on a routine basis where a large number of samples are to analyze. DTPA-Sorbitol has been recommended as a replacement to these cumbersome hot water extraction procedures (Shiffler et al., 2005).

#### **8. Advances in boron analysis**

The spectrophotometric technique using a colorimetric reaction with azomethine-H has been the most extensively tested B determination method for soil and plant samples (Ogner, 1980; Parker & Gardner, 1981; Porter et al., 1981; Lohse, 1982; Garcia et al., 1985; Lee et al., 1987; Chen et al., 1989; Kaplan et al., 1990; Banuelos et al., 1992; Campana et al., 1992; Nogueira et al., 1993). In this type of determination, hot water and 0.5 M HCl have commonly been used as extractants, both for acidic and alkaline soils. The use of these extractants is attributed with certain merits and demerits. For example, the HWE procedure

soil tests have been developed to predict B deficient soils and have not generally been evaluated for their ability to predict soil conditions that produce B toxicity effects in plants. The work reported by Goldberg et al. (2002, 2003) for using shallow groundwater to apply for crops could predict improvement in irrigation efficiency. Such sort of attributes must be incorporated in B investigation techniques/methodologies to take into loop B content of both field grown and crops grown under controlled conditions of potential B toxicity. Boron determination by ICP–MS suffers no spectroscopic interferences, and is considered the most practical and convenient technique for B isotope determination. Among the present technologies, ICP–MS has emerged as the method of choice for determining B concentration and a convenient method for B isotope determination (Sah & Brown, 1997). With the increased use of inductively coupled plasma atomic emission spectrophotometry (ICP) (especially instruments with simultaneous detection capability) soil test laboratories would welcome the need for different extractions for many element analyses. Boron is readily measured with ICP instrumentation. However, the predictions of B deficiencies by soil testing needs to be based on local data and not from broad generalizations from other areas.

Boron (B) is a unique micro mineral nutrient required for normal plant growth and optimum yield of crops. Its deficiency is widespread in alkaline/calcareous, coarse-textured and low organic matter soils in many countries of the world. Annual [fiber (cotton), cereal (rice, maize/corn, wheat), legume/pulse (soybean), oilseed (groundnut/peanut, oilseed rape/canola)], vegetable (potato), and perennial [citrus fruit orchards, alfalfa] crops grown on such soils usually suffer from B deficiency. This paper discusses factors affecting B availability in soils, including parent material, soil pH, texture, clay minerals and organic matter, irrigation sources, nutrient interactions, and plant species. The paper also documents the diagnosis and correction of B deficiency in several important crops in a wide range of soils. Crop yield increases up to 14% each in cotton and wheat, 14-30% in rice, 20% in maize, 58% in soybean, 10% in groundnut, 45% in oilseed rape, 30% in potato, 37% in alfalfa seed and 159% in alfalfa forage are reported with application of B by using appropriate rates, methods (soil or foliar) and sources (such as borax) on B-deficient soils. Application of B fertilizers up to 2.5 kg B ha-1 is recommended to prevent/correct B deficiency in major crops depending on the placement method. The paper also reviews comparative efficiency of various boron extracts under different soil conditions in addition to advances in boron analysis. Among the present technologies, ICP–MS has emerged as the method of choice for determining B concentration and a convenient method for B isotope

Prevention and/or correction of B deficiency in crops on B-deficient soils can have a dramatic effect on yield and produce quality of many crops including fibers, cereals, pulses, oilseeds, vegetables, citrus fruits and alfalfa. Source, rate, formulation, time and method of B application and proper balancing of B with other nutrients in soil all affect crop yield on Bdeficient soils. Both soil and foliar application methods of B are effective in improving crop yield, produce quality, concentration and uptake of B, and economic returns. Soil applied B leaves residual effect for years on succeeding crops grown on B-deficient soils in the same fields. The actual fraction of B fertilizer removed by the crops is only 1-2% of the total applied fertilizer through soil. However, it is very important that research for improving

**9. Summary** 

determination.

embodies several potential sources of error like difficulty to standardize (Novozamsky et al., 1990), time consuming and tedious for routine and reproducible usage (Deabreu et al., 1994). Further, the amount of B extracted is affected by the reflux time (McGeehan et al. 1989), extraction time and temperature (Spouncer et al., 1992). The coloured hot water extracts in some soils may affect B determination. The HWE method has limitations for some soils and B extracted by this method did not correlate with crop responses under some management conditions (Gestring & Soltanpour, 1987; Offiah & Axley, 1988; Mustafa et al., 1993). The use of 0.05 M HCL has eliminated the problems of extraction with hot water. Overall, the colorimetric methods, in general, suffer several interferences, such as sample pH in the range of 6.4 to 7.0 (Carrero et al., 1993), sample colour (McGeehan et al., 1989; Evans & Krahenbuhl, 1994a), nitrate complexes in the wet HNO3 acid digests of plants (Gestring & Soltanpour, 1981a) and the presence Fe, Al, Cu, Zn and Mo (Arruda & Zagatto, 1987). These interferences and lack of sensitivity limit the application of these methods for the samples with low B concentrations and complex matrices.

The reliability of B measurements has improved in the last decade with better instrumentation and analytical methodology (Sah & Brown, 1997). After spectrophotometry, B has been determined utilizing potentiometer, chromatography, flame atomic emission and absorption spectrometry, inductively coupled plasma (ICP) optical emission (OES) and mass spectrometry (MS), and neutron activation analysis using neutron radiography and promptactivation analysis. The extraction with 0.05 M HCl is concerned; it has also worked well for predicting B availability to crop plants in acid soils (Ponnemperuma et al., 1981; Renan & Gupta, 1991). However, Fe extracted with the acid extractant often interferes in B determination by spectrophotometric and ICP–OES methods (Evans & Krahenbuhl, 1994a; Pougnet & Orren, 1986a, 1986b).

There are reports on the use of plasma-source OES for assaying B (Pritchard & Lee, 1984; Nilsson & Jennische, 1986; Lee et al., 1987; Jeffrey & McCallum, 1988; Novozamsky et al., 1990; Goto et al., 1992; Spouncer et al., 1992; Ferrando et al., 1993; Evans & Krahenbuhl, 1994a). Reported detection limits for B are 10 to 15 mg B L-1 in soil solutions and plant digests (Spiers et al., 1990). Boron determination by ICP–OES is also affected by other interfering species, for example, Si (Owens et al., 1982; Din, 1984), Ni, Cr, Al, V, Mn, Ti, Mo and high concentrations of Na (Pougnet & Orren, 1986b; Kavipurapu et al., 1993). Nevertheless, recently, Mehlich-3 has been promoted as a "universal" extractant in a wide variety of soils. But ICP analysis of B following extraction with Mehlich- 3 chemicals has proven difficult because of B contamination within the ICP unit. Secondly, the effects of distilled water, nitric acid and sorbitol solutions used between samples for correcting B contamination has also been diagnosed by Allen et al. (2005). Sorbitol solution is found as the most effective solution to rectify the contamination problem. The contamination problem unique to Mehlich-3 has the tendency to limit the development of Mehlich-3 as the widely accepted extractant. However, no such problem was observed with ICP analysis of B with hot water, pressurized hot water or DTPA-Sorbitol extractions. (Allen et al., 2005). Consequently, pressurized hot water or DTPA-Sorbitol extractions have been proposed as replacement.

Overall, mostly all the above mentioned soil B extractants, provide good correlaton with plant B contents under controlled conditions. However, the efficacy of these extractants should also be tested under field conditions (Goldberg & Chunming, 2007). Historically, B

soil tests have been developed to predict B deficient soils and have not generally been evaluated for their ability to predict soil conditions that produce B toxicity effects in plants. The work reported by Goldberg et al. (2002, 2003) for using shallow groundwater to apply for crops could predict improvement in irrigation efficiency. Such sort of attributes must be incorporated in B investigation techniques/methodologies to take into loop B content of both field grown and crops grown under controlled conditions of potential B toxicity. Boron determination by ICP–MS suffers no spectroscopic interferences, and is considered the most practical and convenient technique for B isotope determination. Among the present technologies, ICP–MS has emerged as the method of choice for determining B concentration and a convenient method for B isotope determination (Sah & Brown, 1997). With the increased use of inductively coupled plasma atomic emission spectrophotometry (ICP) (especially instruments with simultaneous detection capability) soil test laboratories would welcome the need for different extractions for many element analyses. Boron is readily measured with ICP instrumentation. However, the predictions of B deficiencies by soil testing needs to be based on local data and not from broad generalizations from other areas.

#### **9. Summary**

96 Crop Plant

embodies several potential sources of error like difficulty to standardize (Novozamsky et al., 1990), time consuming and tedious for routine and reproducible usage (Deabreu et al., 1994). Further, the amount of B extracted is affected by the reflux time (McGeehan et al. 1989), extraction time and temperature (Spouncer et al., 1992). The coloured hot water extracts in some soils may affect B determination. The HWE method has limitations for some soils and B extracted by this method did not correlate with crop responses under some management conditions (Gestring & Soltanpour, 1987; Offiah & Axley, 1988; Mustafa et al., 1993). The use of 0.05 M HCL has eliminated the problems of extraction with hot water. Overall, the colorimetric methods, in general, suffer several interferences, such as sample pH in the range of 6.4 to 7.0 (Carrero et al., 1993), sample colour (McGeehan et al., 1989; Evans & Krahenbuhl, 1994a), nitrate complexes in the wet HNO3 acid digests of plants (Gestring & Soltanpour, 1981a) and the presence Fe, Al, Cu, Zn and Mo (Arruda & Zagatto, 1987). These interferences and lack of sensitivity limit the application of these methods for the samples

The reliability of B measurements has improved in the last decade with better instrumentation and analytical methodology (Sah & Brown, 1997). After spectrophotometry, B has been determined utilizing potentiometer, chromatography, flame atomic emission and absorption spectrometry, inductively coupled plasma (ICP) optical emission (OES) and mass spectrometry (MS), and neutron activation analysis using neutron radiography and promptactivation analysis. The extraction with 0.05 M HCl is concerned; it has also worked well for predicting B availability to crop plants in acid soils (Ponnemperuma et al., 1981; Renan & Gupta, 1991). However, Fe extracted with the acid extractant often interferes in B determination by spectrophotometric and ICP–OES methods (Evans & Krahenbuhl, 1994a;

There are reports on the use of plasma-source OES for assaying B (Pritchard & Lee, 1984; Nilsson & Jennische, 1986; Lee et al., 1987; Jeffrey & McCallum, 1988; Novozamsky et al., 1990; Goto et al., 1992; Spouncer et al., 1992; Ferrando et al., 1993; Evans & Krahenbuhl, 1994a). Reported detection limits for B are 10 to 15 mg B L-1 in soil solutions and plant digests (Spiers et al., 1990). Boron determination by ICP–OES is also affected by other interfering species, for example, Si (Owens et al., 1982; Din, 1984), Ni, Cr, Al, V, Mn, Ti, Mo and high concentrations of Na (Pougnet & Orren, 1986b; Kavipurapu et al., 1993). Nevertheless, recently, Mehlich-3 has been promoted as a "universal" extractant in a wide variety of soils. But ICP analysis of B following extraction with Mehlich- 3 chemicals has proven difficult because of B contamination within the ICP unit. Secondly, the effects of distilled water, nitric acid and sorbitol solutions used between samples for correcting B contamination has also been diagnosed by Allen et al. (2005). Sorbitol solution is found as the most effective solution to rectify the contamination problem. The contamination problem unique to Mehlich-3 has the tendency to limit the development of Mehlich-3 as the widely accepted extractant. However, no such problem was observed with ICP analysis of B with hot water, pressurized hot water or DTPA-Sorbitol extractions. (Allen et al., 2005). Consequently, pressurized hot water or DTPA-Sorbitol extractions have been proposed as

Overall, mostly all the above mentioned soil B extractants, provide good correlaton with plant B contents under controlled conditions. However, the efficacy of these extractants should also be tested under field conditions (Goldberg & Chunming, 2007). Historically, B

with low B concentrations and complex matrices.

Pougnet & Orren, 1986a, 1986b).

replacement.

Boron (B) is a unique micro mineral nutrient required for normal plant growth and optimum yield of crops. Its deficiency is widespread in alkaline/calcareous, coarse-textured and low organic matter soils in many countries of the world. Annual [fiber (cotton), cereal (rice, maize/corn, wheat), legume/pulse (soybean), oilseed (groundnut/peanut, oilseed rape/canola)], vegetable (potato), and perennial [citrus fruit orchards, alfalfa] crops grown on such soils usually suffer from B deficiency. This paper discusses factors affecting B availability in soils, including parent material, soil pH, texture, clay minerals and organic matter, irrigation sources, nutrient interactions, and plant species. The paper also documents the diagnosis and correction of B deficiency in several important crops in a wide range of soils. Crop yield increases up to 14% each in cotton and wheat, 14-30% in rice, 20% in maize, 58% in soybean, 10% in groundnut, 45% in oilseed rape, 30% in potato, 37% in alfalfa seed and 159% in alfalfa forage are reported with application of B by using appropriate rates, methods (soil or foliar) and sources (such as borax) on B-deficient soils. Application of B fertilizers up to 2.5 kg B ha-1 is recommended to prevent/correct B deficiency in major crops depending on the placement method. The paper also reviews comparative efficiency of various boron extracts under different soil conditions in addition to advances in boron analysis. Among the present technologies, ICP–MS has emerged as the method of choice for determining B concentration and a convenient method for B isotope determination.

Prevention and/or correction of B deficiency in crops on B-deficient soils can have a dramatic effect on yield and produce quality of many crops including fibers, cereals, pulses, oilseeds, vegetables, citrus fruits and alfalfa. Source, rate, formulation, time and method of B application and proper balancing of B with other nutrients in soil all affect crop yield on Bdeficient soils. Both soil and foliar application methods of B are effective in improving crop yield, produce quality, concentration and uptake of B, and economic returns. Soil applied B leaves residual effect for years on succeeding crops grown on B-deficient soils in the same fields. The actual fraction of B fertilizer removed by the crops is only 1-2% of the total applied fertilizer through soil. However, it is very important that research for improving

Ahmad, W.; Ahmad, N.; Ibrahim, M. & Niaz, A. (2004). Boron contents in ground and river

Ahmad, W.; Niaz, A.; Kanwal, S.; Rahmatullah, & Rasheed, M. K. (2009). Role of boron in plant growth: A review. *Journal of Agricultural Research.* Vol. 47, pp. 329-338. Ahmed, N.; Abid, M. & Ahmad, F. (2008). Boron toxicity in irrigated cotton (*Gossypium* 

Aitken, R. L.; Jeffrey, A. J. & Compton, B. L. (1987). Evaluation of selected extractants for

Aitken, R. L. & McCallum, L. E. (1988). Boron toxicity in soil solution. *Australian Journal of* 

Akram, M. S.; Ali, Q.; Athar, H. & Bhatti, A. S. (2006). Ion uptake and distribution in

Ali, S. J. & Monoranjan, R. (1989). Effect of NPK and micronutrient in controlling sterility in

Allen, S. K.; Jolley, V. D.; Webb, B. L.; Shiffler, A. K. & Haby, V. (2005). Challenges of

*Texas Agricultural Expt*. Stn., PO Box 200, Overton, TX 75684-0200, U.S.A. Anonymous. (1998). Micronutrients in Agriculture - Pakistan Perspective. NFDC Pub. No. 4/98. National Fertilizer Development Centre, Islamabad, Pakistan. Anonymous. (2007a). Soybean response to boron fertilization, 2007 [Online].

Anonymous. (2007b). National Institute of Agriculture and Biology (NIAB). Seventh five-

boron in some Queensland soils. *Australian Journal of Soil Research.* Vol. 25, pp. 263-

*Panicum antidotale* Retz. under salt stress. *Pakistan Journal of Botany.* Vol.38, pp. 1661-

Mehlich 3 Extraction for B and Comparison with Other Methods in Boron-Treated Soils. *Brigham Young University Department of Plant and Animal Sciences, University,* 

Available:http://ipni.net/far/farguide.nsf/\$webindex/article=5F51B22A852573F1

year report on research and other activities (6 April 2002 to 5 April 2007).

http://www.statpak.gov.pk/depts/fbs/publications/yearbook2008/yearbook200

http://www.statpak.gov.pk/depts/fbs/publications/yearbook2009/yearbook200

pumping for the spectrophotometric flow-injection determination of boron in

wet acid digestion on the determination of boron in plant tissue. *Communications in* 

Arora, C. L.; Singh, B. & Takkar, P. N. (1985). Secondary and micronutrient deficiency in

Arruda, M. A. Z. & Zagatto, E. A. G. (1987). A simple stopped-flow method with continuous

Ba˜nuelos, G.S.; Cardon, G.; Pflaum, T. & Akohoue, S. (1992). Comparison of dry ashing and

*hirsutum* L.). *Pakistan Journal of Botany.* Vol. 40, pp. 2443-2445.

waters used for irrigation in district Faisalabad. *In: Abstracts 10th International Congress of Soil Science. March 16-19, 2004,* Sindh Agricultural University, Tandojam,

**11. References** 

273.

1669.

Pakistan. 109 pp.

*Soil Research.* Vol. 26, pp. 605–610.

Faisalabad, Pakistan. 67 pp.

8.html [15 May 2010].

9.html [15 May 2010].

crops. *Progressive Farming.* XXI 8: 13.

wheat. *Fertilizer News.* Vol. 34, pp. 35-36.

0001A460FBF6FC76!opendocument [15 May 2010].

Anonymous. (2008). Pakistan statistical year book 2008. [Online] Available:

Anonymous. (2009). Pakistan statistical year book 2009. [Online] Available:

plants. *Analytica Chimica Acta.* Vol. 199, pp. 137–145.

*Soil Science and Plant Analysis.* Vol. 23, pp. 2383–2397.

crop yields must move beyond applications of B based on general recommendations, and that deriving methods to predict site-specific deficiencies (e.g., soil or plant tests) are essential since the potential for B toxicity is large and the difference between deficiency and toxicity is very narrow. This could be especially important if B is applied sequentially to fields over a series of years without knowing the residual effects.

#### **10. Conclusions and future research needs**

Prevention and/or correction of B deficiency in crops on B-deficient soils can have a dramatic effect on yield and produce quality of many crops including fibers, cereals, pulses, oilseeds, vegetables, citrus fruits and alfalfa. An increase in yield of 14% each in cotton and wheat, 14-30% in rice, 20% in maize/corn, 58% in soybean, 10% in groundnut/peanut, 45% in oilseed rape, 30% in potato, 37% in alfalfa seed and 159% in alfalfa forage was observed with B fertilization. Source, rate, formulation, time and method of B fertilizer application, and proper balancing of B with other nutrients in soil all affect crop yield on B-deficient soils. Both soil and foliar application methods of B are effective in improving crop yield, produce quality, concentration and uptake of B, and economic returns. Application of B to rice on B-deficient soils also enhanced milling recovery and head rice recovery, and improved kernels quality traits like stickiness and cooking quality. Zinc application has been found to neutralize toxic effect of B in some crop plants and produced increase in crop yield.

Soil applied B leaves residual effect for years on succeeding crops grown on B-deficient soils in the same fields. The actual fraction of B fertilizer removed by the crops is only 1-2% of the total applied fertilizer through soil. Research on recycling of crops from rotation system rather than mono-cropping culture can generate useful information for B management. Moreover, adaptive research is also a pre-requisite for B management under efficient irrigation systems, e.g., drip, sprinkler and others. Impact of B nutrient use on product quality is needed, especially for high B requirement crops. Moreover, B efficient genotypes for different crops need to be identified and developed for commercial use.

Effect of B fertilizer use in high input system should also be given priority as a futuristic option for the sustainability of crop production, soil quality and environment. Further, management decisions for use of B fertilizers should consider both immediate and longterm effects of B fertilizer on crop yield, produce quality and economic returns. Research is also required, in different agro-ecological zones, to determine the long-term effects of different sources of B on accumulation and distribution of B and its balanced application with other nutrients to investigate its relationship with disease and insect resistance in different crops. However, it is very important that research for improving crop yields must move beyond applications of B based on general recommendations, and that deriving methods to predict site-specific deficiencies (e.g., soil or plant tests) are essential since the potential for B toxicity is large and the difference between deficiency and toxicity is very narrow. This could be especially important if B is applied sequentially to fields over a series of years without knowing the residual effects. It could become a serious problem if B was applied for several years for a tolerant crop and then change to B sensitive crop. Soil test could potentially determine potential problems of excessive accumulations of B in the soil.

#### **11. References**

98 Crop Plant

crop yields must move beyond applications of B based on general recommendations, and that deriving methods to predict site-specific deficiencies (e.g., soil or plant tests) are essential since the potential for B toxicity is large and the difference between deficiency and toxicity is very narrow. This could be especially important if B is applied sequentially to

Prevention and/or correction of B deficiency in crops on B-deficient soils can have a dramatic effect on yield and produce quality of many crops including fibers, cereals, pulses, oilseeds, vegetables, citrus fruits and alfalfa. An increase in yield of 14% each in cotton and wheat, 14-30% in rice, 20% in maize/corn, 58% in soybean, 10% in groundnut/peanut, 45% in oilseed rape, 30% in potato, 37% in alfalfa seed and 159% in alfalfa forage was observed with B fertilization. Source, rate, formulation, time and method of B fertilizer application, and proper balancing of B with other nutrients in soil all affect crop yield on B-deficient soils. Both soil and foliar application methods of B are effective in improving crop yield, produce quality, concentration and uptake of B, and economic returns. Application of B to rice on B-deficient soils also enhanced milling recovery and head rice recovery, and improved kernels quality traits like stickiness and cooking quality. Zinc application has been found to neutralize toxic effect of B in some crop plants and produced increase in crop

Soil applied B leaves residual effect for years on succeeding crops grown on B-deficient soils in the same fields. The actual fraction of B fertilizer removed by the crops is only 1-2% of the total applied fertilizer through soil. Research on recycling of crops from rotation system rather than mono-cropping culture can generate useful information for B management. Moreover, adaptive research is also a pre-requisite for B management under efficient irrigation systems, e.g., drip, sprinkler and others. Impact of B nutrient use on product quality is needed, especially for high B requirement crops. Moreover, B efficient genotypes

Effect of B fertilizer use in high input system should also be given priority as a futuristic option for the sustainability of crop production, soil quality and environment. Further, management decisions for use of B fertilizers should consider both immediate and longterm effects of B fertilizer on crop yield, produce quality and economic returns. Research is also required, in different agro-ecological zones, to determine the long-term effects of different sources of B on accumulation and distribution of B and its balanced application with other nutrients to investigate its relationship with disease and insect resistance in different crops. However, it is very important that research for improving crop yields must move beyond applications of B based on general recommendations, and that deriving methods to predict site-specific deficiencies (e.g., soil or plant tests) are essential since the potential for B toxicity is large and the difference between deficiency and toxicity is very narrow. This could be especially important if B is applied sequentially to fields over a series of years without knowing the residual effects. It could become a serious problem if B was applied for several years for a tolerant crop and then change to B sensitive crop. Soil test could potentially determine potential problems of excessive

for different crops need to be identified and developed for commercial use.

fields over a series of years without knowing the residual effects.

**10. Conclusions and future research needs** 

yield.

accumulations of B in the soil.


Chang, S. S. (1993). Nutritional physiology of boron and the diagnosis and correction of

Chang, S. S.; Huang, W. T.; Lian, S. & Wu, W. L. (1992). Research on soil testing and leaf

Chaudhary, D. R. & L. M. Shukla. (2004). Evaluation of extractants for predicting availability

Chaudhry, E. H.; Timmer, V.; Javed, A. S. & Siddique, M. T. (2007). Wheat response to micronutrients in rain-fed areas of Punjab. *Soil & Environment.* Vol. 26, pp. 97-101. Chaudhry, F. M.; Latif, A.; Rashid, A. & Alam, S. M. (1976). Response of the rice varieties to

Chauhan, R. P. S. & Asthana, A. K. (1981). Tolerance of lentil, barley and oats to boron in

Chen, D.; Lazaro, F.; Decastro, L. & Valcarcel, M. (1989). Direct spectrophotometric

Chiu, T. F. & Chang, S. S. (1985). Diagnosis and correction of boron deficiency in citrus

Communar, G. & Keren, R. (2008). Boron adsorption by soils as affected by dissolved

da Silva, A. R. & de Andrade, J. M. V. (1980). A cultura do trigo nas varzeas de Minas

Dannel, F.; Pfeffer, H.; Walch-Liu, P. & Romheld, V. (2001). Plant nutrition – food security and sustainability of agro-ecosystems. Dordrecht: Kluwer, 162–163.

De Oliveira, R. H.; Milanez, C. R. D.; Moraes-Dallaqua, M. A. & Rosolem, C. A. (2006).

Deabreu, C. A.; Deabreu, M. F, Vanraij, B. & Bataglia, O. C. (1994). Extraction of boron from

Dell, B. &. Huang, L. (1997). Physiological response of plants to low boron. *Plant and Soil*.

growth of cotton. *Journal of Plant Nutrition,* Vol. 29, pp. 2035-2048.

dos Cerrados (CPAC). *Circular Técnica* No. 2: 69 pp. (in Portuguese). Dannel, F.; Pfeffer, H.; Romheld, V. (2000). Characterization of root boron pools, boron

*Australian Journal of Plant Physiology* Vol. 156, pp. 756–761.

*Science and Plant Analysis.* Vol. 25, pp. 3321–3333.

orchard of Taiwan. *Soils and Fertilizers in Taiwan,* Taipei, Taiwan.

*Department of Agriculture and Forestry,* Taiwan. pp.167-195.

*Improvement Station, Taiwan.* pp. 109-122.

*Analysis.* Vol. 35, pp. 267-283.

Vol. 72, pp. 492-499.

Vol. 193, pp. 103–120.

*Scientific Research* Vol. 19, pp. 134-139.

irrigation water. *J. Agric. Sci.* Vol. 97, pp. 75-78.

systems. *Analytica Chimica Acta,* Vol. 226, pp. 221–227.

boron deficiency and toxicity in crops. *Proceedings of the Symposium on Reclamation of the Problem Soils in the Eastern Taiwan* (S. N. Hwang & G. C. Chiang, eds*). Chinese Society of Plant Nutrition and Fertilizer Science and Hwalian District Agricultural* 

diagnosis as guides to fertilization recommendation for the citrus orchards in Taiwan. *Annual Research Reports on Soils and Fertilizers 81. Published by the Provincial* 

of boron to Mustard in arid soils of India. *Communications in Soil Science and Plant* 

field application of micronutrient fertilizers. *Pakistan Journal of Industrial and* 

determination of total boron in soils with ultrasonic leaching in automatic flow

orchard. In: *Seminar on Leaf Diagnosis as a Guide to Orchard Fertilization. Technical Bulletin No. 91.* Food Fertilizer and Technology Center, Taipei, Taiwan. pp. 1-12. Chiu, T. S. & Chang, S. S. (1986). Diagnosis and correction of boron deficiency in the citrus

organic matter from treated sewage effluent. *Soil Science Society of America Journal*.

Gerais-possibilidades e dificuldades. Embrapa, Centro de Pesquisa Agropecuária

uptake and boron translocation in sunflower using the stable isotope 10B and 11B.

Boron deficiency inhibits petiole and peduncle cell development and reduces

soil by microwave heating for ICPAES determination. *Communications in Soil* 


Barber, S. A. (1995). Soil nutrient bioavailability: a mechanistic approach. John Wiley and

Barrow, N. J. (1989). Testing a mechanistic model. X. The effect of pH and electrolyte

Bell, R. W. (1997). Diagnosis and prediction of boron deficiency for plant production. *Plant* 

Berger, K. C. & Truog, E. (1939). Boron determination in soils and plants using the

Bergmann, W. (1988). Ernährungsstörungen bei Kulturpflanzen. Entstehung, visuelle und

Bhatnagar, R. S.; Attri, S. C.; Mathur, G. S. & Chaudhary, R. S. (1979). Boron adsorption

Bhatta, M. R. & Ferrara, G. O. (2005). Wheat sterility induced by boron deficiency in Nepal.

Biggar, J. W. & Fireman, M. (1960). Boron adsorption and release by soils. Soil Science

Bingham, F. T. (1982). Boron. In Methods of Soil Analysis, eds. A. L Page, 431–447. Madison,

Bingham, F. T.; Page, A. L.; Coleman, N. T. & Flach, K. (1971). Boron adsorption

Borkakati, K. & Takkar, P. N. (2000). Forms of boron in acid alluvial and lateritic soils in

Brown, P. H. & Hu, H. (1998). Boron mobility and consequent management. In: *Better Crops.* 

Brown, P. H. & Shelp, B. J. (1997). Boron mobility in plants. *Plant and Soil.* Vol. 193, pp. 85-

Bullock, D. G. & Sawyer, J. E. (1991). Nitrogen, potassium and boron fertilization of canola.

Campana, A. M. G.; Barrero, F. A. & Ceba, M. R. (1992). Spectrofluorimetric determination

Cartwright, B.; Tiller, K. G.; Zarcinas, B. A. & Spouncer, L. R. (1983). The chemical

Chakraborti, S. K. & Barman, P. (2003). Enhancement of yield of wheat genotypes by application of borax in Terai region. *Journal of Interacademicia.* Vol. 7, pp. 256-261. Chang, S. S.; Hu, N. H.; Chen, C. C. & Chu, T. F. (1983). Diagnosis criteria of boron

of boron in soils plants and natural waters with Alizarin red-S. *Analyst*. Vol. 117,

assessment of the boron status of soils. *Australian Journal of Soil Research.* Vol. 21,

deficiency in papaya and the soil boron status of Taitung area (Taiwan). *Chinese* 

analytische diagnose. Fischer Verlag, Jena, Germany.

equilibrium in soils. *Annals of Arid Zone.* Vol. 18, pp. 86-95.

Mountain Development (ICIMOD), Kathmandu, Nepal.

Society of America Proceedings. Vol. 24, pp. 115-120.

concentration on borate sorption by a soil. *Journal of Soil Science.* Vol. 40, pp. 427-

quinalizarin reaction. Industrial and Engineering Chemistry. Analytical Edition.

In: Micronutrients in South and South East Asia, 221-229; P. Andersen, J. K. Tuladhar, K. B. Karki & S. L. Maskey (Eds), International Centre for Integrated

characteristics of selected soils from Mexico and Hawaii. *Soil Science Society of* 

relation to ecosystem and rainfall distribution. In: *Proceedings of International Conference on Managing Resources for Sustainable Agricultural Production in the 21st*

Sons, New York, NY, U.S.A.

*and Soil.* Vol. 193, pp. 149-168.

Vol. 11, pp. 540-545.

Wisconsin, U.S.A.: ASA.

Vol. 82, pp. 28-31.

pp. 1189–1191.

pp. 321-332.

101.

*America Journal.* Vol.35, 546-550.

*Century. Better Crops.* Vol. 2, pp. 127-128.

*Journal of Soil Science.*Vol. 32, pp. 238-252.

*Journal of Production Agriculture.* Vol. 4, pp. 550-555.

435.


Fleet, M. E. L. (1965). Preliminary investigations into the sorption of boron by clay minerals.

Fleming, G. A. (1980). Essential micronutrients. I. Boron and molybdenum. In: Applied soil

Frederickson, A. F. & Reynolds, R. C. Jr. (1959). Clays. In: *Clay Minerals Proceedings*, 203-213,

Fritzsche, R. (1955). On the cork disease of Glocken Apples. *Schweizerische Zeitschrift fur* 

Garcia, I. L.; Cordova, M. H. & Sanchez-Pedrono, C. (1985). Sensitive method for the

Geddes, A. M .W.; Khan, S. M.; Naumann-Etienne, K.; Edwards, R. J. A.; Smith, A. E.;

Gestring, W. D. & Soltanpour, P. N. (1984). Evaluation of the ammonium bicarbonate-DTPA

Gestring, W. D. & Soltanpour, P. N. (1987). Comparison of soil tests for assessing boron toxicity to alfalfa. *Soil Science Society of America Journal.* Vol. 51, pp. 1214–1219. Goldberg, S. (1993). Chemistry and mineralogy of boron in soils. In: U. C. Gupta (Ed.), Boron and its role in crop production, Pages 344, CRC Press, Boca Raton, FL, U.S.A.

Goldberg, S. & Forster, H. S. (1991). Boron sorption on calcareous soils and reference

Goldberg, S. & Glaubig, R. A. (1986). Boron adsorption on California soils. *Soil Science Society* 

Goldberg, S.; Shouse, P. J.; Lesch, S. M.; Grieve, C. M.; Poss, J. A.; Forster, H. S. & Suarez, D.

Gormus, O. (2005). Interactive effect of nitrogen and boron on cotton yield and fiber quality.

Goto, I.; Muramoto, J. & Ninaki, M. T. I. (1992). Application of inductively coupled plasma

Graham, M. J. & Heavner, D. L. (1993). Response of soybean genotypes to boron, zinc and manganese deficiency in tissue culture. *Plant and Soil.* Vol. 150, pp. 307-310. Graham, R. D.; Welch, R. M.; Grunes, D. L.; Cary, E. E. & Norvell, W. A. (1987). Effect of zinc

Grant, C. & Bailey, L. D. (1993). Fertility management in canola production. *Canadian Journal* 

Greenhill, A. W. (1938). Boron deficiency in horticultural crops: recent developments.

L. (2002). Soil boron extractions as indicators of boron content of field-grown crops.

atomic emission spectrometry (ICP-AES) to soil analysis (Part 5) determination of hot water soluble boron in soils by ICP-AES. *Japanese Journal of Soil Science and Plant* 

deficiency on the accumulation of boron and other mineral nutrients in barley. *Soil* 

Goldberg, S. (1997). Reactions of boron with soils. *Plant and Soil.* Vol. 93, pp. 35-48.

*Turkish Journal of Agriculture and Forestry.* Vol. 29 pp. 51-59.

*Science Society of America Journal.* Vol. 51, pp. 652-657.

trace elements, B. E. Davies (Ed.), 155-197, John Wiley and Sons, New York, NY,

spectrophotometric determination of boron in plants and waters using crystal

Bajwa.; K. & Hussain, A. (1989). *Potato atlas of Pakistan: Information on potato production by agro-ecological zones.* 1st edition. Pakistan Agricultural Research

soil test for assessing B availability to alfalfa. *Soil Science Society of America Journal.* 

*Clay Minerals*. Vol. 6, pp. 3-16.

violet. *Analyst*. Vol. 110, pp. 1259.

Council, Islamabad, Pakistan.

calcites. *Soil Science.* Vol. 152, pp. 304–310.

*of America Journal.* Vol 50, pp. 1173-1176.

*Soil Science.* Vol. 167, pp. 720-728.

*Nutrition.* Vol. 63, pp. 53–57.

*of Soil Science.* Vol. 73, pp. 651-670.

*Scientific Horticulture.* Vol. 6, pp. 191-198.

Vol. 48, pp. 96-100.

8th Conf.; Pergamon Press, Oxford, England.

*Obst- und Weinbau*. Vol. 64, No. 11, pp. 193-198.

U.S.A.



Diana, G. (2006). Boron in the soil, from deficit to toxicity. *Informatore Agrario*. Vol. 62, pp.

Din, V. K. (1984). The preparation of iron-free solutions from geological materials for the

Dregne, H. E. & Powers, W. L. (1942). Boron fertilization of alfalfa and other legumes in

Duncan, L. W. & Cohn, E. (1990). Nematode parasites of citrus. In: *Plant parasitic nematodes in* 

Dwivedi, G. K.; Dwivedi, M. & Pal, S. S. (1990). Mode of application of micronutrients in

Eguchi, S. (2000). Effect of boron deficiency on growth, yield and contents of protein and fat

Eguchi, S. & Yamada, Y. (1997). Long term field experiment on the application of slow-

Elrashidi, M. A. & O'Connor, G. A. (1982). Boron sorption and desorption in soils. *Soil* 

Elseewi, A. A. (1974). Some observations on boron in water, soils, and plants at various locations in Egypt. *Alexandria Journal of Agricultural Research.* Vol. 22, pp. 463-473. Evans, C. M. & Sparks, D. L. (1983). On the chemistry and mineralogy of boron in pure and

Evans, L. J. (1987). Retention of boron by agricultural soils from Ontario. *Canadian Journal of* 

Evans, S. & Krahenbuhl, U. (1994a). Boron analysis in biological material – microwave

Ferrando, A. A.; Green, N. R.; Barnes, K. W. & Woodward, B. (1993). Microwave digestion

Fitzpatrick, R. E. & Woodbridge, C. G. (1941). Boron deficiency in Apricots. *Scientific* 

Flannery, R. L. (1985). Understanding boron needs in crop production. *Fertilizer Progress.* 

Fertilizer Institute, Washington, U.S.A. Vol. 16, pp. 41-45.

*sub-tropical and tropical agriculture,* Luc, R.; Sikora, R. A. & Bridge, J. (Eds)*.* CAB

soybean –wheat crop sequence. *Journal of the Indian Society of Soil Science,* Vol. 38,

in grains of soybean (*Glycine max*). *Journal of soil science and plant nutrition.*(Japan).

release boron fertilizer: Part 2. Behaviour of boron in the soil. In: *Proceedings of International Symposium on Boron in Soils and Plants*, R. W. Bell & B. Rerkasem,

in mixed systems: A review. *Communications in Soil Science and Plant Analysis.* Vol.

digestion procedure and determination by different methods. *Fresenius' Journal of* 

preparation and ICP determination of boron in human plasma. *Biological Trace* 

Oregon. *Journal of American Society of Agronomy.* Vol. 34, pp. 902-12.

emission spectrometry. *Analytica Chimica Acta.* Vol. 159, pp. 387–391. Dinesh, K. & Sudkep, S. (2009). Influence of soil applied sulfur and boron on yield and quality parameters of soybean. *Annals of Biology.* Vol. 25, pp. 105-111. Dobermann, A. & Fairhurst, T. (2000). Rice nutrient disorders & nutrient management [Online] Available: http://books.irri.org/9810427425\_content.pdf [May, 15, 2010]. Dordas, C. (2006). Foliar boron application improves seed set, seed yield, and seed quality of

alfalfa. *Agronomy Journal.* Vol. 98, pp. 907-913.

International, 321-346, Wallingford, Oxon, UK.

(Eds.), 49–56, September, 1997, Chiang Mai, Thailand.

*Science Society of America Journal.* Vol. 46, pp. 27-31.

determination of boron (and other elements) by inductively coupled plasma

54-58.

pp. 458-463.

Vol. 71, pp. 171-178.

14, pp. 827-846.

*Soil Science.* Vol. 67, pp. 33-42.

*Analytical Chemistry.* Vol. 349, pp. 454–459.

*Element Research.* Vol. 37, pp. 17–25.

*Agriculture.* Vol. 22, No. 4, pp. 271-273.


Hopkins, B. G.; Jolley, V. D.; Webb, B. L. & Callahan, R. K. (2010). Boron fertilization and

Hossain, A. K. M. Z. & Hossain, M. A. (2004). Effects of aluminum and boron supply on

Hou, J.; Evans, L. J. & Spiers, G. A. (1996). Chemical fractionation of soil boron. I. Method

Hu, Y. S.; Ma, Y. H.; Sun, Y. L. & Guo, G. (1994). Effect of B application on the agronomic

Huang, L. & Ye, Z. (2005). Boron nutrition and chilling tolerance of warm climate crop

Ibrahim, M. E.; Bekheta, M. A.; El-Moursi, A. & Gaafar, N. A. (2009). Effect of arginine, pro-

of cotton plants. *World Journal of Agricultural Sciences*. Vol. 5, pp. 863-870. Iyenger, S. S.; D. C. Martens, & W. P. Miller. (1981). Distribution and plant availability of soil zinc fractions. *Soil Science Society of America Journal.* Vol. 45, pp. 735–739. Jeffrey, A. J. & McCallum, L. E. (1988). Investigation of a hot 0.01 M CaCl2 soil boron

Jin, JI-Yun, D. C. Martens, & L. W. Zelazny. (1988). Plant availability of applied and native boron in soils with diverse properties. *Plant and Soil.* Vol. 105, pp. 127-132. Johnson, G. (2006). *Pakistan citrus industry challenges: Opportunities for Australia-Pakistan collaboration in research, development and extension*. Islamabad, Pakistan. Jones, J. B. (Jr). (2003). Plant mineral nutrition. In: *Agronomic handbook: Management of crops,* 

Kabata-Pendias, A. & Pendias, H. (1992). *Trace elements in soils and plants*, 2nd ed., CRC Press,

Kanwal, S.; Rahmatullah; Aziz, T.; Maqsood, M. A. & Abbas, N. (2008). Critical ratio of

Kao, M. M. & Juang, T. C. (1978). Comparison of boron test methods for sugarcane soils in Taiwan. In: *Report of Taiwan Sugar Experiment Station*, 15-27, Tainan, Taiwan. Kaplan, D. I.; Burkman, W.; Adriano, D. C.; Mills, G. L. & Sajwan, K. S. (1990).

Katyal, J. C. & Randhawa, N. S. (1983). Micronutrients. *FAO Fertilizer and Plant Nutrition* 

Kavipurapu, C. S.; Gupta, K. K.; Dasgupta, P.; Chatterjee, N. & Pandey, L. P. (1993).

Keren, R. & Bingham, F. T. (1985). Boron in water, soils, and plants. *Advances in Soil Sciences.* 

Keren, R. & Communar, G. (2009). Boron sorption on wastewater dissolved organic matter: pH effect. *Soil Science Society of America Journal.* Vol. 73, pp. 2021-2025.

calcium and boron in maize shoot for optimum growth. *Journal of Plant Nutrition,* 

Determination of boron in soils containing inorganic and organic boron sources.

Determination of boron in steels by inductively coupled plasma optical emission

*soils and their fertility,* Pages 325, CRC Pres, Boca Raton, FL, U.S.A.

*Soil Science Society of America Journal.* Vol. 54, pp. 708–714.

Bangladesh. *Soil Science and Plant Nutrition.* Vol. 50, pp. 189-195.

development. *Canadian Journal of Soil Science.* Vol. 76, pp. 485–491.

*Science and Plant Analysis*. Vol. 41, pp. 527-539.

species. *Annals of Botany.* Vol. 96, pp. 755-767.

*and Plant Analysis.* Vol. 19, pp. 663–673.

Boca Raton, FL, U.S.A.

Vol. 31, pp. 1535-1542.

*Bulletin. No. 7.* Rome, Italy.

Vol. 1, pp. 229-276.

spectrometry. *Analusis.* Vol. 21, pp. 21–25.

*Crops (China).* Vol. 16, pp. 43-46.

evaluation of four soil extractants: Russet Burbank potato*. Communications in Soil* 

growth of seedlings among 15 cultivars of wheat (*Triticum aestivum* L.) grown in

traits, yields and oil contents of a double-row rape (*Brassica napus* L.) cultivar. *Oil* 

hexadione-Ca, some macro and micro-nutrients on growth, yield and fiber quality

extraction procedure followed by ICP-AES analysis. *Communications in Soil Science* 


Gupta, S. K. & Stewart, J. W. B. (1975). The extraction and determination of plant-available

Gupta, U. C. (1968). Relationship of total and hot-water soluble boron and fixation of added

Gupta, U. C. (1972). Interaction of boron and lime on barley. *Soil Science Society of America* 

Gupta, U. C. (1983). Boron deficiency and toxicity symptoms for several crops as related to

Gupta, U. C. (1984). Boron nutrition of alfalfa, red clover, and timothy grown on podzol

Gupta, U. C. (1993). Introduction. In: *Boron and its role in crop production,* U. C. Gupta (Ed.),

Gupta, U. C. (2006). Chapter 8. Boron. In: *Hand book of Plant Nutrition*, Barker, A.V. & D. J.

Gupta, U. C.; Jame, Y. M.; Campbell, C. A.; Leyshon, A. J. & Nicholaichuk, W. (1985). Boron

Haby, V. A. & Leonard, A. T. (2000). Alfalfa production on coastal plain soils. In:

Han, S. & Chen, L. S. (2008). Boron deficiency decreases growth and photosynthesis, and

Hatcher, J. T.; Blair, G. Y. & Bower, C. A. (1959). Response of beans to dissolved and

Hatcher, J. T.; Blair, G. Y. & Bower, C. A. (1962). Adjusting soil solutions to specified B

Hazra, C. R. & Tripathi, S. B. (1998). Effect of secondary and micronutrients on yield and

Hill, H. (1936). Minor elements affecting horticultural crops. *Scientific Agriculture.* Vol. 17,

Hill, W. E. & Morrill, L. G. (1975). Boron, calcium and potassium interactions in Spanish peanuts. *Soil Science Society of America Proceedings.* Vol. 39, pp. 80-83. Hingston, F. J. (1964). Reaction between boron and clays. *Australian Journal of Soil Research.* 

Ho, S. B. (2000). Boron deficiency of crops in Taiwan. Department of Agricultural Chemistry,

toxicity and deficiency: A review. *Canadian Journal of Soil Science.* Vol. 65, pp. 381-

*Proceedings/Reports of the American Forage and Grassland Council,* 37th North American Alfalfa Improvement Conference, July 16 19, 2000, Madison, WI, U.S.A. Haby, V. A. & Leonard, A. T. (2005). Sustainable alfalfa production on coastal plain soils of

the United States. *Communications in Soil Science and Plant Analysis.* Vol. 36, pp. 47-

increases starch and hexoses in leaves of citrus seedlings. *Journal of Plant Physiology.* 

tissue boron levels. *Journal of Plant Nutrition.* Vol. 6, pp. 387–395.

soils of eastern. *Canadian Journal of Soil Science.* Vol. 137, pp. 16–22.

Pilbeam (Eds.), 241-277, CRC Press, Boca Raton, FL, USA.

Pages 237, CRC Press, Boca Raton, FL, U.S.A.

absorbed boron. *Soil Science.* Vol. 88, pp. 98-100.

concentrations. *Soil Science.* Vol. 94, pp. 55-57.

National 684 Taiwan University, Taipei 106, Taiwan.

quality of forages. Fert. News 43: 77-82.

Gupta, U. C. (1979a). Boron nutrition of crops. *Advances in Agronomy.* Vol. 31, pp. 273-307. Gupta, U. C. (1979b). Some factors affecting the determination of hot water-soluble boron

boron, to properties of Podzol soils. *Soil Science Society of America Proceedings.* Vol.

from Podzol soils using azomethine-H. *Canadian Journal of Soil Science.* Vol.59, pp.

boron in soils. *Schweiz. Landwirtsch. Forsch.* Vol. 14, pp. 153–159.

32, pp. 45-48.

241-247.

409.

63.

Vol. 165, pp. 1331-1341.

pp. 148-153.

Vol. 2, pp. 83-95.

*Proceedings.* Vol. 36, pp. 332-334.


Lovatt, C. J. (1985). Evolution of xylem resulted in a requirement of boron in the apical

Luo, X. Y.; Peng, Y. C. & Wang, B. Y. (1990). Effect of boron fertilization on yield and quality of groundnut. *Journal of Zhejiang Agricultural Science.* Vol. 1, pp. 30-32. Mackin, J. E. (1986). The free-solution diffusion coefficient of boron: influence of dissolved

Mahler, R. L.; Naylor, D. V. & Fredrichson, M. K. (1984). Hot water extraction of boron from

Malhi, S. S.; Raza, M.; Schoenau, J. J.; Mermut, A. R.; Kutcher, R.; Johnston, A. M. & Gill, K.

Marschner, H. (1995). *Mineral nutrition of higher plants.* 2nd ed. Academic Press. London. UK.

Matsi, T.; Antoniadis, V. & Barbayiannis, N. (2000). Evaluation of the NH4HCO3-DTPA soil

McGeehan, S. L.; Topper, K. & Naylor, D. V. (1989). Sources of variation in hot water

McLarty, H. R. (1940). British Columbia uses boron for fruit. In: *Better Crops*. Vol. 24, No. 4,

Mehmood, E. H.; Kausar, R.; Akram, M. & Shahzad, S. M. (2009). Is boron required to

Mengel, K. & Kirkby, E. A. (1982). *Principles of plant nutrition.* 3rd ed. International Potash

Mengel, K. & Kirkby, E. A. (1987). *Principles of plant nutrition.* 4th ed. International Potash

Mengel, K. & Kirkby, E. A. (2001). Boron. In: *Principles of plant nutrition.* 621-638, Kluwer Academic Publishers (5th ed.) Dordrecht/ Boston/ London, Netherlands. Mezuman, U. & Keren, R. (1981). Boron adsorption by soils using a phenomenological adsorption equation. *Soil Science Society of America Journal.* Vol. 45, pp. 722-726. Midgley, A. R. & Dunklee, D. E. (1947). Boron for Vermont soils and crops. *Exp. Sta. Bull.*

Miley, W. N.; Hardy, G. W. & Sturgis, M. B. (1969). Influence of boron, nitrogen and

Miller, R. O.; B. Vaughan, & J. Kutoby-Amacher. (2000). Extraction of soil boron with DTPA-

soils using sealed plastic pouches. *Communications in Soil Science and Plant Analysis.*

S. (2003). Feasibility of B fertilization for yield, seed quality, and B uptake of canola in north eastern Saskatchewan. *Canadian Journal of Soil Science.* Vol. 83, pp. 99-108. Malik, M. A.; Makhdum, M. I. & Shah, S. I. H. (1992). Cotton response to boron fertilizer in

silt loam soils. In: *Proceedings of s*oil health for sustainable agriculture, 331-336, *3rd National Congress of Soil Science,* March 20-22, 1990, Lahore, Pakistan, Soil Science

test for assessing boron availability to wheat. *Communications in Soil Science and* 

extraction and colorimetric determination of soil boron. *Communications in Soil* 

improve rice growth and yield in saline environment? *Pakistan Journal of Botany.*

*539.* 20 pp. Agricultural Experiment Station, University of Vermont and State

potassium on yield, nutrient uptake and abnormalities of boron. *Agronomy Journal.* 

meristems of vascular plants. *New Phytologist.* Vol. 99, pp. 509-523.

organic matter. *Marine Chemistry.* Vol. 20, pp. 131-140.

Vol. 15, pp. 479-492.

Society of Pakistan.

pp. 8-11; 37-38.

Vol. 61, pp. 9-13.

Vol. 41, pp. 1339-1350.

*Plant Analysis.* Vol. 31, pp. 669-678.

*Science and Plant Analysis.* Vol. 20, pp. 1777–1786.

Institute, Worblaufen-Bern, Switzerland.

Institute, Worblaufen-Bern, Switzerland.

Agricultural College, Burlington, VT, U.S.A.

sorbitol. *Soil and Plant Analysis Spring*: pp. 4–10.

889 pp.


Khan, M. A.; Shaukat, S. S. & Khan, M. A. (2008). Economic benefits from irrigation of maize

Khattak, J. (1995). Micronutrients in Pakistan agriculture. *Project report, Pakistan Agriculture* 

Kirk, G. J. & Loneragan, J. F. (1988). Functional boron requirement for leaf expansion and its

Kormilitsyn, V. F. (1992). Effect of rates of boron, manganese and molybdenum on lucerne yield under irrigation in the Volga region. *Agrokhimiya*. Vol. 8, pp. 94-98. Kumar, D. & Sidhu, S. (2009). Influence of soil applied sulfur and boron on yield and quality

Lancaster, J. D.; Murphy, B. C.; Hurt, B. C. Jr.; Arnold, B. L.; Coats, R. E.; Albritton, R. C. &

Lee, J. J.; van-derWalinga, I.; Manyeki, P. K.; Houba, V. J. G. & Novozamsky, I. (1987).

Li, B.; Li, H.; Kui, W. H.; Chao, M. C.; Jern, W. S.; Li, H. P.; Chu, W. J. & Wang, C. L. (1978).

Liu, G. & Zhang, X. (2005). Effects of combined application of B, Mo and Zn fertilizers on yield and crude protein content of lucerne. *Grassland (China).* Vol. 27, pp. 13-18. Li, R. & Gupta, U. C. (1991). Extraction of soil B for predicting its availability to plants. *Communications in Soil Science and Plant Analysis.* Vol. 22, pp. 1003-1012. Liu Z.; Zhu, Q. Q. & Tong, L. H. (1980). Boron deficient soils and their distribution in China.

Liu Z.; Zhu, Q. Q. & Tong, L. H. (1982a). On the status of microelements in soils and their

Liu Z.; Zhu, Q. Q. & Tong, L. H. (1989). Regularities of content and distribution of boron in

Liu Z.; Zhu, Q. Q.; Tong, L. H.; Xu, J. X. & Yen, C. L. (1982b). Geographical distribution of trace elements deficient soils in China. *Acta Pedologica Sinica.* Vol. 19, pp. 209–223. Lohse, G. (1982). Microanalytical azomethine-H method for boron determination in plant tissue. *Communications in Soil Science and Plant Analysis.* Vol. 13, pp. 127–134. Loomis, W. D. & Durst, R. W. (1992). Chemistry and biology of boron. *Biofactors*. Vol. 3, pp.

Lora, R. S. (1978). Respuesta de los cultivos de clima frio a la aplicación de micronutrientos.

Memorias del V coloquio de suelos. *Suelos Ecuatoriales. SOCIEDAD Colombiana de la* 

*Chemicals and World Food Supplies*, December 1982, Manila, Philippines. Liu Z.; Zhu, Q. Q. & Tong, L. H. (1983). Microelements in the main soils of China. *Soil* 

role in crop production of China. In: *Proceedings of International Conference of* 

parameters of soybean. *Annals of Biology.* Vol. 25, pp. 105-111.

University, Agricultural Experiment Station, Mississippi, U.S.A.

pp. 1091-1098.

*Journal.* Vol. 80, pp. 758-762.

*Analysis.* Vol. 18, pp. 789–802.

*Science.* Vol. 135, pp. 40-46.

*Ciencia del Suelo*. Vol. 9, pp. 183–191.

*Acta Pedologica Sinica.* Vol. 17, pp. 228–239.

soils. *Acta Pedologica Sinica.* Vol. 26, pp. 353–361.

3, pp. 1–19.

229-239.

135 pp.

with treated effluent of waste stabilization ponds. *Pakistan Journal of Botany.* Vol. 40,

*Research Council,* Islamabad and NWFP Agriculture University Peshawar, Pakistan.

use as a critical value for diagnosis of boron deficiency in soybean. *Agronomy* 

Walton, L. (1962). *Boron for cotton.* Bull. 635 pp. State College, Mississippi State

Determination of boron in fresh and in dried plant material by plasma emission spectrometry after extraction with HF-HCl. *Communications in Soil Science and Plant* 

Studies on cause of sterility of wheat. *Journal of Northeastern Agriculture College*. Vol.


Nuttall, W. F.; Ukrainetz, H.; Stewart, J. W. B. & Spurr, D. T. (1987). The effect of nitrogen,

Nyborg, M. & Hoyt, P. B. (1970). Boron deficiency in turnip rape grown on gray wooded

Offiah, O. & Axley, J. H. (1988). Improvement of boron soil test. *Communications in Soil* 

Ogner, G. (1980). Automatic determination of boron in water samples and soil extracts. *Communications in Soil Science and Plant Analysis.* Vol. 11, pp. 1209–1219. Okazaki, E. & Chao, T. T. (1968). Boron adsorption and desorption by some Hawaiian soils.

Owens, J. W.; Gladney, E. S. & Knab, D. (1982). Determination of boron in geological

Parker, D. R. & Gardner, E. H. (1981). The determination of hotwater-soluble boron in some

Parr, A. J. & Loughman, B. C. (1983). Boron and membrane function in plants. In: *Metals and* 

Patel, P. C. & Patel, K. P. (2003). Effects of zinc and boron application on seed yield of

Patil, G. D.; Patil, M. D.; Patil, N. D. & Adsule, R. N. (1987). Effect of boronated

Porter, S. R, Spindler, S. C. & Widdowson, A. E. (1981). An improved automated

H. *Communications in Soil Science and Plant Analysis.* Vol. 12, pp. 461–473. Pougnet, M. A. B. & Orren, M. J. (1986a). Determination of boron by inductively coupled

Pougnet, M. A. B. & Orren, M. J. (1986b). Determination of boron by inductively coupled

Rahmatullah, Badr-uz-Zaman & Salim, M. (1999). Plant utilization and release of boron

Rammah, A. M. & Khedr, M. S. (1984). Response of alfalfa to iron and boron in some

Egyptian soils. *Journal of Plant Nutrition,*Vol. 7, pp. 235-242.

*Journal of Environmental Analytical Chemistry.* Vol. 24, pp. 253–266.

materials by inductively coupled plasma emission spectrometry. *Analytica Chimica* 

acid Oregon soils using a modified azomethine-H procedure. *Communications in* 

*micronutrients: Uptake and utilization by plants*, 87-107, D. A. Robb & W. S. Pierpoint

lucerne (*Medicago sativa* L.). *Journal of the Indian Society of Soil Science. Vol.* 51, pp.

superphosphate, single superphosphate and borax on yield and quality of groundnut. *Journal of Maharashtra Agriculture University.* Vol. 12, pp. 168-170. Ponnamperuma, F. N.; Clayton, M. T. & Lantin, R. S. (1981). Dilute hydrochloric acid as an

extractant for available zinc, copper, and boron in rice soils. *Plant and Soil.* Vol. 61,

colorimetric method for the determination of boron in extracts of soils soil-less peat-based composts plant materials and hydroponic solutions with Azomethine-

plasma atomic emission spectroscopy: Part 1. Method development. *International* 

plasma atomic emission spectroscopy. Part 2. Applications to South African environmental samples. *International Journal of Environmental Analytical Chemistry.* 

distributed in different fractions in calcareous soils. *Arid Soil Res. Rehab.* Vol. 13, pp.

*compestris* L.). *Canadian Journal of Soil Science.* Vol. 67, pp. 545-559.

soils. *Canadian Journal of Soil Science.* Vol. 50, pp. 87-88.

*Soil Science and Plant Analysis.* Vol. 12, pp. 1311–1322.

(Eds.), Academic Press, New York, NY, U.S.A.

*Science and Plant Analysis.* Vol. 19, pp. 1527–1542.

*Soil Science.* Vol. 105, pp. 255-259.

*Acta.* Vol. 135, pp. 169–172.

320-321.

pp. 297-310.

Vol. 24, pp. 267–282.

293-303.

sulphur and boron on yield and quality of rapeseed (*Brassica napus* L. and *Brassica* 


Mishra, L. C. & Shukla, K. N. (1986). Effects of fly ash deposition on growth, metabolism

Misra, R.; Munankarmi, R. C.; Pandey, S. P. & Hobbs, P. R. (1992). Sterility work in wheat at

*Report No. 11. CIMMYT*, 67-71, C. E. Mann & B. Rerkasem (Eds.), Mexico. Miwa, K. & Fujiwara, T. (2010). Boron transport in plants: co-ordinated regulation of

Moraghan, J. T. & Mascagni. H. J. (1991). Environmental and soil factors affecting

Murphy, B. C. & Lancaster, J. J. D. (1971). *Response of cotton to boron.* Agronomy Journal. *Vol.* 

Mustafa, W. A.; Falatah, A. M. & El. Shall, A. A. (1993). Effect of excess boron fertilization on

Nable, R. O.; Banuelos, G. S. & Paull, J. G. (1997). Boron toxicity. *Plant and Soil.* Vol. 193,

Nable, R. O.; Lance, R. C. M. & Cartwright, B. (1990). Uptake of boron and silicon by barley

Nazli, F.; Bibi F.; F.; Gul, M. & Hannan, A. (2010). Effect of graded level of potassium on

Niaz, A.; Ibrahim, M.; Nisar, A. & Anwar, S. A. (2002). Boron contents of light and medium

Niaz, A.; Ranjha, A. M.; Rahmatullah; Hannan, A. & Waqas, M. (2007). Boron status of soils

Nogueira, A. R. A.; Brienza, S. M. B.; Zagatto, E. A. G.; Lima, J. L. F. C. & Araujo, A. N.

Novozamsky, I.; Barrera, L. L.; Houba, V. J.; Vanderlee, J. J. & Eck, R. (1990). Comparison of

contents. *Pakistan Journal of Agricultural Sciences.* Vol. 44, pp. 428-435. Nilsson L G & Jennische P. (1986). Determination of boron in soils and plants. *Swedish* 

Mortvedt, J. J. (1994). Boron diet essential for crops. *Farm Chemicals.* February, 1994. 2 pp. Mortvedt, J. J. & Woodruff, J. R. (1993). Technology and application of boron fertilizers for

transporters. *Annals of Botany.* Vol. 105, pp. 1103–1108.

J. Luxmoore (Ed.), Soil Sci. Soc. Am.; Madison, WI, U.S.A.

*Ecol. Biol.* Vol. 42, 1-13.

Press, Boca Raton, FL, U.S.A.

March 24-27, 2010, Faisalabad, Pakistan.

*Journal of Agricultural Research.* Vol. 16, pp. 97–103

*Analytica Chimica Acta.* Vol. 276, pp. 121–125.

63, pp. 539-540.

71–78.

pp. 181–198.

pp. 83–90.

4, pp. 534–536.

2195.

and dry matter production of maize and soybean. *Environmental Pollution Series A:* 

Tarahara in the eastern Terai of Nepal. In: *Boron deficiency in wheat, Wheat Special* 

micronutrient deficiencies and toxicities. In: *Micronutrients in agriculture*, 371-425, R.

crops. In: Boron and its role in crop production, U. C. Gupta (Ed.), 156-176, CRC,

status and availability of boron in calcareous soils. *Fertilizer Research.* Vol. 36, pp.

genotypes with differing susceptibilities to boron toxicity. *Annals of Botany.* Vol. 66,

quality of potato crop. In: *Abstracts 13th International Congress of Soil Science,* 13 pp.,

textured soils and cotton plants. *International Journal of Agriculture and Biology.* Vol.

as affected by different soil characteristics–pH, CaCO3, organic matter and clay

(1993). Multi-site detection in flow analysis: Part 2. Monosegmented systems with relocating detectors for the spectrophotometric determination of boron in plants*.* 

a hot water and cold 0.01 M CaCl2 extraction procedures for the determination of boron in soil. *Communications in Soil Science and Plant Analysis.* Vol. 21, pp. 2189–


Rerkasem, B.; Bell, R. W.; Lodkaew, S. &. Loneragan, J. F. (1993). Boron deficiency in

Rerkasem, B.; Jamjod, S. & Nirantrayagul, S. (2004). Increasing boron efficiency in many

production on soils low in boron. *Field Crops Research.* Vol. 86, pp. 175-184. Reuter, D. J. & Robinson, J. B. (1986). *Plant analysis an interpretation manual.* Inkata Press,

Russell, E. W. (1973). *Soil condition and plant growth,* 849 pp, 10th ed. Longman Ltd.; London,

Ryan, J.; Miyamoto, S. & Stroehlein, J. L. (1977). Relation of solute and sorbed boron to the boron hazard of irrigation water. *Plant and Soil.* Vol. 47, pp. 253-256. Sah, R. N. & Brown, P. H. (1997). Techniques for boron determination and their application to the analysis of plant and soil samples. *Plant and Soil.* Vol. 193, pp. 15–33. Sakal, R. & Singh, A. P. (1995). Boron research and agricultural production. In: *Micronutrient* 

Sakal, R.; Singh, A. P.; Sinha, R. B. & Bhogal, N. S. (1988). Annual progress reports. ICAR All

Bihar, *Res. Bull., Department of Soil Science*, RAU, Samastipur, Bihar, India. Salinas, R.; Cerda, A.; Romero, M. & Caro, M. (1981). Boron tolerance of pea (*Pisum sativum*).

Sarwar, N. & Mubeen, K. (2009). *Nutrient deficiency in rice crop.* DAWN [Online] Available:24

Schalscha, E. B.; Bingham, F. T.; Galindo, G. G. & Galvan, M. P. (1973). Boron adsorption by

Scott, H. D.; Beasley, S. D. & Thompson, L. F. (1975). Effect of lime on boron transport and uptake by cotton. *Soil Science Society of America Proceedings.* Vol. 39, pp. 1116-1121. Shafiq, M.; Ranjha, A. M.; Yaseen, M.; Mehdi, S. M. & Hannan, A. (2008). Comparison of

Shi, L. & Wang, Y. H. (2009). Inheritance of boron efficiency in oilseed rape. *Pedosphere.* Vol.

Shiffler, A. K.; V. D. Jolley, J. E. Christopherson, B.L. Webb, D. C. Farrer, & V. A. Haby.

*Communications in Soil Science and Plant Analysis.* Vol. 36, pp. 2179–2187. Shorrocks, V. M. (1997). The occurrence and correction of boron deficiency. *Plant and Soil.*

Sillanpaa, M. (1982). Micronutrients and nutrient status of soils, a global study. In: *FAO Soil* 

freundlich and Langmuir adsorption equations for boron adsorption on calcareous

(2005). Pressurized hot water and DTPA-sorbitol as viable alternatives for soil boron extraction. I. Boron-treated soil incubation and efficiency of extraction.

volcanic ash soils in Southern Chile. *Soil Sci.* Vol. 116, pp. 70-76.

soils. *Journal of Agricultural Research.* Vol. 46, pp. 141-148.

http://www.dawn.com/wps/wcm/connect/dawn-content-library/dawn/inpaper-magazine/economic-and-business/nutrient-deficiency-in-rice-crop-489 [15

*Journal of Plant Nutrition.* Vol. 4, pp. 205-217.

289-294.

UK

August.

May 2010].

19, pp. 403-408.

Vol. 193, pp. 121-148.

*Bulletin. No. 48,* Rome, Italy.

Melbourne, Australia.

soybean, peanut and black gram: Symptoms in seeds and differences among soybean cultivars in susceptibility to boron deficiencies. *Plant and Soil*. Vol. 150, pp.

international bread wheat, durum wheat, triticale and barley germplasm will boost

*research and agricultural production*, 1-64, H. L. S. Tandon (Ed.), Fertilizer Development and Consultation Organization, New Delhi, India. ISBN, 8185116601.

India Coordinated Scheme of Micro-and Secondary Nutrients in Soils and crops of


Rashid, A. (1993). Nutritional disorders of rapeseed-mustard and wheat grown in Potohar

Rashid, A. 1994. Nutrient indexing surveys and micronutrient requirement of crops. In:

Rashid, A. (1995). Nutrient indexing of cotton and micronutrient requirement of cotton and

Rashid, A. (2006). *Boron deficiency in soils and crops of Pakistan: Diagnosis and management.* Pakistan Agricultural Research Council (PARC), Islamabad, Pakistan. Viii, 34 pp. Rashid, A. & Akhtar, M. E. (2006). Soil fertility research and nutrient management in

Rashid, A. & Qayyum, F. (1991). *Cooperative research programme on micronutrient status of* 

Rashid, A. & Rayan, J. (2004). Micronutrient constraints to crop production in soils with

Rashid, A.; Muhammad, S. & Rafique, E. (2005). Rice and wheat genotypic variation in

Rashid, A.; Rafique, E. & Bughio, N. (1994). Diagnosing boron deficiency in rapeseed and

Rashid, A.; Rafique, E. and Bughio, N. (1997). Micronutrient deficiencies in rain-fed

Rashid, A.; Yasin, M.; Ali, M. A.; Ahmad, Z. & Ullah, R. (2009). Boron deficiency in rice in

Raven, J. A. (1980). Short- and long-distance transport of boric acid in plants. *New Phytol.*

Raza, M.; Mermut, A. R.; Schoenau, J. J. & Malhi, S. S. (2002). Boron fractionation in some Saskatchewan soils. *Canadian Journal of Soil Science.* Vol. 82, pp. 173-179. Razzaq, A. & Rafiq, M. (1996). Soil classification and survey. In: *Soil Science,* 405-437, A.

Reid, R. J.; Hayes, J. E.; Post, A.; Stangoulis, J. C. & Graham, R. D. (2004). A critical analysis of the causes of boron toxicity in plants. *Plant Cell Environ.* Vol. 27, pp. 1405–1414. Reid, R.; & Fitzpatrick, K. (2009). Influence of leaf tolerance mechanisms and rain on boron toxicity in barley and wheat. *Plant Physiology.* Vol. 151, pp. 413–420. Rerkasem, B. & S. Jamjod. (2004). Boron deficiency in wheat: A review. *Field Crops Research.* 

Research Center (NARC), Islamabad, Pakistan.

Research Center (NARC), Islamabad, Pakistan.

Islamabad, Pakistan.

*Analysis.* Vol. 25, pp. 2883-2897.

Vol. 84, pp. 231-249.

Vol. 89, pp. 173-186.

959-975.

Agricultural Research Center (NARC), Islamabad, Pakistan.

Agricultural Research Center (NARC), Islamabad, Pakistan.

boron use efficiency. *Soil Environ*. Vol. 24, pp. 98-102.

*Science and Plant Analysis.* Vol. 28, pp. 444-454.

Book of Foundation, Islamabad, Pakistan.

(Eds.). Springer-Verlag, Berlin-Heidelberg, Germany.

area. In: *Micronutrient Project Annual Report, 1991-1992,* 25-36, National Agricultural

*Micronutrient Project Annual Report, 1992-1993,11-19,* National Agricultural

groundnut. In: *Micronutrient Project Annual Report, 1994-95,* 13-28, National

Pakistan. In: *Proceedings of Symposium on Balanced Fertilizer Use: Impact on Crop Production,* 90-113, October 30-31, National Fertilizer Development Centre (NFDC),

*Pakistan soils and their role in crop production: Final Report,* 1983-1990. National

Mediterranean type characteristics: A review. *Journal of Plant Nutrition,* Vol. 27, pp.

mustard by plant analysis and soil testing. *Communications in Soil Science and Plant* 

calcareous soils of Pakistan. III. Boron nutrition of sorghum. *Communications in Soil* 

Pakistan: A serious constraint to productivity and grain quality. In: *Salinity and water stress: Improving crop efficiency*, 213-219, M. Ashraf; M. Ozturk and H. R. Athar

Rashid and K. S. Memon (managing authors), E. Bashir & R. Bental (Eds.), National

	- http://www.dawn.com/wps/wcm/connect/dawn-content-library/dawn/inpaper-magazine/economic-and-business/nutrient-deficiency-in-rice-crop-489 [15 May 2010].

Takkar, P.N. & Nayyar, V. K. (1984). Integrated approach to combat micronutrie nt

Tariq, A.; Gill, M. A.; Rahmatullah & Sabir, M. (2004). Mineral nutrition of fruit trees. In:

Tsadilas, C. D.; Yassoglou, C. S.; Cosmas, C. S. & Kallianou, C. H. (1994). The availability of

Tsadilas, C. D.; D. Dimoyiannis, & V. Samaras. (1997). Methods of assessing boron

Turan, M.;. Ketterings, Q. M.; Gunes, A.; Ataoglu, N.; Esring, A.; Bilgili, A. V. & Huang, Y.

US Borax Inc. (1996). The boron bonus canola. US Borax Inc., Valencia, CA. No. 280412/1–

Vaughan, B. & J. Howe. (1994). Evaluation of boron chelates in extracting soil boron. *Communications in Soil Science and Plant Analysis.* Vol. 25, pp. 1071-1084. Wang, K., &. Chen, Q. (2003). Effect of Zn, B, and Mo application on growth and quality of

Warington, K. (1923). The effect of boric acid and borax on the broad bean and certain other

Warncke, D. D. (2005). Ameliorating internal black spot in cranberry bean seed with boron application. *Communications in Soil Science and Plant Analysis.* Vol. 36, pp. 775-781. Wear, J. I. & Patterson, R. M. (1962). Effect of soil pH and texture on the availability of

Welch, R. M. (1995). Micronutrient nutrition of plants. *Critical Reviews in Plant Sciences.* Vol.

Welch, R. M.; Allaway, W. H.; House W. A. & Kubota, J. (1991). Geographic distribution of

Wild, A. & Mazaheri, A. (1979). Prediction of the leaching rate of boric acid under field

Wong, M. (2003). Monitor crops closely for signs of low boron. *Farming Ahead.* No. 135.

water-soluble boron in the soil. *Soil Science Society of America Proceedings.* Vol. 26,

trace element problems. In: *Micronutrients in agriculture,* 31-57, J. J. Mortvedt (Ed.).

http://www.borax.com/agriculture/files/cotton.pdf [May 15 2010]. US Borax Inc. (2009). Functions of boron in plant nutrition [Online] Available: http://www.borax.com/agriculture/files/an203.pdf [August 15 2010]. Vasil, I. K. (1987). Physiology and culture of pollen. In: *Pollen cytology and development*, 127-

174, K. L. Giles & J. Prakash (Eds.), Orlando, FL, U.S.A.

alfalfa. *Soils and Fertilizers. Beijing.* Vol. 3, pp. 24-28.

plants. *Annals of Botany.* Vol. 37, pp. 629–672.

*conditions*, 28-33. Agriculture Research Institute Sariab, Quetta, Pakistan. Tisdale, S. L.; Nelson, W. L.; Beaton, J. D. & Havlin. J. L. (1993). *Soil fertility and fertilizers*. 5th

ed. Macmillan Publishing Company, New York, NY, U.S. A.

properties. *Plant Soil* . Vol. 162, pp. 211– 217.

*Science and Plant Analysis.* Vol. 28, pp. 973-987.

Plant Soil Sci. Vol. 60, pp. 427 – 436.

US Borax Inc. (2002). B in cotton [Online] Available:

Delhi, India.

96.

pp. 344-346.

14, pp. 49-82.

March 2003.

2nd edition, Madison, WI, U.S.A.

conditions. *Geoderma.* Vol. 22, pp. 127–36.

deficiency. In: *Proceedings of FAI Annual Seminar*, PS, Vol. 111, No. 2, pp. 1-16. New

*Proceedings Plant-nutrition management for horticultural crops under water stress* 

soil boron fractions to olive trees and barley and their relationships to soil

availability to kiwifruit plants growing on high boron soils. *Communications in Soil* 

M. (2010). Boron fertilization of Mediterranean aridisols improves lucerne (*Medicago sativa* L.) yields and quality. *Acta Agriculturae Scandinavica,* Section B -


Sillanpaa, M. & Vlek, P. L. G. (1985). Micronutrients and the agroecology of tropical and

Sims, J. R. & Bingham, F. T. (1967). Retention of boron by layer silicates, sesquioxides and

Sims, J. R. & Bingham, F. T. (1968a). Retention of boron by layer silicates, sesquioxides and

Sims, J. R. & Bingham, F. T. (1968b). Retention of boron by layer silicates, sesquioxides and

Singh, A. L. (1994). Micronutrient nutrition and crop productivity in groundnut. In: *Plant* 

Singh, M. V. (2001). Evaluation of micronutrient stocks in different agroecological zones of

Sinha, R. B.; Sakal, R.; Singh, A. P.; Bhogal, N. S. (1991). Response of some field crops to

Smithson, J. B. & Heathcote, R. G. (1976). A new recommendation for the application of

Sotiropoulos, T. (1997). Boron toxicity of kiwifruit orchards in northern Greece. *Acta* 

Soylu, S. & Topal, A. (2004). Yield and yield attributes of durum wheat genotypes as

Spiers, G. A.; Evans, L. J.; Mcgeorge, S. W.; Moak, H. W. & Su, C. (1990). Boron analysis of

Spouncer, L. R.; Nable, R. O. & Cartwright, B. (1992). A procedure for the determination of

Tahir, M.; Hussain, F.; Kausar, M. A. & Bhatti, A. S. (1990). Differential uptake and growth

Takkar, P. N.; Chibba, I. M. & Mehta, S. K. (1989). Twenty years of coordinated research on

*Communications in Soil Science and Plant Analysis.* Vol. 23, pp. 441–453. Stangoulis, J. C.; Reid, R. J.; Brown, P. H. & Graham, R. D. (2001). Kinetic analysis of boron

transport in Chara. *Planta.* Vol. 213, pp. 142–146.

*Science Society of America Proceedings.* Vol. 32, pp. 369-373.

Botanical Publishers, Bikaner, India.

*Agri Newsletter*. Vol. 18, pp. 59-63.

*Horticulture.* Vol. 44, pp. 243-247.

*Sciences.* Vol. 27, pp. 367-373.

India. *Fertilizer News.* Vol. 42, pp. 25-42.

Netherlands.

pp. 728-732.

pp. 364-369.

pp. 342-345.

1077-1106.

1661.

India.

mediterranean regions. In: Micronutrients in tropical food crop production, 151- 167, P. L. G. Vlek, (Ed.), Martinus Nijhoff and W. Junk Publishers, Dordrecht,

soil materials: I. Layer silicates. *Soil Science Society of America Proceedings.* Vol. 31,

soils materials: II. Sesquioxides. *Soil Science Society of America Proceedings.* Vol. 32,

soil materials: III. Iron- and aluminum-coated layer silicates and soil materials. *Soil* 

*productivity under environmental stress,* 67-72, K. Singh & S. S. Purohit (Eds.), Agro

boron application in calcareous soils. *Journal of Indian Society of Soil Science.* Vol. 39,

boronated superphosphate to cotton in northeastern Beune Plateau States. *Samarau* 

affected by boron application in boron-deficient calcareous soils: an evaluation of major Turkish genotypes for boron efficiency. *Journal of Plant Nutrition,*Vol. 27, pp.

soil solutions and plant digests using a photodiode-array equipped ICP spectrometer. *Communications in Soil Science and Plant Analysis.* Vol. 21, pp. 1645–

soluble boron in soils ranging widely in boron concentrations sodicity and pH.

response to micronutrients in various rice cultivars. *Pakistan Journal of Agricultural* 

micronutrient in soils and plants. *Bull. 314*. *Indian Institute of Soil Science*, Bhopal,


http://www.borax.com/agriculture/files/cotton.pdf [May 15 2010].


**Part 2** 

**General** 


**Part 2** 

114 Crop Plant

Wooding, F. J. (1985). Interior Alaska crops respond to boron applications. *Agroborealis*. Vol.

Woodruff, J. R.; Moore, F. W. & Musen, H. L. (1987). Potassium, boron, nitrogen and lime

Wu, M. (1986). A study on boron deficiency in soybean. *Soybean Science.* Vol. 5, pp. 167-174. Yang, Y. H. & Gu, H. J. (2004). Effects of boron on aluminum toxicity on seedlings of two

Zhang, J. H.; Zhang, D. J. Jiang, Z. L. & Liu, C. X. (1986). A study on boron nutrition and

Zhao, D. & Oosterhuis, D. M. (2003). Cotton growth and physiological responses to boron

Zia, M. H.; Ahmad R.; Khaliq, I.; Ahmad, A. & Irshad, M. (2006). Micronutrients status and

Zia, M. S. (1993). Fertilizer use efficiency project and soil fertility. *Agricultural Research Project-II (ARP-II) Annual Report.* 1992-93. NARC, Islamabad, Pakistan.

soybean cultivars. *Water Air & Soil Pollution.* Vol. 154, pp. 239-248.

deficiency. *Journal of Plant Nutrition.*Vol. 26, pp. 855-867.

effects on corn yield and ear leaf nutrient concentrations. *Agronomy Journal.* Vol. 79,

application of boron to groundnut. *Journal of Soil Science. (China)*. Vol. 17, pp. 173-

management in orchards soils: applied aspects. *Soil & Environment.* Vol. 25, pp. 6-

17, pp. 47-49.

pp. 520-524.

176.

16.

**General** 

**6** 

*Brazil* 

José Henrique Cattanio

*Federal University of Pará - UFPA* 

**Leaves Material Decomposition from** 

**Leguminous Trees in an Enriched Fallow** 

In Amazon human activities such as slashing and burning converted large areas of primary forest to intermittently used agricultural land. Thus, the fallow vegetation plays an important role to maintain or restore soil productivity. In the systems with the soil are poor in nutrients and carbon (C) and farmers do not have much subsidies to buy fertilizer, the efficiency with which nutrient in plant residues is used depends on the amount and quality of the organic matter, the rate at which they are mineralized and thus on the time when they are made available relative to crop requirements. It is important to find contrasting litter quality, and mix this organic material with the possibility to alter the pattern of Nitrogen-release and the efficiency of Nitrogen (N) utilization from the residue by a soil microbial biomass and crop

Soil organic matter (SOM) represents a major proportion of the organic carbon within the terrestrial biosphere and plays an important role in soil fertility (Powlson et al. 2001). An accumulation of organic matter is not only beneficial to soil functions related to agriculture, favouring growth of biomass, promoting and facilitating carbonation processes, reducing erosion and favouring pedogenesis, and developing organic matter-rich horizons recovering degraded or contaminated soils, but also represents a sequestration of C from atmospheric CO2 (Macías and Arbestain 2010). In contrast, management practices (e.g. slash and burn system in Amazon region) leading to a decline in SOM content release CO2, the major greenhouse gas (Powlson et al. 2001). SOM also has a range of other environmental functions such as water retention and the regulation of trace greenhouse gases between land

Fallow trees affect the soil by their litter deposition in terms of quantity and quality, root activity and changes in microclimate brought about by the leaf canopy. However, the intensification of land use, by small farmers in the tropic, has drastically reduced the fallow period with a decline in soil productivity and environmental quality, resulting in a progressive deterioration of natural resources. Therefore, the soil quality has to be restored in shorter time. Decline in soil productivity and environmental quality and progressive deterioration of natural resources in the tropics have led to a search for new methods to sustain crop production via more efficient nutrient cycling. In Northeastern of Pará (Brazil) the Amazon region was occupied by an intensive colonization process over the last century until today.

system where other sources of mineral N such as fertilizer are limited or excluded.

**1. Introduction** 

surface and the atmosphere.

## **Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow**

José Henrique Cattanio *Federal University of Pará - UFPA Brazil* 

#### **1. Introduction**

In Amazon human activities such as slashing and burning converted large areas of primary forest to intermittently used agricultural land. Thus, the fallow vegetation plays an important role to maintain or restore soil productivity. In the systems with the soil are poor in nutrients and carbon (C) and farmers do not have much subsidies to buy fertilizer, the efficiency with which nutrient in plant residues is used depends on the amount and quality of the organic matter, the rate at which they are mineralized and thus on the time when they are made available relative to crop requirements. It is important to find contrasting litter quality, and mix this organic material with the possibility to alter the pattern of Nitrogen-release and the efficiency of Nitrogen (N) utilization from the residue by a soil microbial biomass and crop system where other sources of mineral N such as fertilizer are limited or excluded.

Soil organic matter (SOM) represents a major proportion of the organic carbon within the terrestrial biosphere and plays an important role in soil fertility (Powlson et al. 2001). An accumulation of organic matter is not only beneficial to soil functions related to agriculture, favouring growth of biomass, promoting and facilitating carbonation processes, reducing erosion and favouring pedogenesis, and developing organic matter-rich horizons recovering degraded or contaminated soils, but also represents a sequestration of C from atmospheric CO2 (Macías and Arbestain 2010). In contrast, management practices (e.g. slash and burn system in Amazon region) leading to a decline in SOM content release CO2, the major greenhouse gas (Powlson et al. 2001). SOM also has a range of other environmental functions such as water retention and the regulation of trace greenhouse gases between land surface and the atmosphere.

Fallow trees affect the soil by their litter deposition in terms of quantity and quality, root activity and changes in microclimate brought about by the leaf canopy. However, the intensification of land use, by small farmers in the tropic, has drastically reduced the fallow period with a decline in soil productivity and environmental quality, resulting in a progressive deterioration of natural resources. Therefore, the soil quality has to be restored in shorter time.

Decline in soil productivity and environmental quality and progressive deterioration of natural resources in the tropics have led to a search for new methods to sustain crop production via more efficient nutrient cycling. In Northeastern of Pará (Brazil) the Amazon region was occupied by an intensive colonization process over the last century until today.

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 119

Different organic materials decompose at contrasting rates because they are decomposed differentially by catabolic enzymes produced by saprophytic organisms (Linkins et al. 1984). Furthermore, decomposition rates are affected by nutrient and lignin content of litter (Moorhead et al. 1996), because the initial lignin-to-N and the lignin + polyphenolto-N ratios are correlated well with the N-mineralization or N accumulation (Constantinides and Fownes 1994; Janssen 1996; Handayanto et al. 1997). In the other hand, the SOM in the organo-mineral fraction of some soils is relatively protected against mineralization and therefore does not immediately influence crop yields in the shortterm

The decomposition of organic matter is the key process in soil-plant N cycle (Barraclough 1997), principally governing the availability of this nutrient to crop growth. The chains of processes are very complex, as ammonia NH4+, the initial product of N mineralization, can be consumed by several processes (plant uptake, nitrification, immobilization and volatilization). Heterotrophic bacteria involved in the mineralization-immobilization turnover reactions between inorganic and organic pools of N compete more effectively for

The efficiency with which N in plant residues is used depends on the rate at which they are mineralized and thus on the time when they are made available relative to crop requirements. The present work aims to determine whether, with contrasting legume litter quality in terms of N mineralization, by mixing this organic material of different quality, it will be possible to alter the pattern of N release and the efficiency of utilization of N from

In this way four different legume species (*Acacia mangium* Willd., *A. angustissima* Kuntze, *Sclerolobium paniculatum* Vogel and *Inga edulis* Mart.) each used in enrich the fallow were compared with natural fallow vegetation, which is a mixture of different species, and poor

 Within this experiment the following points are essential: a) the impact of enriched legume material in soil N mineralization; b) the use of mineralization with the use of contrasting litter quality; c) the influence of organic material quality on soil microbial

After identifying the contrasting species, two laboratory decomposition experiments with two different techniques will be used to elaborate the effect that mixing these organic materials of different quality has on the pattern of N release and the efficiency of utilization

The first decomposition experiment was made using soil incorporated legume leaf material from the two contrasting species, and their mixture, with 15N at natural abundance and fertilized with enriched 15N-urea fertilizer (conventional isotope dilution technique). In parallel, one experiment with the same species and mixture of legume with previously enriched 15N and fertilized with 14N-urea was carried out (pre-labeling plant

biomass in terms of N mineralization, immobilization and consumption.


(Mapfumo et al. 2007).

NH4+ than for nitrate NO3

**2. State of the art** 

material).

soil without added organic material.

the residue by a soil microbial biomass and catch crop.

of N from the residue by a soil microbial biomass.

The region was to be utilized, initially by clearing the forests for timber and later by the use of the land for subsistence agriculture, based on slash and burn agriculture. In the context of a bilateral German-Brazilian project ("Secondary Forests and Fallow Vegetation in Eastern Amazon – Function and Management", SHIFT project) slash and mulch system are being recommended to realize fire-free land clearing by cutting and chopping the fallow vegetation and leaving mulch layer on site (Denich et al. 2005). In addition, the fallow vegetation is enriched with fast-growing legume trees to support the mulching effect by increasing biomass production and nitrogen input during the fallow period.

The purpose of this technique is to maintain soil organic matter and assure a slow and continuous release of nutrients, improve moisture retention, reduces excessive soil heating and runoff, reduce soil erosion, and prevent weed seed germination (Denich et al. 2005). Hence, mulching may improve flexibility in planting date to cope with unreliable rain due to conserved soil moisture.

The rate of decomposition and the amount of N-mineralization from organic material determines the short-term benefits of tree residues for plant nutrition (Jensen et al. 1995). If burning is to be abandoned, then the synchronization of nutrient release from organic material and nutrient uptake by plants (Addiscott et al. 1991, Myers et al. 1994), accompanying the competition between plant and microorganisms for nutrients (Cattanio et al. 2008), will be the core problem in applied tropical soil biology research. Yield losses in field trials of the SHIFT project have shown that yield losses in mulch practices are evident as compared to burned treatments (Kato et al 1999). The same authors showed that yield losses were eliminated with fertilizer application, indicating nutrient competition with decomposers and/or an unfavorable nutrient release pattern as compared to crop demand was a problem.

The structure and decomposability of leaf litter varies to a large degree, thus affecting the rate of nutrient cycling and the nutrient availability in soil (Priha and Smolander 1997). Soluble C, which includes metabolic and storage C, is of high quality and is primarily responsible for promoting microbial growth and activity. Large amounts of soluble C but little soluble N and P in decomposing plant residues induce net immobilization (Cattanio et al. 2008). The challenge resides in sustaining crop production while maintaining soil fertility through supply and efficient management of organic residues (Isaac et al. 2000). Biederbeck et al. (1994) suggested that it may be possible to manipulate the timing and quality of litter input through appropriate management of mixed stands to improve the synchrony of nutrient release with crop requirements.

Some works in litter manipulated with mixtures was done by Meentemeyer (1978), Melillo et al. (1982), Anderson et al. (1983), Gallardo and Merino (1993), Vitousek et al. (1994), Hobbie (2000), Lonrez et al (2000). But studies with mixtures in soil litter decomposition were scarce (Franagan and van Cleve 1983). Blair et al. (1990) found in litterbags containing mixed residues that there were significantly greater initial releases of N and lower subsequent N immobilization than predicted, and they suggest that it resulted from differences in the decomposer community originated from the mixtures of varied litter resource quality. In the same way, Handayanto et al. (1997), Kuo and Sainju (1998), Zimmer (2002) and Cattanio et al. (2008) showed that soil N-mineralization rate of prunings could be manipulated by mixing different quality materials.

Different organic materials decompose at contrasting rates because they are decomposed differentially by catabolic enzymes produced by saprophytic organisms (Linkins et al. 1984). Furthermore, decomposition rates are affected by nutrient and lignin content of litter (Moorhead et al. 1996), because the initial lignin-to-N and the lignin + polyphenolto-N ratios are correlated well with the N-mineralization or N accumulation (Constantinides and Fownes 1994; Janssen 1996; Handayanto et al. 1997). In the other hand, the SOM in the organo-mineral fraction of some soils is relatively protected against mineralization and therefore does not immediately influence crop yields in the shortterm (Mapfumo et al. 2007).

The decomposition of organic matter is the key process in soil-plant N cycle (Barraclough 1997), principally governing the availability of this nutrient to crop growth. The chains of processes are very complex, as ammonia NH4+, the initial product of N mineralization, can be consumed by several processes (plant uptake, nitrification, immobilization and volatilization). Heterotrophic bacteria involved in the mineralization-immobilization turnover reactions between inorganic and organic pools of N compete more effectively for NH4+ than for nitrate NO3- (Jansson 1958; Jenkinson et al. 1985; Schimel et al. 1989).

The efficiency with which N in plant residues is used depends on the rate at which they are mineralized and thus on the time when they are made available relative to crop requirements. The present work aims to determine whether, with contrasting legume litter quality in terms of N mineralization, by mixing this organic material of different quality, it will be possible to alter the pattern of N release and the efficiency of utilization of N from the residue by a soil microbial biomass and catch crop.

### **2. State of the art**

118 Crop Plant

The region was to be utilized, initially by clearing the forests for timber and later by the use of the land for subsistence agriculture, based on slash and burn agriculture. In the context of a bilateral German-Brazilian project ("Secondary Forests and Fallow Vegetation in Eastern Amazon – Function and Management", SHIFT project) slash and mulch system are being recommended to realize fire-free land clearing by cutting and chopping the fallow vegetation and leaving mulch layer on site (Denich et al. 2005). In addition, the fallow vegetation is enriched with fast-growing legume trees to support the mulching effect by

The purpose of this technique is to maintain soil organic matter and assure a slow and continuous release of nutrients, improve moisture retention, reduces excessive soil heating and runoff, reduce soil erosion, and prevent weed seed germination (Denich et al. 2005). Hence, mulching may improve flexibility in planting date to cope with unreliable rain due

The rate of decomposition and the amount of N-mineralization from organic material determines the short-term benefits of tree residues for plant nutrition (Jensen et al. 1995). If burning is to be abandoned, then the synchronization of nutrient release from organic material and nutrient uptake by plants (Addiscott et al. 1991, Myers et al. 1994), accompanying the competition between plant and microorganisms for nutrients (Cattanio et al. 2008), will be the core problem in applied tropical soil biology research. Yield losses in field trials of the SHIFT project have shown that yield losses in mulch practices are evident as compared to burned treatments (Kato et al 1999). The same authors showed that yield losses were eliminated with fertilizer application, indicating nutrient competition with decomposers and/or an unfavorable nutrient release pattern as compared to crop demand

The structure and decomposability of leaf litter varies to a large degree, thus affecting the rate of nutrient cycling and the nutrient availability in soil (Priha and Smolander 1997). Soluble C, which includes metabolic and storage C, is of high quality and is primarily responsible for promoting microbial growth and activity. Large amounts of soluble C but little soluble N and P in decomposing plant residues induce net immobilization (Cattanio et al. 2008). The challenge resides in sustaining crop production while maintaining soil fertility through supply and efficient management of organic residues (Isaac et al. 2000). Biederbeck et al. (1994) suggested that it may be possible to manipulate the timing and quality of litter input through appropriate management of mixed stands to improve the synchrony of

Some works in litter manipulated with mixtures was done by Meentemeyer (1978), Melillo et al. (1982), Anderson et al. (1983), Gallardo and Merino (1993), Vitousek et al. (1994), Hobbie (2000), Lonrez et al (2000). But studies with mixtures in soil litter decomposition were scarce (Franagan and van Cleve 1983). Blair et al. (1990) found in litterbags containing mixed residues that there were significantly greater initial releases of N and lower subsequent N immobilization than predicted, and they suggest that it resulted from differences in the decomposer community originated from the mixtures of varied litter resource quality. In the same way, Handayanto et al. (1997), Kuo and Sainju (1998), Zimmer (2002) and Cattanio et al. (2008) showed that soil N-mineralization rate of prunings could be

increasing biomass production and nitrogen input during the fallow period.

to conserved soil moisture.

was a problem.

nutrient release with crop requirements.

manipulated by mixing different quality materials.

In this way four different legume species (*Acacia mangium* Willd., *A. angustissima* Kuntze, *Sclerolobium paniculatum* Vogel and *Inga edulis* Mart.) each used in enrich the fallow were compared with natural fallow vegetation, which is a mixture of different species, and poor soil without added organic material.

 Within this experiment the following points are essential: a) the impact of enriched legume material in soil N mineralization; b) the use of mineralization with the use of contrasting litter quality; c) the influence of organic material quality on soil microbial biomass in terms of N mineralization, immobilization and consumption.

After identifying the contrasting species, two laboratory decomposition experiments with two different techniques will be used to elaborate the effect that mixing these organic materials of different quality has on the pattern of N release and the efficiency of utilization of N from the residue by a soil microbial biomass.

The first decomposition experiment was made using soil incorporated legume leaf material from the two contrasting species, and their mixture, with 15N at natural abundance and fertilized with enriched 15N-urea fertilizer (conventional isotope dilution technique). In parallel, one experiment with the same species and mixture of legume with previously enriched 15N and fertilized with 14N-urea was carried out (pre-labeling plant material).

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 121

content at the end of laboratory incubation period. The initial nutrient content in the organic amendment (wood + leaf material) for different treatments and soils (without added organic matter) is shown in Table 1. In this experiment *I. edulis* and *A. mangium* had an initially higher N content than the other single-legume treatments. But the leaf + wood material added to soil from *S. paniculatum* and *A. mangium* showed an initially higher P concentration. For these two important chemical elements, the mixture of two legumes

Species OM N C P Lignin Cellulose Phenol

*A. mangium* **14.15**(0.14) **0.11**(0.001) **6.84**(0.07) **0.004 2.61**(0.03) **5.95**(0.06) **1.50**(0.02) m\*e¥ **14.33**(0.14) **0.14**(0.003) **6.90**(0.07) **0.007 3.16**(0.04) **5.76**(0.05) **1.03**(0.01) *I. edulis* **14.11**(0.04) **0.17**(0.001) **6.77**(0.02) **0.009 3.59**(0.01) **5.42**(0.01) **0.52**(0.00) *S. paniculatum* **14.16**(0.11) **0.11**(0.001) **6.83**(0.05) **0.004 3.15**(0.03) **3.72**(0.03) **0.82**(0.01) p\*a§ **14.30**(0.14) **0.15**(0.002) **6.97**(0.07) **0.007 3.10**(0.03) **4.53**(0.04) **1.26**(0.01) *A. angustissima* **14.26**(0.22) **0.18**(0.005) **7.01**(0.11) **0.008 3.00**(0.05) **5.27**(0.08) **1.67**(0.03) Fallow **9.50**(0.06) **0.08**(0.001) **4.68**(0.03) **0.004 2.51**(0.02) **3.91**(0.03) **1.05**(0.01)

Table 1. Organic matter added to Amazon sandy soil and the nutrient (selected) content and material quality for different treatments in a laboratory incubation experiment. The number

After 128 days of incubation the significant difference between legume species and mixture were found only for the *S. paniculatum* treatment, which showed the lowest total-N concentration founded in soil. This same species showed a higher initial C-to-N ratio and lignin concentration. In this way, legume species and fallow treatment had a significant positive correlation (r2 = 0.59, P < 0.01) with the total-N losses and initial C-to-N ratio. The total-N losses1 decrease in the following order: *S. paniculatum* > Fallow vegetation > *A. mangium* = *A. angustissima* > soil > *I. edulis*2. The mixture of *S. paniculatum* and *A. angustissima* leaf material showed a significant inhibitory effect in the total remaining nitrogen, and only 10.8% of total-N was lost. However, the other mixture did not exhibit

Soil-nitrogen mineralization without added organic matter more than doubled the amount of mineral N in the soil founded during the incubation period (Figure 1). This control treatment showed a rapid initial increase after it became steadier. In contrast, the amended soils showed immobilization-consumption of the native soil-N, reversing into a release of N after about a month for the legume-amended soil, particularly if m\*e mixture or *S. paniculatum* were present. The fallow amended soil was slower in immobilizing soil-N, but

1Losses mean the differences of total N concentration in soil between begin and end of incubation period.

2">" symbol represents statistical differences at P < 0.05, and "=" no statistical difference.

mg g-1 soil

showed an intermediate concentration in comparison to the single species.

Soil **1.27 16.73 0.001**

represents Mean (standard deviation), with n = 21.

statistical differences in relation to the single species.

**3.2 Nitrogen mineralization and immobilization** 

continued to immobilize over the entire incubation period.

¥ In this study, m\*e represents a mixture of *A. mangium* and *I. edulis* (50:50 w/w).

**§** In this study, p\*a represents a mixture of *S. paniculatum* and *A. angustissima* (50:50 w/w).

 Within this experiment the following points are essential: a) the use of contrasting litter quality may improve N-mineralization in terms of the rate at which they are mineralized; b) the quantification the real amount of N stored in the soil microbial biomass; c) the quantification of N-mineralization and immobilization through the use of labeling techniques. These isotope dilution techniques have the objective to quantify the proportion of N that comes from fertilizer or organic matter and is immobilized by soil microbial biomass.

To assess further whether N recovery by rice could be accurately predicted from relationships between pruning-material quality and N mineralization-immobilization, a greenhouse pot experiment was conducted in which the two isotope techniques were used with the same contrasting materials and their mixture, and 15N uptake by rice was measured.

 Within this experiment the following points are essential: a) whether the use of contrasting litter quality may improve N-mineralization rate and thus the time when they make N available to the crop; b) the quantification of N competition between rice and soil microbial biomass; c) the quantification of N-mineralization and immobilization through the use of two techniques of isotope dilution which allow the quantification of the proportion of N coming from fertilizer or organic matter immobilized by soil microbial biomass and used by rice.

One of the hypothesis of this work is that with the elimination of the burning of biomass and the addition of organic matter as mulch, nitrogen immobilization in mulch by microorganisms will be increased and lead to a decrease in the quality of SOM. To confirm this hypothesis a field experiment whit litterbags from different legume treatment was conducted.

 Within this experiment the following points are essential: a) quantifying mulch decomposition during the field incubation on the litter; b) assessing nitrogen and carbon mineralization from mulch system; c) mulch nutrient retention during the field incubation; d) predict of N mineralization.

In generally this study is intended to answer the following questions:


#### **3. Identify contrasting leguminous decomposition on leaf and wood material incorporated in the soil**

#### **3.1 Biochemical characteristics of the plant material**

In the Brazilian Amazon soil the decomposition of contrasting amended material with regard to the measure quality characteristics show in all treatments a decrease in total-N

 Within this experiment the following points are essential: a) the use of contrasting litter quality may improve N-mineralization in terms of the rate at which they are mineralized; b) the quantification the real amount of N stored in the soil microbial biomass; c) the quantification of N-mineralization and immobilization through the use of labeling techniques. These isotope dilution techniques have the objective to quantify the proportion of N that comes from fertilizer or organic matter and is immobilized by

To assess further whether N recovery by rice could be accurately predicted from relationships between pruning-material quality and N mineralization-immobilization, a greenhouse pot experiment was conducted in which the two isotope techniques were used with the same contrasting materials and their mixture, and 15N uptake by rice was

 Within this experiment the following points are essential: a) whether the use of contrasting litter quality may improve N-mineralization rate and thus the time when they make N available to the crop; b) the quantification of N competition between rice and soil microbial biomass; c) the quantification of N-mineralization and immobilization through the use of two techniques of isotope dilution which allow the quantification of the proportion of N coming from fertilizer or organic matter

One of the hypothesis of this work is that with the elimination of the burning of biomass and the addition of organic matter as mulch, nitrogen immobilization in mulch by microorganisms will be increased and lead to a decrease in the quality of SOM. To confirm this hypothesis a field experiment whit litterbags from different legume treatment was

 Within this experiment the following points are essential: a) quantifying mulch decomposition during the field incubation on the litter; b) assessing nitrogen and carbon mineralization from mulch system; c) mulch nutrient retention during the field

Can we regulating N release through the use of mixing residues from legume tree

Can we fulfill the crop demands with organic matter fertilization (mulching) using

What happens with the use of mulch system in terms of N-mineralization and

**3. Identify contrasting leguminous decomposition on leaf and wood material** 

In the Brazilian Amazon soil the decomposition of contrasting amended material with regard to the measure quality characteristics show in all treatments a decrease in total-N

Is N immobilization affected by legume tree material and therefore by mixtures?

immobilized by soil microbial biomass and used by rice.

In generally this study is intended to answer the following questions:

material with different patterns of N mineralization?

**3.1 Biochemical characteristics of the plant material** 

incubation; d) predict of N mineralization.

materials from enriched fallows?

immobilization?

**incorporated in the soil** 

soil microbial biomass.

measured.

conducted.

content at the end of laboratory incubation period. The initial nutrient content in the organic amendment (wood + leaf material) for different treatments and soils (without added organic matter) is shown in Table 1. In this experiment *I. edulis* and *A. mangium* had an initially higher N content than the other single-legume treatments. But the leaf + wood material added to soil from *S. paniculatum* and *A. mangium* showed an initially higher P concentration. For these two important chemical elements, the mixture of two legumes showed an intermediate concentration in comparison to the single species.


¥ In this study, m\*e represents a mixture of *A. mangium* and *I. edulis* (50:50 w/w).

**§** In this study, p\*a represents a mixture of *S. paniculatum* and *A. angustissima* (50:50 w/w).

Table 1. Organic matter added to Amazon sandy soil and the nutrient (selected) content and material quality for different treatments in a laboratory incubation experiment. The number represents Mean (standard deviation), with n = 21.

After 128 days of incubation the significant difference between legume species and mixture were found only for the *S. paniculatum* treatment, which showed the lowest total-N concentration founded in soil. This same species showed a higher initial C-to-N ratio and lignin concentration. In this way, legume species and fallow treatment had a significant positive correlation (r2 = 0.59, P < 0.01) with the total-N losses and initial C-to-N ratio. The total-N losses1 decrease in the following order: *S. paniculatum* > Fallow vegetation > *A. mangium* = *A. angustissima* > soil > *I. edulis*2. The mixture of *S. paniculatum* and *A. angustissima* leaf material showed a significant inhibitory effect in the total remaining nitrogen, and only 10.8% of total-N was lost. However, the other mixture did not exhibit statistical differences in relation to the single species.

#### **3.2 Nitrogen mineralization and immobilization**

Soil-nitrogen mineralization without added organic matter more than doubled the amount of mineral N in the soil founded during the incubation period (Figure 1). This control treatment showed a rapid initial increase after it became steadier. In contrast, the amended soils showed immobilization-consumption of the native soil-N, reversing into a release of N after about a month for the legume-amended soil, particularly if m\*e mixture or *S. paniculatum* were present. The fallow amended soil was slower in immobilizing soil-N, but continued to immobilize over the entire incubation period.

 1Losses mean the differences of total N concentration in soil between begin and end of incubation period.

<sup>2&</sup>quot;>" symbol represents statistical differences at P < 0.05, and "=" no statistical difference.

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 123

Total mineral nitrogen in leguminous-amended soil was significantly different with time (P < 0.01) for the two species used in the mixtures, and for the interaction species and time (P < 0.01). The higher differences were found in the beginning of the incubation period and the final inorganic N content in legume-amended soil ranged from 2.0 to 6.1 mg N kg-1 soil compared with 1.6 mg N kg-1 soil in the fallow-amended soil and 20.7 mg N kg-1 soil in the control soil. Thus, at the end of incubation period, mineral nitrogen decreased in the following order: Soil as control > *S. paniculatum* > *I. edulis* > *A. mangium* > *A. angustissima* >

The decomposition patterns of the mixture and total N-mineralization did not reflect the simple mean of the decomposition patterns of single-species organic matter. The m\*e mixture showed a higher increase in total N mineral at the end of the experiment, and that of the single species was comparatively lower in the same period. In the same way a\*p mixture did not amount to the arithmetic mean of the N-mineralization in of the two single

In these sandy soils from Amazon, initial nitrification was significantly higher for soil without added organic matter, *I. edulis* and Fallow treatment. The *I. edulis* treatment

concentration at the end of the experiment; the other treatments showed a NO3--N

m\*e


**p\*a**

**N-NH4 + N-NO3 -**

**Incubation period (days)**

Fig. 2. Concentration of mineral N as influenced by incorporated wood and leaves from different legumes species in comparison with fallow vegetation (Fallow) and soil with initial

organic matter (Soil) as control. Bars represent standard error of the mean.


incubation (Figure 2). Only *I. edulis* and the m\*e mixture showed a small increase in NO3


*I. edulis*


*A. angustissima*


**Soil**



presented a significantly higher consumption of NO3-

consumption and/or denitrification.

*A. mangium*


*S. paniculatum*


**Fallow**


natural fallow.

specie

**µg N g-1 soil**

Fig. 1. Soil inorganic N concentration as affected by incorporated wood and leaves from different legumes species in comparison with fallow vegetation (Fallow) and soil with initial organic matter. In all graphics the m\*e and p\*a symbol correspond to *A. mangium* and *I. edulis* mixture (50:50 w/w), and *A. angustissima* and *S. paniculatum* mixture (50:50 w/w), respectively. Bars represent standard error of the mean.


*A. mangium I. edulis* m\*e Fallow Soil


*A. angustissima S. paniculatum* a\*p Fallow Soil

Fig. 1. Soil inorganic N concentration as affected by incorporated wood and leaves from different legumes species in comparison with fallow vegetation (Fallow) and soil with initial organic matter. In all graphics the m\*e and p\*a symbol correspond to *A. mangium* and *I. edulis* mixture (50:50 w/w), and *A. angustissima* and *S. paniculatum* mixture (50:50 w/w),

0

0

respectively. Bars represent standard error of the mean.

5

10

15

**N-min (µg N g-1 soil)**

20

25

5

10

15

**N-min (µg N g-1 soil)**

20

25

Total mineral nitrogen in leguminous-amended soil was significantly different with time (P < 0.01) for the two species used in the mixtures, and for the interaction species and time (P < 0.01). The higher differences were found in the beginning of the incubation period and the final inorganic N content in legume-amended soil ranged from 2.0 to 6.1 mg N kg-1 soil compared with 1.6 mg N kg-1 soil in the fallow-amended soil and 20.7 mg N kg-1 soil in the control soil. Thus, at the end of incubation period, mineral nitrogen decreased in the following order: Soil as control > *S. paniculatum* > *I. edulis* > *A. mangium* > *A. angustissima* > natural fallow.

The decomposition patterns of the mixture and total N-mineralization did not reflect the simple mean of the decomposition patterns of single-species organic matter. The m\*e mixture showed a higher increase in total N mineral at the end of the experiment, and that of the single species was comparatively lower in the same period. In the same way a\*p mixture did not amount to the arithmetic mean of the N-mineralization in of the two single specie

In these sandy soils from Amazon, initial nitrification was significantly higher for soil without added organic matter, *I. edulis* and Fallow treatment. The *I. edulis* treatment presented a significantly higher consumption of NO3 --N after approximately 4 days of incubation (Figure 2). Only *I. edulis* and the m\*e mixture showed a small increase in NO3 --N concentration at the end of the experiment; the other treatments showed a NO3 --N consumption and/or denitrification.

#### **Incubation period (days)**

Fig. 2. Concentration of mineral N as influenced by incorporated wood and leaves from different legumes species in comparison with fallow vegetation (Fallow) and soil with initial organic matter (Soil) as control. Bars represent standard error of the mean.

Treatment

(P < 0.05).

different treatments.

14.4, 10.0 and 9.8 µg N- NO3-

symbol showed statistical difference (P < 0.05).

Fallow vegetation3.

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 125

*A. mangium* 1.00a -0.21a -0.15a -0.18a

m\*e 0.73b -0.16c -0.05c 2.76a

*I. edulis* 2.25a -0.95b -1.26b -0.68b

*S. paniculatum* 1.37b -0.87c -0.46c 2.79a

p\*a 0.42ab 0.23b 0.34b 1.63a

*A. angustissima* 1.40a -0.16b -0.25b 0.15b

Fallow 2.34a -1.64b -1.50b -1.56b

LSD≤0.05 0.67 0.68 0.80 1.16

Least significant difference (LSD) to compare treatments in the same sampling time.

particularly so for the fallow and *I. edulis* i.e., -3.96 and -2.93 µg NH4

*paniculatum* showed a higher increase when mixed with *A. angustissima*.

<sup>Φ</sup> Apparent net N-ammonification (*a*) was calculated by subtracting soil NH4+-N in the time *n* (*tn*) from soil NH4+-N in the initial time (*t0*). ¥ Values within a line that are followed by different letters are significantly different with the Tukey test

Table 2. Apparent net N-ammonification, a (µg NH4+-N g-1 soil)Φ as a function of time for

The m\*e mixture, *S. paniculatum* and p\*a mixture showed a significantly higher increase in ammonification at the end of incubation period with 2.03, 1.42 and 1.21 µg NH4+-N g-1 soil, respectively (Table 2). All other treatments showed a decrease in N-ammonification,

The mixture of these two legume species was significantly better mineralizable than the single species. This interaction was not observed with the other mixture, where *S* 

The treatments with amendment showed a significant difference (P < 0.05) in N nitrification during the incubation period (Table 3). Fallow vegetation, *I. edulis* and *A. angustissima* showed a significantly higher initial nitrification, with m\*e, p\*a mixture and *S. paniculatum* showing intermediate values, respectively (Table 3). However, a significantly higher nitrification decrease was found in the fallow, *I. edulis* and *A. angustissima*, with variations of

initial N nitrification. At the end of the incubation period the mineralization decreased in the following order: *S*. *paniculatum* ≥ *A. mangium* ≥ m\*e = p\*a > *I. edulis* ≥ *A. angustissima* >

3 "≥" and "=" symbols means that the treatments did not show statistical difference (P > 0.05), and ">"

g-1 soil, respectively. *A. mangium* showed a significant lower

Incubation period (days)¥

0 16 64 128

+-N g-1 soil, respectively.

Calculation of net ammonification (*a*, Figure 3) and net nitrification (*n*), in order to have a comparative parameters between treatments, was performed by subtracting soil NH4 +-N in the time x (tx) from soil NH4+-N in the initial time (t0) and NO3- -N in the time x (tx) from soil NO3- -N in the initial time (t0), respectively. Apparent microorganism NH4+-Nimmobilization (*i*) was calculated by subtracting soil NH4+-N microbial biomass in the organic amendment treatment from NH4+-N microbial biomass in the control treatment (soil without organic amendment) (Jensen 1997), using the fumigation-extraction method. NO3 -- N consumption (*c*) was calculated by subtracting soil NO3- -N in the control treatment from soil NO3 --N in the organic amendment treatment. This is based on the assumption that the mineralization and losses of indigenous soil N were similar in control and residue-treated soils (Jensen 1997).

Fig. 3. Flows of soil nitrogen indicated by: (a) apparent N-ammonification, (r) apparent Nremineralization, (n) apparent N-nitrification, (i) apparent NH4 +-N immobilization, (c) apparent NO3--N consumption, (d) denitrification. Adapted from Stark and Schimel (2001).

Apparent ammonification (a) was significantly different (P < 0.05) during the incubation period for the majority of treatments (Table 2). Only A. mangium organic material did not show a significant difference in N-ammonification between sampling times during the incubation period. The fallow and I. edulis treatments showed a significantly higher initial N-ammonification. *A. angustissima*, S. paniculatum and A. mangium presented an intermediate initial N-ammonification; with the mixture of two legume species presenting a significantly lower initial nitrogen ammonification (Table 2).

Calculation of net ammonification (*a*, Figure 3) and net nitrification (*n*), in order to have a comparative parameters between treatments, was performed by subtracting soil NH4

+-N in the initial time (t0) and NO3-

soil NO3--N in the organic amendment treatment. This is based on the assumption that the mineralization and losses of indigenous soil N were similar in control and residue-treated

N-NH4

a n

Fig. 3. Flows of soil nitrogen indicated by: (a) apparent N-ammonification, (r) apparent Nremineralization, (n) apparent N-nitrification, (i) apparent NH4+-N immobilization, (c)

Apparent ammonification (a) was significantly different (P < 0.05) during the incubation period for the majority of treatments (Table 2). Only A. mangium organic material did not show a significant difference in N-ammonification between sampling times during the incubation period. The fallow and I. edulis treatments showed a significantly higher initial N-ammonification. *A. angustissima*, S. paniculatum and A. mangium presented an intermediate initial N-ammonification; with the mixture of two legume species presenting a

significantly lower initial nitrogen ammonification (Table 2).


r


the time x (tx) from soil NH4

soils (Jensen 1997).

soil

apparent NO3

organic-N

N consumption (*c*) was calculated by subtracting soil NO3-

NO3-

+-N in




<sup>+</sup> N-NO3

**microbial biomass-N**

i c


d


<sup>Φ</sup> Apparent net N-ammonification (*a*) was calculated by subtracting soil NH4+-N in the time *n* (*tn*) from

soil NH4+-N in the initial time (*t0*). ¥ Values within a line that are followed by different letters are significantly different with the Tukey test (P < 0.05).

Least significant difference (LSD) to compare treatments in the same sampling time.

Table 2. Apparent net N-ammonification, a (µg NH4+-N g-1 soil)Φ as a function of time for different treatments.

The m\*e mixture, *S. paniculatum* and p\*a mixture showed a significantly higher increase in ammonification at the end of incubation period with 2.03, 1.42 and 1.21 µg NH4+-N g-1 soil, respectively (Table 2). All other treatments showed a decrease in N-ammonification, particularly so for the fallow and *I. edulis* i.e., -3.96 and -2.93 µg NH4 +-N g-1 soil, respectively. The mixture of these two legume species was significantly better mineralizable than the single species. This interaction was not observed with the other mixture, where *S paniculatum* showed a higher increase when mixed with *A. angustissima*.

The treatments with amendment showed a significant difference (P < 0.05) in N nitrification during the incubation period (Table 3). Fallow vegetation, *I. edulis* and *A. angustissima* showed a significantly higher initial nitrification, with m\*e, p\*a mixture and *S. paniculatum* showing intermediate values, respectively (Table 3). However, a significantly higher nitrification decrease was found in the fallow, *I. edulis* and *A. angustissima*, with variations of 14.4, 10.0 and 9.8 µg N- NO3 g-1 soil, respectively. *A. mangium* showed a significant lower initial N nitrification. At the end of the incubation period the mineralization decreased in the following order: *S*. *paniculatum* ≥ *A. mangium* ≥ m\*e = p\*a > *I. edulis* ≥ *A. angustissima* > Fallow vegetation3.

<sup>3 &</sup>quot;≥" and "=" symbols means that the treatments did not show statistical difference (P > 0.05), and ">" symbol showed statistical difference (P < 0.05).

NO3-

fallow treatment.

treatment (soil without organic amendment).

incubation period and treatment.

Treatment

(P < 0.05).

soil NO3-

(P < 0.05).

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 127

*A. mangium* 0.76b 0.38b 1.90b 5.22a m\*e 6.46a 2.61b 2.59b 2.28b *I. edulis* 4.15a 1.24c 3.34ab 2.59b *S. paniculatum* 1.16ab 0.31b 0.85ab 2.34a p\*a 5.92a 1.02c 3.28b 3.44b *A. angustissima* 0.95ab 0.19c 0.90b 1.40a Fallow 1.72ab 2.44a 0.65b 1.33ab LSD≤0.05 0.96 0.21 0.25 1.26 <sup>Φ</sup> Apparent microorganism NH4+-N immobilization (*i*) was calculated by subtracting soil NH4+-N microbial biomass in the organic amendment treatment from NH4+-N microbial biomass in the control

¥ Values within a line that are followed by different letters are significantly different with the Tukey test

Table 4. Apparent net N-microbial immobilization, i (µg NH4+-N g-1 soil)Φ as a function of

*A. mangium* 7.20d 14.13c 19.33b 21.24a m\*e 4.81c 13.54b 18.97a 19.41a *I. edulis* 1.84c 8.29b 17.57a 18.99a *S. paniculatum* 7.05d 13.90c 19.14b 20.79a p\*a 6.27d 14.04c 19.15b 20.87a *A. angustissima* 3.06d 14.02c 19.21b 21.27a Fallow 0.61d 9.35c 17.90b 20.91a LSD≤0.05 0.93 1.06 0.25 0.37

0 16 64 128


Least significant difference (LSD) to compare treatments in the same sampling time.

Treatment Incubation period (days)¥

<sup>Φ</sup> Apparent net consumption (*c*) was calculated by subtracting soil NO3-

function of incubation period and treatment.


Table 5. Apparent Net microbial consumption of NO3--N, c (µg NO3--N g-1 soil)Φ as a

Least significant difference (LSD) to compare treatments in the same sampling time.

¥ Values within a line that are followed by different letters are significantly different with the Tukey test


Incubation period (days)¥

0 16 64 128


<sup>Φ</sup> Net N nitrification (*n*) was calculated by subtracting soil NO3- -N in the time *n* (*tn*) from soil NO3- -N in the initial time (*t0*).

¥ Values within a line that are followed by different letters are significantly different with the Tukey test (P < 0.05).

Least significant difference (LSD) to compare treatments in the same sampling time.

Table 3. Net N nitrification, *n* (µg NO3--N g-1 soil) <sup>Φ</sup> as a function of time for different treatment.

Differences between the treatments were observed for NH4+-N immobilization at the beginning of the experiment (P < 0.05) (Table 4). Control treatment4 decreased NH4+-N immobilization from 1.69 µg NH4+-N g-1 soil in the time 0 to 0.27 µg NH4 +-N g-1 soil at the end of the incubation period. The m\*e, p\*a mixture and *I. edulis* treatments presented a significantly higher initial NH4+-N immobilization (Table 4), and the treatments showed a lower initial C-to-N ratio in comparison to *A. mangium* and *S. paniculatum* (Table 1). *A. angustissima* also showed a low initial C-to-N ratio (38.9), which did not explain the low initial N immobilization, but this treatment had a higher initial phenol content (Table 1), which contributed to the low initial N-immobilization (Mafongoya et al. 1998).

Only the m\*e and p\*a mixtures as well as the *I. edulis* significantly decreased (P < 0.05) in net NH4+-N immobilization at the end of incubation period. *A. mangium* showed a smaller initial net NH4+-N immobilization and a significantly higher (P < 0.01) increase in the net NH4 +-N immobilization at the end of incubation period. Between 64 and 128 days of the incubation period, NH4 +-N immobilization was not significantly different (P > 0.05) for the m\*e, *I. edulis*, p\*a, *A. angustissima*, and Fallow treatments. However, for this same period, *A. mangium* and *S. paniculatum* experienced a stronger increase in NH4+-N immobilization, with 3.32 and 1.18 µg NH4+-N g-1 soil, respectively.

As with the net immobilization, the microbial consumption of NO3- -N was noticeably different (P < 0.01) between treatments at the beginning of the incubation period (Table 5). The strong differences in net consumption at the beginning of the experiment may reflect the differences in organic material quality. The *A. mangium*, *S. paniculatum* and p\*a mixture treatment showed a significantly higher initial NO3 --N consumption, and fallow vegetation showed the lowest value at the same time. All treatments showed a significant elevation in

<sup>4</sup>Control treatment mean soil without added organic material.

A. mangium 0.90a -0.76b -0.74b -0.62b m\*e 3.29a -2.56b -2.78b -1.18b *I. edulis* 6.26a -0.28b -4.35c -3.73c *S. paniculatum* 1.05a -0.68b -0.71b -0.32b p\*a 1.83a -1.60b -1.50b -1.18b *A. angustissima* 5.04a -4.79b -4.77b -4.79b Fallow 7.49a -2.57b -5.91c -6.89c LSD≤0.05 0.93 1.32 1.28 1.06

¥ Values within a line that are followed by different letters are significantly different with the Tukey test

Differences between the treatments were observed for NH4+-N immobilization at the beginning of the experiment (P < 0.05) (Table 4). Control treatment4 decreased NH4+-N

end of the incubation period. The m\*e, p\*a mixture and *I. edulis* treatments presented a significantly higher initial NH4+-N immobilization (Table 4), and the treatments showed a lower initial C-to-N ratio in comparison to *A. mangium* and *S. paniculatum* (Table 1). *A. angustissima* also showed a low initial C-to-N ratio (38.9), which did not explain the low initial N immobilization, but this treatment had a higher initial phenol content (Table 1),

Only the m\*e and p\*a mixtures as well as the *I. edulis* significantly decreased (P < 0.05) in net NH4+-N immobilization at the end of incubation period. *A. mangium* showed a smaller initial net NH4+-N immobilization and a significantly higher (P < 0.01) increase in the net NH4+-N immobilization at the end of incubation period. Between 64 and 128 days of the incubation period, NH4+-N immobilization was not significantly different (P > 0.05) for the m\*e, *I. edulis*, p\*a, *A. angustissima*, and Fallow treatments. However, for this same period, *A. mangium* and *S. paniculatum* experienced a stronger increase in NH4+-N immobilization, with

different (P < 0.01) between treatments at the beginning of the incubation period (Table 5). The strong differences in net consumption at the beginning of the experiment may reflect the differences in organic material quality. The *A. mangium*, *S. paniculatum* and p\*a mixture treatment showed a significantly higher initial NO3--N consumption, and fallow vegetation showed the lowest value at the same time. All treatments showed a significant elevation in

0 16 64 128



+-N g-1 soil at the


Treatment Incubation period (days)¥

Least significant difference (LSD) to compare treatments in the same sampling time.

immobilization from 1.69 µg NH4+-N g-1 soil in the time 0 to 0.27 µg NH4

which contributed to the low initial N-immobilization (Mafongoya et al. 1998).

+-N g-1 soil, respectively.

4Control treatment mean soil without added organic material.

As with the net immobilization, the microbial consumption of NO3-

Table 3. Net N nitrification, *n* (µg NO3--N g-1 soil) <sup>Φ</sup> as a function of time for different

<sup>Φ</sup> Net N nitrification (*n*) was calculated by subtracting soil NO3-

the initial time (*t0*).

3.32 and 1.18 µg NH4

(P < 0.05).

treatment.


NO3- -N consumption during the incubation period. But *I. edulis* showed a significantly lower NO3--N consumption (P < 0.01) at the beginning of the experiment following by fallow treatment.

<sup>Φ</sup> Apparent microorganism NH4+-N immobilization (*i*) was calculated by subtracting soil NH4+-N microbial biomass in the organic amendment treatment from NH4+-N microbial biomass in the control treatment (soil without organic amendment).

¥ Values within a line that are followed by different letters are significantly different with the Tukey test (P < 0.05).

Least significant difference (LSD) to compare treatments in the same sampling time.

Table 4. Apparent net N-microbial immobilization, i (µg NH4+-N g-1 soil)Φ as a function of incubation period and treatment.


<sup>Φ</sup> Apparent net consumption (*c*) was calculated by subtracting soil NO3- -N in the control treatment from soil NO3- -N in the organic amendment treatment in the same incubation period.

¥ Values within a line that are followed by different letters are significantly different with the Tukey test (P < 0.05).

Least significant difference (LSD) to compare treatments in the same sampling time.

Table 5. Apparent Net microbial consumption of NO3--N, c (µg NO3--N g-1 soil)Φ as a function of incubation period and treatment.

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 129

All treatments showed a strong initial increase in CO2-C production, which resulted in a high slope of the accumulative CO2-C curve (Figure 4). It was conform the increase in the

Soil N mineral decreased with increasing CO2 production and microbial biomass C. In contrast, N immobilizations by microorganism increased with an increase in CO2 production due to added organic C, and with a decrease of the N concentrations. This suggests that N dynamics in the legumes amended treatments were highly correlated with

> 01234567 **N-min (µg N-min g-1 soil)**

Fig. 5. The relationship between mineral N (N-min) and the extractable organic C resulting from treatment with different legumes, mixture and fallow at the end of incubation period (128 days). The symbols correspond to Fallow (○), *A. angustissima* (×), *A. mangium* (), p\*a

Since N immobilization is limited by substrate availability in a broad range of ecosystems and soil types, soil organic C concentration would strongly influence N immobilization (Barrett and Burke 2000). The correlation between nitrogen mineralization and carbon mineralization suggests that rapid stabilization of nitrogen is facilitated by an active microbial community and the availability of a readily mineralizable organic substrate. Critical levels of C and plant nutrients, which limit the enzyme activities of microbial decomposition, were found to be important for determining nutrient release (Seneviratne 2000). Soil microbial biomass has been defined as an indicator of soil fertility, long before changes is soil organic matter occur (Powlson and Brookes 1987). Microbial nitrogen NH4+ immobilization and NO3- consumption appears to be an important variable that needs to be

taken into account in studying organic matter decomposition and N mineralization.

mixture (), *I. edulis* (), m\*e mixture (▲), *S. paniculatum* (△) treatment.

y = 6.698x + 47.326

r 2 = 0.588

initial microbial C, NO3- consumption and the high decrease in the initial N mineral.

organic C dynamics (Figure 5).

**Organic C (µg C g-1 soil)**

At the end of the experiment, a significantly lower apparent NO3 - -N consumption was found in *I. edulis* and m\*e mixture (P < 0.05), in comparison the other treatment (Table 10). Only for these two treatments, NO3 - -N consumption remained constant after 64 days of incubation. The significantly highest consumption of 20.3 µg NO3 - -N g-1 soil was found in the Fallow treatment.

In this study the data indicate that the decline of mineral-N was strongly influenced by immobilization and consumption of mineral N by the microflora. Microbial consumption of NO3--N was of a greater magnitude than NH4 +-N immobilization, thus indicate that the decreases observed in net N-mineralization were due to increasing microbial consumption of N. However, immobilization into soil organic matter (SOM) may be attributed to the apparent net N-mineral loss (Bending et al. 1998).

As was shown by Verchot et al. (2001), the results demonstrate that the patterns of Nmineralization are dependent upon differences between microbial production and consumption. These processes are reliant on organic matter quality. Small changes in mineralization, nitrification, immobilization and consumption may possibly have a large impact on soil N availability for the crop system.

The principal loss of C from SOM is through respiration during decomposition (Woomer et al. 1994). All legume-amended soils showed significantly higher cumulative CO2 production than fallow-amended soil and the control treatment (soil) (Figure 4). The cumulative CO2 production was significantly higher for mixtures in comparison with the individual species.

Fig. 4. Accumulative C-CO2 (µg C g-1 soil day-1) production during the incubation period for treatments with different organic matter soil added in comparison with soil without added organic matter as control (Soil).

*I. edulis* and m\*e mixture (P < 0.05), in comparison the other treatment (Table 10). Only for these


In this study the data indicate that the decline of mineral-N was strongly influenced by immobilization and consumption of mineral N by the microflora. Microbial consumption of NO3--N was of a greater magnitude than NH4+-N immobilization, thus indicate that the decreases observed in net N-mineralization were due to increasing microbial consumption of N. However, immobilization into soil organic matter (SOM) may be attributed to the

As was shown by Verchot et al. (2001), the results demonstrate that the patterns of Nmineralization are dependent upon differences between microbial production and consumption. These processes are reliant on organic matter quality. Small changes in mineralization, nitrification, immobilization and consumption may possibly have a large

The principal loss of C from SOM is through respiration during decomposition (Woomer et al. 1994). All legume-amended soils showed significantly higher cumulative CO2 production than fallow-amended soil and the control treatment (soil) (Figure 4). The cumulative CO2 production was significantly higher for mixtures in comparison with the individual species.

> 0 20 40 60 80 100 120 140 **Incubation period (days)**

Fig. 4. Accumulative C-CO2 (µg C g-1 soil day-1) production during the incubation period for treatments with different organic matter soil added in comparison with soil without added





*A. mangium* m\*e *I. edulis S. paniculatum*

*A. angustissima* Fallow Soil

p\*a

At the end of the experiment, a significantly lower apparent NO3

two treatments, NO3

0

organic matter as control (Soil).

50

100

150

200

**CO2-C µg g-1 soil day-1**

250

300

350


significantly highest consumption of 20.3 µg NO3

apparent net N-mineral loss (Bending et al. 1998).

impact on soil N availability for the crop system.

All treatments showed a strong initial increase in CO2-C production, which resulted in a high slope of the accumulative CO2-C curve (Figure 4). It was conform the increase in the initial microbial C, NO3- consumption and the high decrease in the initial N mineral.

Soil N mineral decreased with increasing CO2 production and microbial biomass C. In contrast, N immobilizations by microorganism increased with an increase in CO2 production due to added organic C, and with a decrease of the N concentrations. This suggests that N dynamics in the legumes amended treatments were highly correlated with organic C dynamics (Figure 5).

Fig. 5. The relationship between mineral N (N-min) and the extractable organic C resulting from treatment with different legumes, mixture and fallow at the end of incubation period (128 days). The symbols correspond to Fallow (○), *A. angustissima* (×), *A. mangium* (), p\*a mixture (), *I. edulis* (), m\*e mixture (▲), *S. paniculatum* (△) treatment.

Since N immobilization is limited by substrate availability in a broad range of ecosystems and soil types, soil organic C concentration would strongly influence N immobilization (Barrett and Burke 2000). The correlation between nitrogen mineralization and carbon mineralization suggests that rapid stabilization of nitrogen is facilitated by an active microbial community and the availability of a readily mineralizable organic substrate. Critical levels of C and plant nutrients, which limit the enzyme activities of microbial decomposition, were found to be important for determining nutrient release (Seneviratne 2000). Soil microbial biomass has been defined as an indicator of soil fertility, long before changes is soil organic matter occur (Powlson and Brookes 1987). Microbial nitrogen NH4 + immobilization and NO3- consumption appears to be an important variable that needs to be taken into account in studying organic matter decomposition and N mineralization.

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 131

Mafagoya et al. (1998) identified three types of soluble constituents that result in interactions between organic materials: a) compounds that contain available carbon as a substrate, b) compounds that contain readily available N, and c) soluble polyphenols, which can complex with proteins, rendering them resistant to microbial assault. The results showed in this experiment confirm that the carbon availably and soluble polyphenols may be the important

**3.3 Decomposition of contrasting leguminous leaf material and gross N dynamics in** 

A decomposition study of 15N-labeled plant material (*S. paniculatum, I. edulis*, and mixture, p\*e) with contrasting litter quality was conducted to assess the rates of mineralization and immobilization, using rice as an indicator plant (Table 7). N-urea fertilizer (3.92 N mg pot-1 with N at natural abundance) and 15N-labeled leguminous organic material from *S. paniculatum* and *I. edulis* with 2.02% N and 0.392 atom % 15N, and 1.93% N and 0.390 atom % 15N, respectively, were used to find the amount of mineral-N coming from organic matter decomposition, and the extent of competition between microorganisms, soil + organic

*S. paniculatum*<sup>15</sup> 50.62(0.10) 70.66(0.002) 1.62(0.001) 0.08(0.002) 6.36(0.012)

p\*e15 50.62(0.11) 70.48(0.001) 1.64(0.001) 0.08(0.001) 6.40(0.009)

*I. edulis*15 50.52(0.09) 71.00(0.014) 1.69(0.006) 0.08(0.002) 6.61(0.010)

<sup>Φ</sup> species name with *14* had leaf with nitrogen at natural abundance and was fertilized with 5.34 atom % 15N. Species name with *15* had leaf with enriched 15N (both species at 0.39 % 15N in leaf material) and was

Total rice biomass and total N in rice was affected by N from the legume leaves in the amended soil during the incubation period (Figure 6). Initial rice dry matter was not statistically different at the time of transplantation. Although the N uptake by rice in the p\*e mixture treatment did not differ statistically from that in the *I. edulis* treatment at the end of incubation period, it was significantly higher than the *S. paniculatum* and the control treatments (Figure 6). All treatments showed a significantly (P < 0.01) higher total N content in comparison with the control. But the concentration of nitrogen in seedlings of rice were significantly when are used the interaction between two contrasting leguminous material as

Table 7. Amount of organic matter (OM) mixed with soil in a plastic pot and the total 14N and 15N-excess added with leaf and fertilizer in greenhouse experiment. The numbers

Control 50.68(0.13) 0.08(0.001)

Soil Leaf N N-fertilizer 15N

g mg g-1 soil µg g-1 soil

parameters that result in interaction between contrasting organic material.

**soil using rice as an indicator plant** 

matter fixation, and rice absorption.

fertilized with fertilizer at natural abundance.

represent mean(standard deviation), n = 12.

Treatment

a source of nitrogen.

The best predictor of N-mineralization was phenol + lignin when all treatments were included in the analyses (Table 6), followed by phenol + lignin-to-N ratios and lignin. Initial N + P-to-phenol ratios were highly correlated with cumulative N-mineralization for leguminous species and mixtures (Table 6).


<sup>Φ</sup> All treatment means legumes, mixture and fallow treatments.

<sup>Ω</sup> mixture means mixture of two legume species.

<sup>δ</sup> Single legume means legumes species without mixtures treatments. The \* is P ≤ 0.05, \*\* is P ≤ 0.01

Table 6. Correlation coefficients relating the cumulative amount of N-mineralization to initial chemical properties in the treatments.

Soil-incorporated plant lignins degrade to polyphenol, which, with the other plant and microbial polyphenol, become the main constituents of recalcitrant N, containing humic polymers (Haynes 1986). Lignin intertwines also with the cell wall, physically protecting cellulose and other cell wall constituents from degradation (Chesson 1997).

Polyphenols include a range of compounds differing in size, solubility, and reactivity. Also, polyphenol can serve as a carbon substrate for decomposers (Mafongoya et al. 1998) but in general they inhibit the growth or function of decomposers and the other organisms (Swift et al. 1981, Zucker 1982). Defense compounds, including phenolics and terpenoids can also influence rates of litter decomposition, by means of direct inhibitor effects on saprophytic organisms (Palm and Sanchez 1991). Condensed tannins, also known as proanthocyanidins, are the polyphenol most noted for their effects on decomposition and nutrient dynamics. This results from their reactions with proteins and nitrogen (Myers et al. 1994; Mafongoya et al. 1998).

This experiment confirms that the resource quality and mixture of contrasting resource quality affect the N-mineralization and -immobilization processes during decomposition. In agreement with Palm (1995), the following factors must be considered when choosing parameters to describe plant quality: a) the processes of decomposition and N release are controlled by different parameters; b) the critical parameters will depend on the time frame of the crop need; and c) the importance of certain parameters change with the type and the mixture of the plant material. The ultimate aim is to identify robust parameters that predict decomposition and nutrient release.

The decomposition patterns and N-mineralization of the mixture were not the arithmetic mean of the decomposition patterns of the component organic material. In this case, there are interactions between components principally in terms of the rate of decomposition and N release, which was demonstrated in N-mineralization (net and total mineralization), microbial biomass C and extractable organic C.

The best predictor of N-mineralization was phenol + lignin when all treatments were included in the analyses (Table 6), followed by phenol + lignin-to-N ratios and lignin. Initial N + P-to-phenol ratios were highly correlated with cumulative N-mineralization for

Phenol + Lignin Phenol N + P

N N Phenol

Lignin

<sup>Ω</sup> -0.813\* -0.798\* -0.768\* -0.830\* 0.950\*\* Single legumes δ -0.788\* -0.871\* -0.868\* -0.803\* 0.916\*

<sup>δ</sup> Single legume means legumes species without mixtures treatments. The \* is P ≤ 0.05, \*\* is P ≤ 0.01 Table 6. Correlation coefficients relating the cumulative amount of N-mineralization to

cellulose and other cell wall constituents from degradation (Chesson 1997).

Soil-incorporated plant lignins degrade to polyphenol, which, with the other plant and microbial polyphenol, become the main constituents of recalcitrant N, containing humic polymers (Haynes 1986). Lignin intertwines also with the cell wall, physically protecting

Polyphenols include a range of compounds differing in size, solubility, and reactivity. Also, polyphenol can serve as a carbon substrate for decomposers (Mafongoya et al. 1998) but in general they inhibit the growth or function of decomposers and the other organisms (Swift et al. 1981, Zucker 1982). Defense compounds, including phenolics and terpenoids can also influence rates of litter decomposition, by means of direct inhibitor effects on saprophytic organisms (Palm and Sanchez 1991). Condensed tannins, also known as proanthocyanidins, are the polyphenol most noted for their effects on decomposition and nutrient dynamics. This results from their reactions with proteins and nitrogen (Myers et al. 1994; Mafongoya et

This experiment confirms that the resource quality and mixture of contrasting resource quality affect the N-mineralization and -immobilization processes during decomposition. In agreement with Palm (1995), the following factors must be considered when choosing parameters to describe plant quality: a) the processes of decomposition and N release are controlled by different parameters; b) the critical parameters will depend on the time frame of the crop need; and c) the importance of certain parameters change with the type and the mixture of the plant material. The ultimate aim is to identify robust parameters that predict

The decomposition patterns and N-mineralization of the mixture were not the arithmetic mean of the decomposition patterns of the component organic material. In this case, there are interactions between components principally in terms of the rate of decomposition and N release, which was demonstrated in N-mineralization (net and total mineralization),

leguminous species and mixtures (Table 6).

<sup>Ω</sup> mixture means mixture of two legume species.

initial chemical properties in the treatments.

decomposition and nutrient release.

microbial biomass C and extractable organic C.

Legume and mixture

al. 1998).

Object of analyze Lignin Phenol +

All treatment Φ -0.812\* -0.826\* -0.819\*

<sup>Φ</sup> All treatment means legumes, mixture and fallow treatments.

Mafagoya et al. (1998) identified three types of soluble constituents that result in interactions between organic materials: a) compounds that contain available carbon as a substrate, b) compounds that contain readily available N, and c) soluble polyphenols, which can complex with proteins, rendering them resistant to microbial assault. The results showed in this experiment confirm that the carbon availably and soluble polyphenols may be the important parameters that result in interaction between contrasting organic material.

#### **3.3 Decomposition of contrasting leguminous leaf material and gross N dynamics in soil using rice as an indicator plant**

A decomposition study of 15N-labeled plant material (*S. paniculatum, I. edulis*, and mixture, p\*e) with contrasting litter quality was conducted to assess the rates of mineralization and immobilization, using rice as an indicator plant (Table 7). N-urea fertilizer (3.92 N mg pot-1 with N at natural abundance) and 15N-labeled leguminous organic material from *S. paniculatum* and *I. edulis* with 2.02% N and 0.392 atom % 15N, and 1.93% N and 0.390 atom % 15N, respectively, were used to find the amount of mineral-N coming from organic matter decomposition, and the extent of competition between microorganisms, soil + organic matter fixation, and rice absorption.


<sup>Φ</sup> species name with *14* had leaf with nitrogen at natural abundance and was fertilized with 5.34 atom % 15N. Species name with *15* had leaf with enriched 15N (both species at 0.39 % 15N in leaf material) and was fertilized with fertilizer at natural abundance.

Table 7. Amount of organic matter (OM) mixed with soil in a plastic pot and the total 14N and 15N-excess added with leaf and fertilizer in greenhouse experiment. The numbers represent mean(standard deviation), n = 12.

Total rice biomass and total N in rice was affected by N from the legume leaves in the amended soil during the incubation period (Figure 6). Initial rice dry matter was not statistically different at the time of transplantation. Although the N uptake by rice in the p\*e mixture treatment did not differ statistically from that in the *I. edulis* treatment at the end of incubation period, it was significantly higher than the *S. paniculatum* and the control treatments (Figure 6). All treatments showed a significantly (P < 0.01) higher total N content in comparison with the control. But the concentration of nitrogen in seedlings of rice were significantly when are used the interaction between two contrasting leguminous material as a source of nitrogen.

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 133

Fig. 7. 14N+15N and 15N recovery in rice for soil treatment with 15N-leaf material of two

matter).

different legumes species and mixture, and 14N-urea fertilizer in comparison with soil without added leaf material and fertilized with 14N-urea, for different incubation periods. The 15N is expressed as the atom % 15N excess abundance above the background (0.3663 atom %).

The N concentration in rice increased until 10 days after transplanting (Table 8) and, during this phase of rice growth, the legume treatments did not differ from the control (P > 0.05). At the end of the incubation period, *S. paniculatum* showed a significantly (P < 0.01) lower N final concentration in rice (11.6 mg N g-1 dry matter) in comparison with the control (17.4 mg N g-1 dry matter), *I. edulis* (18.0 mg N g-1 dry matter) and p\*e mixture (18.2 mg N g-1 dry

The results with leaf enriched with 15N reveal that most of the N absorbed by rice (Table 9), came from the soil, but the interaction between the two leguminous plants was provided to

more N for rice after 50 days of incubation (Figure 7).

Fig. 6. Rice dry matter (g pot-1, boxes) and total N in plant material (mg N pot-1, lines) growing in soil with leaf-14N legume material and mixture using enriched urea-15N fertilizer. Control was soil that only included urea-14N fertilizer. Bars represent standard error of the mean.

Treatment with the mixture of two legumes species showed a high recovery of N and 15N in comparison to the two legume species (Figure 7). Recovery of 15N from rice was significantly higher (P < 0.001) in the *S. paniculatum* treatment than with the *I. edulis* treatment. In contrast, the recovery of total N was higher for the *I. edulis* than for *S. paniculatum*.

0 5 10 50

0

5

10

15

**N in Rice (mg pot-1)**

20

25

Incubation period (days)

Fig. 6. Rice dry matter (g pot-1, boxes) and total N in plant material (mg N pot-1, lines) growing in soil with leaf-14N legume material and mixture using enriched urea-15N fertilizer. Control was soil that only included urea-14N fertilizer. Bars represent standard

contrast, the recovery of total N was higher for the *I. edulis* than for *S. paniculatum*.

Treatment with the mixture of two legumes species showed a high recovery of N and 15N in comparison to the two legume species (Figure 7). Recovery of 15N from rice was significantly higher (P < 0.001) in the *S. paniculatum* treatment than with the *I. edulis* treatment. In

*S. paniculatum* p\*e *I. edulis* Control

0

error of the mean.

0,5

**Rice biomass (g pot-1)**

1

1,5

Fig. 7. 14N+15N and 15N recovery in rice for soil treatment with 15N-leaf material of two different legumes species and mixture, and 14N-urea fertilizer in comparison with soil without added leaf material and fertilized with 14N-urea, for different incubation periods. The 15N is expressed as the atom % 15N excess abundance above the background (0.3663 atom %).

The N concentration in rice increased until 10 days after transplanting (Table 8) and, during this phase of rice growth, the legume treatments did not differ from the control (P > 0.05). At the end of the incubation period, *S. paniculatum* showed a significantly (P < 0.01) lower N final concentration in rice (11.6 mg N g-1 dry matter) in comparison with the control (17.4 mg N g-1 dry matter), *I. edulis* (18.0 mg N g-1 dry matter) and p\*e mixture (18.2 mg N g-1 dry matter).

The results with leaf enriched with 15N reveal that most of the N absorbed by rice (Table 9), came from the soil, but the interaction between the two leguminous plants was provided to more N for rice after 50 days of incubation (Figure 7).

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 135

In the *I. edulis* and p\*e mixture treatments, a significantly fast initial N microbial immobilization was observed, which increased toward the end of the experiment. However, at the end of the experiments with this species, a decrease in N immobilization was observed. After five days of incubation, the *S. paniculatum* treatment exhibited an approximately constant N immobilization. The immobilization of N by soil microorganisms seems to explain why most of the N absorbed by the rice comes from the ground. The experiment showed that most of the N released from the leaf material, through the process

Nitrogen derived from residues (Ndfr; Hood, 2001) of different 15N-legume leaf material was not statistically different between treatments until five days of incubation (Table 9). At ten days, a significantly higher amount of total N in rice came from the *I. edulis* (34.1%) and p\*e mixture (31.9%) than from the *S. paniculatum* (25.2%). However, at the end of the incubation period, N in rice originating from the *S. paniculatum* leaf material increased by 11.4% in comparison with a decrease of 3.4% and 2.5% for the p\*e mixture and *I. edulis*

N derived from residue (Ndfr, %)

*S. paniculatum* 8.62 21.5 25.2 36.2 p\*e 8.87 21.7 31.9 28.6 *I. edulis* 7.66 22.4 34.1 31.6 LSD 1.21NS 0.92NS 2.18\*\* 0.66\*\* N derived from residue (Ndfr, mg) *S. paniculatum* 0.08 0.42 1.17 2.99 p\*e 0.11 0.52 1.55 4.18 *I. edulis* 0.06 0.60 2.31 3.45 LSD 0.03\* 0.07\*\* 0.10\*\* 0.73\* N recovered from residue (Nrfr, %) *S. paniculatum* 0.10 0.51 1.45 3.68 p\*e 0.14 0.64 1.91 5.16 *I. edulis* 0.08 0.75 2.90 4.32 LSD 0.03\* 0.03\*\* 0.13\*\* 0.84\*

Table 9. Nitrogen derived from residue (NdfR) in % and mg, and N recovered from residue (NrfR, %), according to Hoods (2001), during the greenhouse incubation period in soil

The amount of N derived from residues increased significantly for all treatments during the incubation period (Table 9). The higher increase in N recovered from the added leaf material was found in the p\*e mixture treatment, which showed an increase of 4.07 mg N in comparison

With this higher increase, the total N recovered from added legume leaf material was significantly higher for the p\*e mixture than the other treatments (Table 9). However, for the first 10 days of the incubation period, the *I. edulis* treatment showed the higher percentage

with 3.39 and 2.91 mg N for the *I. edulis* and *S. paniculatum* treatments, respectively.

0 5 10 50

of decomposition, was immobilized by soil microorganisms (Figure 8).

treatments, respectively, reflecting the slowly biodegradability of *S. paniculatum*.

Treatment Incubation period (days)

treated with 15N-leaf legume and 14N-urea fertilizer.


LSD compares different treatment at the same sampling time; NS is not significant; \* = P < 0.05

Table 8. Nitrogen concentration in rice (mg N g-1 dry matter) for different treatment and control during the incubation time (days). Control was soil without added leaf material and fertilized with N-urea.

Nitrogen microbial immobilization estimated by the fumigation-extraction method showed a contrasting pattern between differently materials (Figure 8 A and B). Generally at the beginning of the experiment, the microbial biomass-N with the small amount of added leaf material behaved exactly opposite to the experiment with the larger amount of leaf material added. The control treatment initially showed a faster decrease in microbial biomass-N.

Fig. 8. A) Nitrogen microbial biomass (µg N-microbial biomass g-1 soil) during the greenhouse incubation period (days) for samples with leaf material, and (B) 15N microbial biomass (µg 15N-microbial biomass g-1 soil) samples with 15N enriched leaf material and fertilized with urea-14N. Soil treatment (as control) was soil without added leaf material and fertilized with urea-14N. The bars represent Standard error of the mean

*S. paniculatum* 11.2 18.0 25.3 11.6 p\*e 14.3 19.2 27.8 18.2 *I. edulis* 14.2 19.6 30.4 18.0 Control 11.6 23.4 31.7 17.4 LSD 3.17NS 1.54\* 6.40NS 6.45\*

Table 8. Nitrogen concentration in rice (mg N g-1 dry matter) for different treatment and control during the incubation time (days). Control was soil without added leaf material and

Fig. 8. A) Nitrogen microbial biomass (µg N-microbial biomass g-1 soil) during the greenhouse incubation period (days) for samples with leaf material, and (B) 15N microbial biomass (µg 15N-microbial biomass g-1 soil) samples with 15N enriched leaf material and fertilized with urea-14N. Soil treatment (as control) was soil without added leaf material and

fertilized with urea-14N. The bars represent Standard error of the mean

Nitrogen microbial immobilization estimated by the fumigation-extraction method showed a contrasting pattern between differently materials (Figure 8 A and B). Generally at the beginning of the experiment, the microbial biomass-N with the small amount of added leaf material behaved exactly opposite to the experiment with the larger amount of leaf material added. The control treatment initially showed a faster decrease in microbial biomass-N.

LSD compares different treatment at the same sampling time; NS is not significant; \*

N concentration (mg N g-1 rice dry matter) 0 5 10 50

= P < 0.05

Treatment / Incubation

fertilized with N-urea.

period (days)

In the *I. edulis* and p\*e mixture treatments, a significantly fast initial N microbial immobilization was observed, which increased toward the end of the experiment. However, at the end of the experiments with this species, a decrease in N immobilization was observed. After five days of incubation, the *S. paniculatum* treatment exhibited an approximately constant N immobilization. The immobilization of N by soil microorganisms seems to explain why most of the N absorbed by the rice comes from the ground. The experiment showed that most of the N released from the leaf material, through the process of decomposition, was immobilized by soil microorganisms (Figure 8).

Nitrogen derived from residues (Ndfr; Hood, 2001) of different 15N-legume leaf material was not statistically different between treatments until five days of incubation (Table 9). At ten days, a significantly higher amount of total N in rice came from the *I. edulis* (34.1%) and p\*e mixture (31.9%) than from the *S. paniculatum* (25.2%). However, at the end of the incubation period, N in rice originating from the *S. paniculatum* leaf material increased by 11.4% in comparison with a decrease of 3.4% and 2.5% for the p\*e mixture and *I. edulis* treatments, respectively, reflecting the slowly biodegradability of *S. paniculatum*.


Table 9. Nitrogen derived from residue (NdfR) in % and mg, and N recovered from residue (NrfR, %), according to Hoods (2001), during the greenhouse incubation period in soil treated with 15N-leaf legume and 14N-urea fertilizer.

The amount of N derived from residues increased significantly for all treatments during the incubation period (Table 9). The higher increase in N recovered from the added leaf material was found in the p\*e mixture treatment, which showed an increase of 4.07 mg N in comparison with 3.39 and 2.91 mg N for the *I. edulis* and *S. paniculatum* treatments, respectively.

With this higher increase, the total N recovered from added legume leaf material was significantly higher for the p\*e mixture than the other treatments (Table 9). However, for the first 10 days of the incubation period, the *I. edulis* treatment showed the higher percentage

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 137

higher microbial biomass-15N concentration for the *S. paniculatum* treatment indicated intensive microbial activity leading to decomposition of recalcitrant leaf material in this treatment. However, after fertilizer had been added, the 15N-excess immobilization in the soil increased to 155% of the initial concentration in the *I. edulis* treatment and 105% in the p\*e mixture treatment, whereas the *S. paniculatum* treatment showed a decrease of 92% in the microbial biomass- 15N concentration. At the end of the experiment, all legume treatments showed a significant decrease in the microbial biomass-15N, and only the *S. paniculatum* showed a significantly higher microbial 15N-concentration in comparison with

The decrease in soil mineral nitrogen over time was attributable not only to rice plant uptake, but also to considerable microbial immobilization. The use of a mixture of two contrasting litter qualities (called p\*e mixture) improved the nitrogen recovered by rice, which was derived from urea-fertilizer by 35.1% and 41.3% of total fertilized N in comparison with *S. paniculatum* and *I. edulis* treatments, respectively. In the same way, the rice recovery of N derived from the legume material in the p\*e mixture improved by 73.3% compared with the *S. paniculatum* treatment, but was 0.9% lower than that of the *I. edulis*

Moreover, the use of a larger amount of leaf material resulted in a higher increase in N immobilization and mineral-N in the soil, depending on the quality of the leaf material. The total cumulative nitrogen mineralization increased 90.9%, 10.3%, and 18.8% for the *S. paniculatum*, p\*e mixture and *I. edulis* treatments, respectively. In the same way, the increases in cumulative microbial biomass-N during the incubation period were 109%, 190% and

The p\*e mixture treatment showed an intermediate cumulative soil microbial- 15N immobilization, higher cumulative rice biomass and total N, and higher recovery of 15N from urea fertilizer. This indicates that the interaction of two different leguminous species increases the nitrogen absorption by rice through the increase in mineral-N and the decrease

Decline in soil productivity and environmental quality and progressive deterioration of natural resources in the tropics have led to a search for new methods to sustain crop production via more efficient nutrient cycling. In agricultural ecosystems in the tropics with limited access to fertilizers, plant residues are often used to meet the N requirements of annual food crops (Constantinides and Fownes 1994). The added organic materials are potentially important sources of N, C and P in crop production, especially for resourcespoor farmers on tropical agricultural land. In order to successfully manage organic materials, the release and uptake of N by crops must be identified (Hood 2002). But the predictions of net N-mineralization are in many cases unreliable because net Nmineralization is affected by N immobilization and remineralization and losses (Stark and

The slash and burn system destroys the above-ground biomass of the fallow vegetation including the litter by burning, which causes loss of nutrients through volatilization and leaching of free nutrients in ash by rainfall. The losses of nitrogen by volatilization and leaching can reach 95-98% (Mackensen et al. 1996). Cerri et al. (1991) observed a reduction of 25% in soil carbon content two years after a plot was cleared, burned and a satisfactorily

control (Table 10).

treatment.

344%, respectively.

Schimel 2001).

of gross microbial-N immobilization.

(3.65%) of total N recovered from the added leaf material in comparison with the p\*e mixture (2.55%) and *S. paniculatum* treatments (1.96%).

Lower rates of 15N-recovery could be due to mineralization-immobilization turnover (Thönnissen et al. 2000). The 15N-release from the legume residue into the soil inorganic pool could be exchanged for 14N in microbial biomass, which could lead to a lower 15N-recovery. On the other hand, lower rates for 15N recovery than for total N may result partly from an overestimation of apparent total N recovery and partly from the importance of soil conditions, in terms of C-quality, during the rapid degradation of 15N-labeled material.

A high (14N+15N)-microbial biomass was observed at the beginning of the greenhouse experiment for the p\*e mixture (Table 10) in comparison to the other treatments. All treatments showed an increase in N absorption from microorganisms during the first five days. This increase was very high for the *I. edulis* treatment, which showed an increase of 500% of the initial concentration, followed by the p\*e mixture (27.0%), *S. paniculatum* (10.0%) and control (8.8%).


LSD compares treatment at the same sampling time; \* = P < 0.05; \*\* = P < 0.01; \*\*\* = P < 0.001.

Table 10. (14N+15N)-microbial biomass (µg 14N+15N g-1 soil) and 15N-microbial biomass (µg 15N excess g-1 soil) in soil amended with 14N-legume leaf material and enriched 15N-urea fertilizer with rice as an indicator plant. Control was soil without added leaf material and fertilized with N-urea. The 15N excess is expressed as the atom % 15N excess abundance above the background (0.3663 atom %).

The high N-microbial biomass level remained high in the *I. edulis* and p\*e mixture treatments for the first 10 days of the greenhouse experiment (Table 10). However, at the end of the incubation period, these treatments showed a significantly higher decrease in the microbial biomass-(14N+15N), whereas the *S. paniculatum* treatment displayed a significantly high increase in the concentration of 14N+15N in the microbial biomass.

In all treatments, a significantly higher microbial biomass-15N was observed during the first 10 days of the incubation period in comparison with the control (Table 10). The initially

(3.65%) of total N recovered from the added leaf material in comparison with the p\*e

Lower rates of 15N-recovery could be due to mineralization-immobilization turnover (Thönnissen et al. 2000). The 15N-release from the legume residue into the soil inorganic pool could be exchanged for 14N in microbial biomass, which could lead to a lower 15N-recovery. On the other hand, lower rates for 15N recovery than for total N may result partly from an overestimation of apparent total N recovery and partly from the importance of soil conditions, in terms of C-quality, during the rapid degradation of 15N-labeled material.

A high (14N+15N)-microbial biomass was observed at the beginning of the greenhouse experiment for the p\*e mixture (Table 10) in comparison to the other treatments. All treatments showed an increase in N absorption from microorganisms during the first five days. This increase was very high for the *I. edulis* treatment, which showed an increase of 500% of the initial concentration, followed by the p\*e mixture (27.0%), *S. paniculatum* (10.0%)

(14N+15N)-microbial biomass (µg 14N+15N g-1 soil)

*S. paniculatum* 3.8b 4.2c 5.1c 5.5a p\*e 12.9a 16.4b 14.1b 2.8b *I. edulis* 3.2b 19.3a 17.5a 1.3c Control 4.7b 5.1c 2.7d 1.0c LSD 1.451\*\*\* 0.915\*\*\* 0.310\*\*\* 0.298\*\*\* 15N-microbial biomass (µg 15N excess g-1 soil) *S. paniculatum* 0.01618a 0.00084b 0.00134a 0.00041a p\*e 0.00180b 0.00316a 0.00369a 0.00021ab *I. edulis* 0.00123b 0.00308a 0.00314a 0.00026ab Control 0.00002c 0.00004c 0.00001b 0.00001b LSD 0.0018\*\*\* 0.00070\*\* 0.00307\* 0.00025\*

Table 10. (14N+15N)-microbial biomass (µg 14N+15N g-1 soil) and 15N-microbial biomass (µg 15N excess g-1 soil) in soil amended with 14N-legume leaf material and enriched 15N-urea fertilizer with rice as an indicator plant. Control was soil without added leaf material and fertilized with N-urea. The 15N excess is expressed as the atom % 15N excess abundance

The high N-microbial biomass level remained high in the *I. edulis* and p\*e mixture treatments for the first 10 days of the greenhouse experiment (Table 10). However, at the end of the incubation period, these treatments showed a significantly higher decrease in the microbial biomass-(14N+15N), whereas the *S. paniculatum* treatment displayed a significantly

In all treatments, a significantly higher microbial biomass-15N was observed during the first 10 days of the incubation period in comparison with the control (Table 10). The initially

high increase in the concentration of 14N+15N in the microbial biomass.

0 5 10 50

= P < 0.05; \*\* = P < 0.01; \*\*\* = P < 0.001.

Treatment Incubation period (days)

LSD compares treatment at the same sampling time; \*

above the background (0.3663 atom %).

mixture (2.55%) and *S. paniculatum* treatments (1.96%).

and control (8.8%).

higher microbial biomass-15N concentration for the *S. paniculatum* treatment indicated intensive microbial activity leading to decomposition of recalcitrant leaf material in this treatment. However, after fertilizer had been added, the 15N-excess immobilization in the soil increased to 155% of the initial concentration in the *I. edulis* treatment and 105% in the p\*e mixture treatment, whereas the *S. paniculatum* treatment showed a decrease of 92% in the microbial biomass- 15N concentration. At the end of the experiment, all legume treatments showed a significant decrease in the microbial biomass-15N, and only the *S. paniculatum* showed a significantly higher microbial 15N-concentration in comparison with control (Table 10).

The decrease in soil mineral nitrogen over time was attributable not only to rice plant uptake, but also to considerable microbial immobilization. The use of a mixture of two contrasting litter qualities (called p\*e mixture) improved the nitrogen recovered by rice, which was derived from urea-fertilizer by 35.1% and 41.3% of total fertilized N in comparison with *S. paniculatum* and *I. edulis* treatments, respectively. In the same way, the rice recovery of N derived from the legume material in the p\*e mixture improved by 73.3% compared with the *S. paniculatum* treatment, but was 0.9% lower than that of the *I. edulis* treatment.

Moreover, the use of a larger amount of leaf material resulted in a higher increase in N immobilization and mineral-N in the soil, depending on the quality of the leaf material. The total cumulative nitrogen mineralization increased 90.9%, 10.3%, and 18.8% for the *S. paniculatum*, p\*e mixture and *I. edulis* treatments, respectively. In the same way, the increases in cumulative microbial biomass-N during the incubation period were 109%, 190% and 344%, respectively.

The p\*e mixture treatment showed an intermediate cumulative soil microbial- 15N immobilization, higher cumulative rice biomass and total N, and higher recovery of 15N from urea fertilizer. This indicates that the interaction of two different leguminous species increases the nitrogen absorption by rice through the increase in mineral-N and the decrease of gross microbial-N immobilization.

Decline in soil productivity and environmental quality and progressive deterioration of natural resources in the tropics have led to a search for new methods to sustain crop production via more efficient nutrient cycling. In agricultural ecosystems in the tropics with limited access to fertilizers, plant residues are often used to meet the N requirements of annual food crops (Constantinides and Fownes 1994). The added organic materials are potentially important sources of N, C and P in crop production, especially for resourcespoor farmers on tropical agricultural land. In order to successfully manage organic materials, the release and uptake of N by crops must be identified (Hood 2002). But the predictions of net N-mineralization are in many cases unreliable because net Nmineralization is affected by N immobilization and remineralization and losses (Stark and Schimel 2001).

The slash and burn system destroys the above-ground biomass of the fallow vegetation including the litter by burning, which causes loss of nutrients through volatilization and leaching of free nutrients in ash by rainfall. The losses of nitrogen by volatilization and leaching can reach 95-98% (Mackensen et al. 1996). Cerri et al. (1991) observed a reduction of 25% in soil carbon content two years after a plot was cleared, burned and a satisfactorily

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 139

leaf material + fertilizer

leaf material + fertilizer

**8.3**(0.44) **54.5**(0.84) **3.6**(0.24) **19.3**(3.48) **9.7**(0.08) **51.7**(0.80) **1.1**(0.14) **18.2**(1.82) **7.1**(0.11) **57.7**(1.23) **1.2**(0.10) **19.4**(1.62) **2.7**(0.09) **52.0**(1.66) **2.6**(0.17) **14.2**(2.61) Total **27.8 215.9 8.5 71.1** 

(days) fertilizer fertilizer **13.1**(0.16) **17.8**(2.04) **1.3**(0.25) **4.8**(0.36) **14.8**(0.26) **12.9**(0.95) **4.9**(0.25) **1.8**(0.41) **16.0**(0.09) **13.3**(0.10) **2.2**(0.08) **0.7**(0.55) **20.4**(0.13) **12.7**(0.18) **1.3**(0.01) **4.6**(1.37) Total **64.3 56.7 9.7 11.9** 

Table 11. Nitrogen-mineralΦ (µg N g-1 soil) and N-microbial biomassΨ (µg N g-1 soil) for incorporated leaf+wood material in comparison with incorporated leaf material+Urea as fertilizer, during the incubation time and total. The numbers represent mean (Standard

Microbial immobilization of labelled nitrogen was unaffected by rice plant growth, but was strongly affected by organic matter and nitrogen addition. Immobilization of nitrogen by organic matter decomposers was determined primarily by the amount and accessibility of available nitrogen. Differences in nitrogen immobilization by decomposers of the legume organic matter were greatest between N treatments, but were also affected by mixture of

Rice growth and nitrogen accumulation closely reflected the differences in chemical composition and mineralization between the residues and their mixture. Approximately the same amount of nitrogen was added in all legume treatments, and yet the amount of

**2.7**(0.04) **19.5**(1.81) **0.6**(0.50) **4.8**(1.33) **0.5**(0.14) **15.5**(1.42) **0.8**(0.06) **5.2**(1.88) **0.7**(0.11) **13.4**(0.28) **0.3**(0.09) **6.3**(3.38) **1.1**(0.12) **31.5**(1.46) **0.1**(0.07) **5.5**(0.71) Total **5.0 79.9 1.8 21.8** 

**N-microbial biomass (µg N g-1 soil)** 

Total N-microbial biomass (µg N g-1 soil)

Total N-microbial biomass (µg N g-1 soil)

leaf material + fertilizer

leaf material + fertilizer

leaf + wood material

leaf + wood material

**(µg N g-1 soil)** 

(µg N g-1 soil)

(µg N g-1 soil)

leaf + wood material

leaf + wood material

<sup>Φ</sup> Nitrogen-mineral was examined with steam distillation procedure. <sup>Ψ</sup> N-microbial biomass was measured with fumigation-extraction procedure.

*<sup>S</sup>***.** *paniculatum* **N-mineral** 

*I. edulis* Total N-mineral

**Control** Total N-mineral

Incubation period

Incubation period

Incubation period

(days)

(days)

error).

two contrasting legume materials

managed pasture established. However, Kato (1998) showed no reduction in carbon content in the mineral soil in the slash and burn system, and attributed this to the biomass accumulated by the rice crop.

The concept of pools of organic matter that differ in their susceptibilities to microbial decomposition and their longevity in soil has provided a basis for understanding the dynamic nature of soil organic matter and how nutrient availability is influenced by management practices and changes in the soil environment (Stevenson and Cole 1999). Our study showed that the patterns of the added organic carbon need to be taken into consideration. This was supported by the strong correlation between nitrogen dynamics in the contrasting legumes-amended soil and soil microbial biomass and organic carbon, found in this study.

Dissolved organic substances contribute to plant nutrition with nitrogen. Due to their water solubility there is a considerable risk that leaching of these substances will result in enhanced soil degradation. The dynamics of dissolved organic substances is influenced by the quantity and the quality of soil organic matter, the sorption characteristics of the soils and the microbial activity. All these parameters are modified by land use.

When immobilization and mobilization processes of N in soil are managed, it is important to quantify the real amount of N stored in the soil microbial biomass (Joergensen and Mueller 1996). Transient immobilization of soil N in the microbial biomass may contribute to improved conservation of soil N sources (Jensen 1997).

The higher initial concentrations of soil inorganic nitrogen in the high-N treatments would unlikely increase nitrogen immobilization significantly in the absence of added organic matter because nitrogen immobilization is generally limited by available carbon (Recous et al. 1988, Bremer and Kuikman 1997). Thus, fertilizer added as urea-N did not lead to differences in total mineral-N and microbial biomass-N in soil without added organic material during the incubation period (Table 11). Urea increased total microbial biomass-N and decreased total mineral-N, which suggests that fertilizer increased microbial biomass and thus nitrogen consumption by soil microorganism.

On the other hand, the increase in soil organic carbon and nitrogen due to added legume leaf + wood material resulted in a decrease in N-min and microbial biomass in comparison with control treatment (Table 11). Assuming that the fumigation-extraction method did not measure the fungal N-absorption, resulting in a underestimation of N-immobilization, and that the mineralization and losses of indigenous soil nitrogen were similar in the control and residue-treated soil, the real nitrogen immobilization and consumption was 63.2 and 37.7 µg N g-1 soil for *S. paniculatum* and *I. edulis* treatment, respectively. This means that the nitrogen in microbial biomass (microbial nitrogen immobilization and consumption) was approximately 9.3 and 1.0 times more than the mineral-N found in the same treatments, respectively.

The withdrawal of the wood material and added nitrogen as fertilizer yielded a strong increase in N-mineralization in comparison with wood material only and control. This suggests that these soils in this Amazon region are very nitrogen and carbon limiting and the microbial competition is very intensive.


managed pasture established. However, Kato (1998) showed no reduction in carbon content in the mineral soil in the slash and burn system, and attributed this to the biomass

The concept of pools of organic matter that differ in their susceptibilities to microbial decomposition and their longevity in soil has provided a basis for understanding the dynamic nature of soil organic matter and how nutrient availability is influenced by management practices and changes in the soil environment (Stevenson and Cole 1999). Our study showed that the patterns of the added organic carbon need to be taken into consideration. This was supported by the strong correlation between nitrogen dynamics in the contrasting legumes-amended soil and soil microbial biomass and organic carbon, found

Dissolved organic substances contribute to plant nutrition with nitrogen. Due to their water solubility there is a considerable risk that leaching of these substances will result in enhanced soil degradation. The dynamics of dissolved organic substances is influenced by the quantity and the quality of soil organic matter, the sorption characteristics of the soils

When immobilization and mobilization processes of N in soil are managed, it is important to quantify the real amount of N stored in the soil microbial biomass (Joergensen and Mueller 1996). Transient immobilization of soil N in the microbial biomass may contribute to

The higher initial concentrations of soil inorganic nitrogen in the high-N treatments would unlikely increase nitrogen immobilization significantly in the absence of added organic matter because nitrogen immobilization is generally limited by available carbon (Recous et al. 1988, Bremer and Kuikman 1997). Thus, fertilizer added as urea-N did not lead to differences in total mineral-N and microbial biomass-N in soil without added organic material during the incubation period (Table 11). Urea increased total microbial biomass-N and decreased total mineral-N, which suggests that fertilizer increased microbial biomass

On the other hand, the increase in soil organic carbon and nitrogen due to added legume leaf + wood material resulted in a decrease in N-min and microbial biomass in comparison with control treatment (Table 11). Assuming that the fumigation-extraction method did not measure the fungal N-absorption, resulting in a underestimation of N-immobilization, and that the mineralization and losses of indigenous soil nitrogen were similar in the control and residue-treated soil, the real nitrogen immobilization and consumption was 63.2 and 37.7 µg N g-1 soil for *S. paniculatum* and *I. edulis* treatment, respectively. This means that the nitrogen in microbial biomass (microbial nitrogen immobilization and consumption) was approximately 9.3 and 1.0 times more than the mineral-N found in the same treatments,

The withdrawal of the wood material and added nitrogen as fertilizer yielded a strong increase in N-mineralization in comparison with wood material only and control. This suggests that these soils in this Amazon region are very nitrogen and carbon limiting and

and the microbial activity. All these parameters are modified by land use.

improved conservation of soil N sources (Jensen 1997).

and thus nitrogen consumption by soil microorganism.

the microbial competition is very intensive.

accumulated by the rice crop.

in this study.

respectively.


<sup>Φ</sup> Nitrogen-mineral was examined with steam distillation procedure.

<sup>Ψ</sup> N-microbial biomass was measured with fumigation-extraction procedure.

Table 11. Nitrogen-mineralΦ (µg N g-1 soil) and N-microbial biomassΨ (µg N g-1 soil) for incorporated leaf+wood material in comparison with incorporated leaf material+Urea as fertilizer, during the incubation time and total. The numbers represent mean (Standard error).

Microbial immobilization of labelled nitrogen was unaffected by rice plant growth, but was strongly affected by organic matter and nitrogen addition. Immobilization of nitrogen by organic matter decomposers was determined primarily by the amount and accessibility of available nitrogen. Differences in nitrogen immobilization by decomposers of the legume organic matter were greatest between N treatments, but were also affected by mixture of two contrasting legume materials

Rice growth and nitrogen accumulation closely reflected the differences in chemical composition and mineralization between the residues and their mixture. Approximately the same amount of nitrogen was added in all legume treatments, and yet the amount of

Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 141

researchers must better understand soil organisms, their functions and their interactions with the chemical and physical environment. Many aspects of soil biology and ecology are worthy of research in view of their fundamental scientific interest and their role in

The high correlation with the ratio of N + P-to-phenol and N-mineralization implies that the plant nutrients which limit microbial action govern decomposition and nutrient release. This is likely associated with the formation of stable polymers with many forms of N binding N

The slash-and-mulch systems with thick mulch mats need to be improved for the synchronization of nutrient release from organic material and nutrient uptake by crop systems. The use of contrasting plant material in terms of litter quality, C reduction in the vegetation with the selective removal of wood, and soil incorporation of fallow residues need to be further tested. On the other hand, the increase of agriculture in the Amazon region cannot be done by the increase of deforestation and the scope of an increase in fertilizer use is limited. Thus, the intensification and improvement of currently managed land would have to be attempted. Fallow-mulch system is a considerable challenge and calls for more research. The agricultural policy in the Amazon region could promote organic agriculture with incentives to production and facilitating the commercialization of Amazon

Addiscott, T. M., Whitmore, A. P. and Powlson, D. S. (1991). Farming fertilizers and the

Anderson, J. M., Proctor, J. and Vallack, H. W. (1983). "Ecological Studies in Four

Barraclough, D. (1997). "The direct or MIT route for nitrogen immobilization: a 15N mirror

Barrett, J. E. and Burke, I. C. (2000). "Potential nitrogen immobilization in grassland soils

Bending, G. D., Turner, M. K. and Burns, I. G. (1998). "Fate of nitrogen from crop residues as

Biederbeck, V. O., Janzen, H. H., Campbell, C. A. and Zentner, R. P. (1994). "Labile soil

Blair, J. M., Parmelee, R. W. and Beare, M. H. (1990). "Decay Rates, Nitrogen Fluxes, and

Bremer, E. and Kuikman, P. (1997). "Influence of competition for nitrogen in soil on net

mineralization of nitrogen." Plant and Soil 190(1): 119-126.

Contrasting Lowland Rain Forests in Gunung Mulu National Park, Sarawak: III. Decomposition Processes and Nutrient Losses from Leaf Litter." Journal of Ecology

image study with Leucine and Glycine." Soil Biology and Biochemistry 29(1): 101-

across a soil organic matter gradient." Soil Biology and Biochemistry 32: 1707-1716.

affected by biochemical quality and the microbial biomass." Soil Biology and

organic matter as influenced by cropping practices in an arid environment." Soil

Decomposer Communities of Single- and Mixed-Species Foliar Litter." Ecology

Nitrate Problem. Wallingford, UK, CAB International.

ecosystem functioning.

organic agricultural products.

71: 503-527.

71(5): 1976–1985.

Biochemistry 30(14): 2055-2065.

Biology and Biochemistry 26(12): 1647-1656.

108.

**5. References** 

released to the soil by the incorporated organic matter.

nitrogen accumulated differed with the legume quality. The use of a mixture of two contrasting litter qualities improved the rice recovery of nitrogen derived from ureafertilizer and the rice recovery of nitrogen derived from legume material.

This study showed that the quality and the quantity of organic carbon presented an important factor affecting soil nitrogen mineralization and immobilization. Changes in soil carbon substrates influenced the dynamics of soil inorganic nitrogen because of the importance of labile carbon in the microbial immobilization and consumption of nitrogen. Compton and Boone (2002) showed that the light fraction of soil organic matter incorporated more 15N than the heavy fraction per unit of carbon, which indicated that not simply the amount but the composition of organic matter controls its function as a site for N incorporation.

Soil microbial biomass immobilizes a higher amount of the residue N mulched or incorporated into the soil and this needs to be taken into consideration. On the other hand, soil microbial biomass immobilization is a labile repository of nitrogen, their turnover and remineralization may conserve this N in the system and release this N for later plant use.

Legume-enriched mulch material had different patterns of mulch decomposition and nutrient release. As was pointed out earlier by Constantinides and Fownes (1994), Fox et al. (1990), the contents of N, lignin and polyphenol are the principal chemical factors controlling degradability of plant material. The high correlation with the ratio of N + P-tophenol and N-mineralization implies that the plant nutrients which limit microbial action govern decomposition and nutrient release. This is likely associated with the formation of stable polymers with many forms of N binding N released to the soil by the incorporated organic matter.

#### **4. Conclusion**

The use of fast-growing legumes for fallow enrichment, as for example *A*. *mangium*, does not necessarily translate directly in a CO2 sequestration, because the fast decomposition rate. Species, contrasting in lignin and polyphenol concentration with higher N- and P-content must be used for enrichment fallow. The *I. edulis* and *S. paniculatum*, two Amazon species, showed greater promise as enrichment candidates, because of their high organic C input in combination with low losses of OM.

Since immobilization of N is generally determined by the amount of decomposable carbon present in the soil rather than by the amount of inorganic N, the addition of compost showing a wide C-to-N ratio accelerates N-immobilization through increased microbial activity. Contrasting leguminous species had different patterns of net N-mineralization and immobilization in comparison with the single species. The use of two contrasting leguminous species increased the nitrogen absorption by rice through the increase of mineral N and decrease of microbial N-immobilization.

Soil microbial biomass immobilizes a higher amount of the residue N mulched or incorporated into the soil and this needs to be taken into consideration. On the other hand, soil microbial biomass immobilization is a labile repository of nitrogen, their turnover and remineralization may conserve N in the system and release this N for later plant use. Managing soil biological processes is a key aspect of sustainable development. The researchers must better understand soil organisms, their functions and their interactions with the chemical and physical environment. Many aspects of soil biology and ecology are worthy of research in view of their fundamental scientific interest and their role in ecosystem functioning.

The high correlation with the ratio of N + P-to-phenol and N-mineralization implies that the plant nutrients which limit microbial action govern decomposition and nutrient release. This is likely associated with the formation of stable polymers with many forms of N binding N released to the soil by the incorporated organic matter.

The slash-and-mulch systems with thick mulch mats need to be improved for the synchronization of nutrient release from organic material and nutrient uptake by crop systems. The use of contrasting plant material in terms of litter quality, C reduction in the vegetation with the selective removal of wood, and soil incorporation of fallow residues need to be further tested. On the other hand, the increase of agriculture in the Amazon region cannot be done by the increase of deforestation and the scope of an increase in fertilizer use is limited. Thus, the intensification and improvement of currently managed land would have to be attempted. Fallow-mulch system is a considerable challenge and calls for more research. The agricultural policy in the Amazon region could promote organic agriculture with incentives to production and facilitating the commercialization of Amazon organic agricultural products.

#### **5. References**

140 Crop Plant

nitrogen accumulated differed with the legume quality. The use of a mixture of two contrasting litter qualities improved the rice recovery of nitrogen derived from urea-

This study showed that the quality and the quantity of organic carbon presented an important factor affecting soil nitrogen mineralization and immobilization. Changes in soil carbon substrates influenced the dynamics of soil inorganic nitrogen because of the importance of labile carbon in the microbial immobilization and consumption of nitrogen. Compton and Boone (2002) showed that the light fraction of soil organic matter incorporated more 15N than the heavy fraction per unit of carbon, which indicated that not simply the amount but the composition of organic matter controls its function as a site for N

Soil microbial biomass immobilizes a higher amount of the residue N mulched or incorporated into the soil and this needs to be taken into consideration. On the other hand, soil microbial biomass immobilization is a labile repository of nitrogen, their turnover and remineralization may conserve this N in the system and release this N for later plant use.

Legume-enriched mulch material had different patterns of mulch decomposition and nutrient release. As was pointed out earlier by Constantinides and Fownes (1994), Fox et al. (1990), the contents of N, lignin and polyphenol are the principal chemical factors controlling degradability of plant material. The high correlation with the ratio of N + P-tophenol and N-mineralization implies that the plant nutrients which limit microbial action govern decomposition and nutrient release. This is likely associated with the formation of stable polymers with many forms of N binding N released to the soil by the incorporated

The use of fast-growing legumes for fallow enrichment, as for example *A*. *mangium*, does not necessarily translate directly in a CO2 sequestration, because the fast decomposition rate. Species, contrasting in lignin and polyphenol concentration with higher N- and P-content must be used for enrichment fallow. The *I. edulis* and *S. paniculatum*, two Amazon species, showed greater promise as enrichment candidates, because of their high organic C input in

Since immobilization of N is generally determined by the amount of decomposable carbon present in the soil rather than by the amount of inorganic N, the addition of compost showing a wide C-to-N ratio accelerates N-immobilization through increased microbial activity. Contrasting leguminous species had different patterns of net N-mineralization and immobilization in comparison with the single species. The use of two contrasting leguminous species increased the nitrogen absorption by rice through the increase of

Soil microbial biomass immobilizes a higher amount of the residue N mulched or incorporated into the soil and this needs to be taken into consideration. On the other hand, soil microbial biomass immobilization is a labile repository of nitrogen, their turnover and remineralization may conserve N in the system and release this N for later plant use. Managing soil biological processes is a key aspect of sustainable development. The

fertilizer and the rice recovery of nitrogen derived from legume material.

incorporation.

organic matter.

**4. Conclusion** 

combination with low losses of OM.

mineral N and decrease of microbial N-immobilization.


Leaves Material Decomposition from Leguminous Trees in an Enriched Fallow 143

Jensen, B. K., Jensen, E. S. and Magid, J. (1995). "Decomposition of 15N-labelled rye grass in

Jensen, E. S. (1997). "Nitrogen immobilization and mineralization during initial

Joergensen, R. G. and Mueller T. (1996). "The fumigation-extraction method to estimate soil

Kato, M. S. A., Kato, O. R., Denich, M. and Vlek, P. L. G. (1999). "Fire-free alternatives to

Kato, O. R. (1988). Fire-free Land Preparation as an Alternative to Slash-and-burn

Kuo, S. and Sainju, U. M. (1998). "Nitrogen mineralization and availability of mixed

Linkins, A. E., Melillo, J. M. and Sinsabaugh, R. L. (1984). Factors affecting cellulase activity

Lonrez, K., Preston, C. M., Raspe, S., Morrison, I. K. and Feger, K. H. (2000). "Litter

Mackensen, J., Hölscher, D., Klinge, R. and Fölster, H. (1996). "Nutrient transfer to the

Macías, F. and M. C. Arbestain (2010). "Soil carbon sequestration in a changing global

Mafongoya, P. L., Giller, K. E. and Palm, C.A. (1998). "Decomposition and nitrogen release patterns of tree prunings and litter." Agroforestry Systems 38(1-3): 77-97. Mapfumo, P., Mtambanengwe, F., and Vanlauwe, B. (2007). "Organic matter quality and

Meentemeyer, V. (1978). "Macroclimate the lignin control of litter decomposition rates."

Melillo, J.M., Aber, J.D. and Muratore, J.F. (1982). "Nitrogen and lignin control of hardwood

Moorhead, D. L., Sinsabaugh, A. E., Linkins, A. E. and Reynolds, J. F. (1996).

Myers, R. J. K., Palm C. A., Cuevas, E., Gunatilleke, I. U. N. and Brossard, M. (1994). The

environment." Mitig Adapt Strateg Glob Change 15: 511–529.

leaf litter decomposition dynamics." Ecology 63(3): 621-626.

soils in Zimbabwe." Plant and Soil 296(1-2): 137–150

Nitrogen Leaching in Ecological Agriculture: 221-228.

fertilizers." Field Crops Research 62(2-3): 225-237.

Soils. 24: 39-44.

28(1): 33-37.

University: 132.

579.

of Soils 26: 346-353.

and Biochemistry 32: 779-792.

Management 86: 121-128.

Ecology 59(3): 465–472.

Total Environment 183: 137-149.

soils from a long-term field experiment with different manuring strategies."

decomposition of 15N-labelled pea and barley residues." Biology and Fertility of

microbial biomass: calibration of the kEN value." Soil Biology and Biochemistry

slash-and-burn for shifting cultivation in the eastern Amazon region: the role of

Agriculture in the Bragantina Region, Eastern Amazon: Crop Performance and Nitrogen Dynamics. Dissertation submitted for the degree of Doctor of Agricultural Sciences of the Facultay of Agricultural Sciences. Goettingen, George-August-

leguminous and non-leguminous cover crop residues in soil." Biology and Fertility

in terrestrial and aquatic ecosystems. Current Perspectives in Microbial Ecology. M.J. Klug and C.A. Reddy. Washington, American Society for Microbiology: 572-

decomposition an humus characteristics in Canadian and German spruce ecosystems: information from tannin analysis and 13C CPMAS NMR." Soil Biology

atmosphere by burning of debris in eastern Amazonia." Forest Ecology and

management effects on enrichment of soil organic matter fractions in contrasting

"Decomposition processes: modelling approaches and applications." The Science of

synchronization of nutrient mineralization and plant nutrient demand. Biological


Cattanio, J.H., Kuehne, R., Vlek, P.L.G. (2008). "Organic material decomposition and

Cerri, C. C., Volkoff, B. and Andreaux, F. (1991). "Nature and behaviour of organic matter in

Chesson, A. (1997). Plant degradation by ruminants: parallels with litter decomposition in

Compton, J. E. and Boone, R. D. (2002). "Soil nitrogen transformations and the role of light

Constantinides, M. and Fownes, J. H. (1994). "Nitrogen mineralization from leaves and litter

Denich, M., Vlek, P.L.G., Abreu-Sa, T. D., Vielhauer, K., Luecke, W. (2005). "A concept for

Fox, R., Myers, R. and Vallis, J. K. (1990). "The nitrogen mineralization rate of legume

Franagan, P. W. and van Cleve, K. (1983). "Nutrient cycling in relation to decomposition and

Gallardo, A. and Merino, J. (1993). "Leaf Decomposition in Two Mediterranean Ecosystems of Southwest Spain: Influence of Substrate Quality." Ecology 74(1): 152–161. Handayanto, E., Cadisch, G. and Giller, K. E. (1997). Regulating N Mineralization from Plant

Haynes, R. J. (1986). The decomposition process: Mineralization, immobilization, humus

Hobbie, S. E. (2000). "Interactions between litter lignin and soil nitrogen availability during

Hood, R. (2001). "Evaluation of a new approach to the nitrogen-15 isotope dilution

Isaac, L., Wood, C. W. and Shannon, D. (2000). "Decomposition and nitrogen release of

Janssen, B. H. (1996). "Nitrogen mineralization in relation to C:N ratio and decomposability

Jansson, S. L. (1958). "Tracer studies on nitrogen transformations in soil with special

Jenkinson, D. S., Fox, R. H. and Rayner, J. H. (1985). "Interaction between fertilizer nitrogen and soil nitrogen: The so-called priming effect." Journal of Soil Science 36: 425-444.

Amazon." Revista Brasileira Ciencia do Solo 32: 1073-1086.

Manaus." Forest Ecology and Management 38: 247-257.

K.E. Giller (eds.). Wallingford, UK, CAB International: 47-66.

concentrations." Soil Biology and Biochemistry 26(1): 49-55.

Agriculture, Ecosystems and Environment 110(1-2): 43–58.

and Soil 129: 251-259.

International: 175-185.

Journal 92: 501-511.

(ed.). Orlando, FL., Academic Press: 52-176.

of organic materials." Plant and Soil 191(1): 39-45.

Agricultural College of Sweden 24: 101-361.

and Fertility of Soils 34: 156-161.

13: 795-817.

nutrient dynamics in a mulch system enriched with leguminous trees in the

soils under natural forest, and after deforestation, burning and cultivation, near

soils. Drive by Nature: Plant Litter Quality and Decomposition. G. Cadisch and

raction organic matter in forest soils." Soil Biology and Biochemistry 34(7): 933-943.

of tropical plants: Relationship to nitrogen, lignin and soluble polyphenol

the development of fire-free fallow management in the Eastern Amazon, Brazil."

residues in soil as influenced by polyphenol, lignin, and nitrogen contents." Plant

organic-matter quality in taiga ecosystems." Canadian Journal of Forest Research

Residues by Manipulation of Quality. Driven by Nature: Plant Quality and Decomposition. G. Cadisch and K.E. Giller (eds.), Wallingford, UK. CAB

formation and degradation. Mineral Nitrogen in the Plant-Soil System. R. J. Haynes

leaf litter decomposition in a Hawaiian Montane Forest." Ecosystems 3(4): 484-494.

technique, to estimate crop N uptake from organic residues in the field." Biology

prunings from hedgerow species assessed fro alley dropping in Haiti." Agronomy

attention to mineralization-immobilization relationships." Annals of the Royal


**7** 

*India* 

**Plant Research** 

**Comparative Analyses of Extracellular Matrix** 

*National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi,* 

Within their social milieu, cells are petite and deformable, enclosed in a flimsy plasma membrane which swerves from their default spherical shape to more polar shapes due to the local deposition, complex interactions and the remodelling of the extracellular matrix (ECM). Consequently, multicellularity has evolved, albeit independently in plants and animals. Although animals are truly multicellular, plants are supracellular organisms because their immobile cells divide via phragmoplast-based incomplete cytokinesis, which results in the formation of cytoplasmic cell-to-cell channels known as plasmodesmata (Baluska et al., 2003). The ECM in plants, often referred as the cell wall, is integrated into the apoplast—a structurally coherent superstructure extending throughout the plant body. In lieu, plant cells are not fully separated and both the plasma membrane and endoplasmic reticulum traverse cellular borders through plasmodesmata (Baluska et al., 2003; Fincher, G. 2009.). The ECM is a fundamental component of the microenvironment of both animal and plant cells that has been substantially expanded during evolution. Throughout the plant kingdom, the formation and regulation of the ECM architecture has been shown to have the potential to influence many conduits of development, position-dependent differentiation, patterning and totipotent cell niches, besides environmental stress response and pathobiology (Brownlee & Berger, 1995; Degenhardt & Gimmer, 2000; Wilson, 2010). Furthermore, it has been reported that the ECM plays an important morphoregulatory role during somatic embryogenesis and organogenesis in plants, besides its pivotal role in cellular osmo- and volume-regulation (Šamaj et al., 1999; Rose et al., 2004). The plant ECM has biomechanical and morphogenetic functions with the immense ability to turn cells into hydraulic machines which establish a crucial functional difference between cell walls and other cellular surface structures. It encloses the cell hermetically and constrains the hydrostatic pressure evoked by osmotic gradients between the cell and its environment which controls cellular osmo- and volume-regulation (Peters et al., 2000; Cosgrove, D. J. 2005). Plasticity in the ECM allows the cellular uptake of massive amounts of water into

**1. Introduction** 

 \*

Corresponding author

**Proteome: An Under-Explored Area in** 

Kanika Narula, Eman Elagamey, Asis Datta, Niranjan Chakraborty and Subhra Chakraborty\*

Management of Tropical Soil Fertility. P.L. Woomer and M.J. Swift (eds.). Chichester, UK, Wiley-Sayce Publication.: 81-116.


## **Comparative Analyses of Extracellular Matrix Proteome: An Under-Explored Area in Plant Research**

Kanika Narula, Eman Elagamey, Asis Datta, Niranjan Chakraborty and Subhra Chakraborty\* *National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, India* 

#### **1. Introduction**

144 Crop Plant

Palm, C. A. (1995). "Contribution of agroforestry trees to nutrient requirements of

Palm, C. A. and Sanchez, P. A. (1991). "Nitrogen release from the leaves of some tropical

Powlson, D. S. and Brookes, P.C. (1987). "Measurement of soil microbial biomass provides

Powlson, D. S., Hirsch, P. R. and Brookes, P. C. (2001). "The role of soil microorganisms in

Priha, O. and Smolander, A. (1997). "Microbial biomass and activity in soil and litter under

Recous, S., Fresneau, C., Faurie, G. and Mary, B. (1988). "The fate of labeled 15N urea and

Schimel, J. P., Jackson, L. E. and Firestone, M. K. (1989). "Spatial and temporal effects on

Seneviratne, G. (2000). "Litter quality and nitrogen release in tropical agriculture: a

Stark, J. M. and Schimel, J. (2001). "Errors in `Overestimation of gross N transformation rates in grassland soils." Soil Biology and Biochemistry 33(10): 1433-1435. Stevenson, F. J. and Cole, M. A. (1999). Cycles of Soil : Carbon, Nitrogen, Phosphorus,

Swift, M. J., Russell-Smith, A. and Perfect, T. J. (1981). "Decomposition and Mineral-Nutrient

Thönnissen, C., Midmorea, D. J., Ladha, J. K., Olk, D. C. and Schmidhalter, U. (2000).

tropical vegetable production systems." Agronomy Journal 92: 253-260. Verchot, L. V., Holmes, Z., Mulon, L., Groffman, P. M. and Lovett, G. M. (2001). "Gross vs

between forest types." Soil Biology and Biochemistry 33(14): 1889-1901. Vitousek, P. M., Turner, D. R., Parton, W. J. and Sanford, R. L. (1994). "Litter Decomposition

Woomer, P. L., Martin, A., Albrecht, A., Resck, D. V. S. and Scharpenseel, H. M. (1994). The

Zimmer, M. (2002). "Is decomposition of woodland leaf litter influenced by its species

Zucker, W. V. (1982). "How aphids choose leaves: the roles of phenolics in host selection by

Chichester, UK, Wiley-Sayce Publication.: 81-116.

Biochemistry 23(1): 83-88.

Agroecosystems 61(1-2): 41-51.

soil." Plant and Soil 112: 205-214.

intercropped plants." Agroforestry Systems 30: 105-124.

incorporation." Soil Biology and Biochemistry 19: 159-164.

afforestation sites." Biology and Fertility of Soils 24(1): 45-51.

grassland." Soil Biology and Biochemistry 21: 1059-1066.

synthesis." Biology and Fertility of Soils. 31: 60-64.

Sulfur, Micronutrients. New York, NY, Wiley. Pp 427.

Nigeria." Journal of Ecology 69(3): 981-995.

Models." Ecology 75(2): 418–429.

Sussex, UK, John Wiley & Sons: 47-80.

a Galling Aphid." Ecology 63(4): 972-981.

richness?" Soil Biology and Biochemistry 34(2): 277-284.

Management of Tropical Soil Fertility. P.L. Woomer and M.J. Swift (eds.).

legumes as affected by their lignin and polyphenolic contents." Soil Biology and

an early indication of changes in total soil organic matter due to straw

soil organic matter conservation in the tropics." Nutrient Cycling in

Pinus sylvestris, Picea abies and Betula pendula at originally similar field

ammonium nitrate applied to a winter wheat crop: Nitrogen transformation in

plant-microbial competition for inorganic nitrogen in a California annual

Dynamics of Plant Litter in a Regenerating Bush-Fallow in Sub-Humid Tropical

"Legume decomposition and nitrogen release when applied as green manures to

net rates of N mineralization and nitrification as indicators of functional differences

on the Mauna Loa Environmental Matrix, Hawai: Patterns, Mechanisms, and

importance and management of soil organic matter in the tropics. P. L. Woomer and M. J. Swift (eds.). The Biological Management of Tropical Soil Fertility. West Within their social milieu, cells are petite and deformable, enclosed in a flimsy plasma membrane which swerves from their default spherical shape to more polar shapes due to the local deposition, complex interactions and the remodelling of the extracellular matrix (ECM). Consequently, multicellularity has evolved, albeit independently in plants and animals. Although animals are truly multicellular, plants are supracellular organisms because their immobile cells divide via phragmoplast-based incomplete cytokinesis, which results in the formation of cytoplasmic cell-to-cell channels known as plasmodesmata (Baluska et al., 2003). The ECM in plants, often referred as the cell wall, is integrated into the apoplast—a structurally coherent superstructure extending throughout the plant body. In lieu, plant cells are not fully separated and both the plasma membrane and endoplasmic reticulum traverse cellular borders through plasmodesmata (Baluska et al., 2003; Fincher, G. 2009.). The ECM is a fundamental component of the microenvironment of both animal and plant cells that has been substantially expanded during evolution. Throughout the plant kingdom, the formation and regulation of the ECM architecture has been shown to have the potential to influence many conduits of development, position-dependent differentiation, patterning and totipotent cell niches, besides environmental stress response and pathobiology (Brownlee & Berger, 1995; Degenhardt & Gimmer, 2000; Wilson, 2010). Furthermore, it has been reported that the ECM plays an important morphoregulatory role during somatic embryogenesis and organogenesis in plants, besides its pivotal role in cellular osmo- and volume-regulation (Šamaj et al., 1999; Rose et al., 2004). The plant ECM has biomechanical and morphogenetic functions with the immense ability to turn cells into hydraulic machines which establish a crucial functional difference between cell walls and other cellular surface structures. It encloses the cell hermetically and constrains the hydrostatic pressure evoked by osmotic gradients between the cell and its environment which controls cellular osmo- and volume-regulation (Peters et al., 2000; Cosgrove, D. J. 2005). Plasticity in the ECM allows the cellular uptake of massive amounts of water into

<sup>\*</sup> Corresponding author

Comparative Analyses of Extracellular

perspectives of the ECM's functions.

**1.1 Exploring the sink and link in ECM** 

Matrix Proteome: An Under-Explored Area in Plant Research 147

In this report, we begin by summarising the essential and unique features of the ECM and we discuss recent findings concerning the regulation and biochemistry of it, with specific emphasis on the fundamental role of ECM proteins in development, environmental stress and signalling by analysing the ECM's proteomes. Furthermore, we report here the comparative analysis of ECM proteomes towards crop specificity, organ-based, developmental and environmental adaptations based on our own findings, the available literature and databases focusing on ECM proteins in view of the current understanding and

Ubiquitously present, the ECM/cell wall is composed of different molecules with diverse functions to meet the specialised requirements of different tissues. It is a dynamic milieu, having homeostatic properties and a reservoir for bioactive molecules, such as carbohydrates and proteins. Long before the determination of comprehensive chemical differences between plant and animal ECMs, Boerhaave proposed in the early 18th century that fermented plant material which is rich in carbohydrate is acidic whereas putrefied animal material which is rich in protein is basic (ammoniacal) (Rose, 2003). It alludes only briefly to the differences and similarities between the ECMs of higher plants and animals. Consequently, proteins are largely responsible for the chemical transformation properties that distinguish plants from animals. The ECM in higher plants and higher animals consists of a mixture of fibrous and amorphous components. In higher animals, a protein-based collagen - elastin, a fibronectin fibre matrix is infiltrated by mucopolysaccharides, peptidoglycans and calcium phosphate, whereas in higher plants, non-protein cellulose and protein-based extensin fibre matrices are infiltrated by a varied assortment of non-nitrogenous hemicelluloses, pectins and lignins and, to a much smaller extent (on a mass basis), by various structural proteins and enzymes (Irshad et al., 2008). Similarities in ECM design may be apparent as it is likely that ancient functional protein domains and carbohydrate backbones have been used in a variety of arrangements and combinations to affect the function of convergent biological structures. On the contrary, as stated by Darwin the "web of wall molecules have a long evolutionary history," and it is therefore relevant that different family members show highly regulated and specific patterns of the expression of ECM components in an evolutionary context. In addition to protein heterogeneity and the presence of various metals as linkers, carbohydrate compositions can vary between cell types and even within one wall of a given cell, suggesting that the cell wall serves as a sink of variability in terms of macromolecules or microelements. On a fresh mass basis, the vegetative growth of all organisms (70-90% water) is predominantly owing to water uptake, but on a dry mass basis the vegetative growth of plants differs markedly from that of animals (Rose, 2003). During differentiation, plant cells increase in size from typically 102 mm3 (volume of a meristematic cell) to up to 107 mm3 (e.g., a xylem vessel). This increase in cellular volume requires the addition of building materials in the form of cell wall polymers and membranes. While new cell wall materials are incorporated, the existing material is deformed and stretched mechanically. The force for this deformation is supplied by the turgor pressure (Geitmann, 2010). The ECM serves as the first line mediator in cell signalling to perceive and transmit extra- and intercellular signals in many cellular pathways. ECM proteins constitute more than just a structural framework but they also play a variety of roles in growth and development, defence against environmental stresses as well as giving structural support.

a central vacuole while rigidity in the ECM determines the conductance of enormous amounts of water and dissolved solutes through vascular bundles. The secretion of an ECM by one cell can also influence the neighbouring cells, conceivably the best exemplified paracrine interaction known in the plant kingdom (for a review, see Brownlee, 2002). Beyond their paramount importance in the generation of form, cell walls are frequently considered 'growth-controlling' (Wolf et al., 2009). Cells devoid of the ECM inevitably lose their polar shape and the loss of cellular polarisation prevents cell-to-cell interactions and communication. The ECM/cell wall is evolutionary and inherently bestowed with information that can be both stored and relayed to cell interior via templating processes. It serves as the first line mediator in cell signalling for perceiving and transmitting extra- and intercellular signals in many cellular pathways. Communication between the cytoplasm and the cell wall is necessary and evident because of events such as cell expansion (Cosgrove, 1997, Schröder, F et al 2009), mechanical stress (Kumar et al., 2006; Telewski, 2006), environmental perturbation (Gail McLean et al., 1997; Thelen, J. and Peck, S. 2007) and pathogen infection (Hammond-Kosack & Jones, 1996) which lead to altered biosynthesis and the modification of wall components and downstream cytoplasmic events. In addition, it can act as a substrate for migration and has also been recognised as a surrogate for providing inputs into cell behaviour (Hall et al., 2002), although the available data is rather scarce for higher plants and critical linker molecules between the cytoskeleton and the ECM are still missing. Thus, the ECM/cell wall primarily serves a dual function, as a cell support system and for signalling during development and stress. The ECM/cell wall must therefore be dynamic as cells divide and elongate, modulating its composition and architecture during its synthesis and after it has been deposited. The wall function is a multi-step, complex process and the underlying mechanisms governing these steps are not fully understood.

Proteome research holds promise of understanding the molecular basis of the ECM function using an unbiased comparative and differential approach. We and others have identified several hundred plant proteins that include both predicted and non-canonical ECM components, presumably associated with a variety of cellular functions; viz. cell wall modification, signal transduction, cellular transport, metabolism, cell defence and rescue, all of which impinge on the complexity of ECM proteins in crop plants (Bhushan et al., 2006; Telewski, 2006). In recent years, reports have also been published focusing on changes in the ECM proteome in varied cellular events (Jones et al., 2004; Irshad et al., 2008, Bhushan et al., 2007, Cheng et al., 2009; Pandey, et al., 2010, Bhushan et al., 2011). The proteins that have been identified reveal the presence of complex regulatory networks that function in this organelle. Currently, we are focusing on disease-responsive ECM proteomes in order to understand the ECM-related pathobiology in plants. Although over the past few years there have been rapid advances in cell wall proteome research, the study of the complexity of ECM proteins remained secondary, irrespective of the fact that they correspond to about 10% of the ECM's mass and are comprised of several hundred different molecules with diverse functions. Moreover, a vast array of post-translational modifications to these proteins adds diversity to the structure and ligand-binding properties of matrix components, leading to their differential activity. Therefore, characterisations of the ECM proteome in plants hold the promise of increasing our understanding about the gene's function.

In this report, we begin by summarising the essential and unique features of the ECM and we discuss recent findings concerning the regulation and biochemistry of it, with specific emphasis on the fundamental role of ECM proteins in development, environmental stress and signalling by analysing the ECM's proteomes. Furthermore, we report here the comparative analysis of ECM proteomes towards crop specificity, organ-based, developmental and environmental adaptations based on our own findings, the available literature and databases focusing on ECM proteins in view of the current understanding and perspectives of the ECM's functions.

#### **1.1 Exploring the sink and link in ECM**

146 Crop Plant

a central vacuole while rigidity in the ECM determines the conductance of enormous amounts of water and dissolved solutes through vascular bundles. The secretion of an ECM by one cell can also influence the neighbouring cells, conceivably the best exemplified paracrine interaction known in the plant kingdom (for a review, see Brownlee, 2002). Beyond their paramount importance in the generation of form, cell walls are frequently considered 'growth-controlling' (Wolf et al., 2009). Cells devoid of the ECM inevitably lose their polar shape and the loss of cellular polarisation prevents cell-to-cell interactions and communication. The ECM/cell wall is evolutionary and inherently bestowed with information that can be both stored and relayed to cell interior via templating processes. It serves as the first line mediator in cell signalling for perceiving and transmitting extra- and intercellular signals in many cellular pathways. Communication between the cytoplasm and the cell wall is necessary and evident because of events such as cell expansion (Cosgrove, 1997, Schröder, F et al 2009), mechanical stress (Kumar et al., 2006; Telewski, 2006), environmental perturbation (Gail McLean et al., 1997; Thelen, J. and Peck, S. 2007) and pathogen infection (Hammond-Kosack & Jones, 1996) which lead to altered biosynthesis and the modification of wall components and downstream cytoplasmic events. In addition, it can act as a substrate for migration and has also been recognised as a surrogate for providing inputs into cell behaviour (Hall et al., 2002), although the available data is rather scarce for higher plants and critical linker molecules between the cytoskeleton and the ECM are still missing. Thus, the ECM/cell wall primarily serves a dual function, as a cell support system and for signalling during development and stress. The ECM/cell wall must therefore be dynamic as cells divide and elongate, modulating its composition and architecture during its synthesis and after it has been deposited. The wall function is a multi-step, complex process and the underlying mechanisms governing these steps are not fully

Proteome research holds promise of understanding the molecular basis of the ECM function using an unbiased comparative and differential approach. We and others have identified several hundred plant proteins that include both predicted and non-canonical ECM components, presumably associated with a variety of cellular functions; viz. cell wall modification, signal transduction, cellular transport, metabolism, cell defence and rescue, all of which impinge on the complexity of ECM proteins in crop plants (Bhushan et al., 2006; Telewski, 2006). In recent years, reports have also been published focusing on changes in the ECM proteome in varied cellular events (Jones et al., 2004; Irshad et al., 2008, Bhushan et al., 2007, Cheng et al., 2009; Pandey, et al., 2010, Bhushan et al., 2011). The proteins that have been identified reveal the presence of complex regulatory networks that function in this organelle. Currently, we are focusing on disease-responsive ECM proteomes in order to understand the ECM-related pathobiology in plants. Although over the past few years there have been rapid advances in cell wall proteome research, the study of the complexity of ECM proteins remained secondary, irrespective of the fact that they correspond to about 10% of the ECM's mass and are comprised of several hundred different molecules with diverse functions. Moreover, a vast array of post-translational modifications to these proteins adds diversity to the structure and ligand-binding properties of matrix components, leading to their differential activity. Therefore, characterisations of the ECM proteome in plants hold the promise of increasing our understanding about the gene's

understood.

function.

Ubiquitously present, the ECM/cell wall is composed of different molecules with diverse functions to meet the specialised requirements of different tissues. It is a dynamic milieu, having homeostatic properties and a reservoir for bioactive molecules, such as carbohydrates and proteins. Long before the determination of comprehensive chemical differences between plant and animal ECMs, Boerhaave proposed in the early 18th century that fermented plant material which is rich in carbohydrate is acidic whereas putrefied animal material which is rich in protein is basic (ammoniacal) (Rose, 2003). It alludes only briefly to the differences and similarities between the ECMs of higher plants and animals. Consequently, proteins are largely responsible for the chemical transformation properties that distinguish plants from animals. The ECM in higher plants and higher animals consists of a mixture of fibrous and amorphous components. In higher animals, a protein-based collagen - elastin, a fibronectin fibre matrix is infiltrated by mucopolysaccharides, peptidoglycans and calcium phosphate, whereas in higher plants, non-protein cellulose and protein-based extensin fibre matrices are infiltrated by a varied assortment of non-nitrogenous hemicelluloses, pectins and lignins and, to a much smaller extent (on a mass basis), by various structural proteins and enzymes (Irshad et al., 2008). Similarities in ECM design may be apparent as it is likely that ancient functional protein domains and carbohydrate backbones have been used in a variety of arrangements and combinations to affect the function of convergent biological structures. On the contrary, as stated by Darwin the "web of wall molecules have a long evolutionary history," and it is therefore relevant that different family members show highly regulated and specific patterns of the expression of ECM components in an evolutionary context. In addition to protein heterogeneity and the presence of various metals as linkers, carbohydrate compositions can vary between cell types and even within one wall of a given cell, suggesting that the cell wall serves as a sink of variability in terms of macromolecules or microelements. On a fresh mass basis, the vegetative growth of all organisms (70-90% water) is predominantly owing to water uptake, but on a dry mass basis the vegetative growth of plants differs markedly from that of animals (Rose, 2003). During differentiation, plant cells increase in size from typically 102 mm3 (volume of a meristematic cell) to up to 107 mm3 (e.g., a xylem vessel). This increase in cellular volume requires the addition of building materials in the form of cell wall polymers and membranes. While new cell wall materials are incorporated, the existing material is deformed and stretched mechanically. The force for this deformation is supplied by the turgor pressure (Geitmann, 2010). The ECM serves as the first line mediator in cell signalling to perceive and transmit extra- and intercellular signals in many cellular pathways. ECM proteins constitute more than just a structural framework but they also play a variety of roles in growth and development, defence against environmental stresses as well as giving structural support.

Comparative Analyses of Extracellular

components (Sakurai et al., 1998).

**2. Methodology and strategy** 

**1.1.2 ECM proteins: Cross talk in signalling and stress** 

Matrix Proteome: An Under-Explored Area in Plant Research 149

ECM senses and physiologically responds to environmental stress via signalling pathways. Signalling events are clearly not linear and induce many different reactions, including stressrelated processes that crosstalk with hormone signalling pathways. It is known that cell wall stress provokes a transient depolarised distribution of the cell wall biosynthetic enzyme glucan synthase and its regulatory subunit RHO1, possibly as a mechanism to repair general damage to the wall. Both environmental and patho-stress are thought to cause wall weakening which in turn transduces a signal to the interior of the cell as a homeostatic mechanism to repair the wall. Various kinases mediate the stress-induced synthesis of ECM proteins to combat cell wall interfering factors, such as pathogens, osmotic stress, dehydration and other environmental stresses. Recently, it has been found that wall-associated kinase (*WAK)* expression was induced when *Arabidopsis* plants were infected with a pathogen or stimulated by exogenous SA or its analogue INA. *WAK1* mRNA induction requires the positive regulator NPR1/NIM1 (Cheng et al., 2009). It provides a direct link between a protein kinase that could mediate signals from the ECM to the events that are precipitated by pathogen infection. It also suggests that while pathogen infection induces protective hangs in cells, these changes can be detrimental if certain cellular components, such as WAK1, are not present in sufficient amounts (Jones et al., 2004). In osmotic, salinity and dehydration stress, the expansion ability of the cell wall decreases. Correlated with this weakening was a substantial decrease in the proportion of crystalline cellulose in the primary cell wall while the amount of insoluble proteins (such as HRGPs) associated with the wall was increased relative to other wall

We have compared the ECM proteome of six plants, namely *Arabidopsis thaliana*, *Cicer arietinum Medicago sp*, *Oryza sativa*, *Zea mays* and *Brassica napus*. The *modus operandi* in investigating the cell wall proteomes of available crops was the extensive literature and availability of relevant databases (wallprotDB, Phosida, UniPro, ProtAnnDB pep2proandSwissprot) search. The CWPs identified in these works were classified into functional categories. This classification is only tentative, since the biological role of many of the proteins identified has not been established experimentally. Furthermore, we applied a cross-species comparison on the available datasets. When analysing proteomes within the specified group of plants, a logical strategy was used to maximise efficiency and the overall comparative results. Thus, it was imperative to first evaluate the available proteomes, followed by an analysis of organ-specific proteomes of the model plant *Arabidopsis*. Once the organspecific differential proteomes of the model plant were analysed, we then tentatively evaluated the developmental proteomics of rice at various leaf stages so as to understand the acquisition of major pathways involved in the development of the cereal. We then moved on to assessing the stress-responsive plant proteomes in order to understand the overlap and specificity amongst different environmental and patho-stress. These comparative studies were customised for specific protein families. For example, when the environmental stressresponsive proteomes were compared, the parallel analysis of the proteomes of different clades of vascular plants were performed, viz. *Arabidopsis* vs. maize for osmotic stress, and chick pea vs. rice for dehydration. Similarly, in case of patho-stress, *Arabidopsis* and *Brassica* proteomes were compared. It is to be noted that protein consensus can be obtained across any

combination of proteomes based on the type of extraction procedure.

#### **1.1.1 The ECM protein sink: A dynamic framework for multiple functions**

Earlier it was believed that ECM proteins were large and complex, with multiple distinct domains, and were highly conserved among the different taxa (Hall & Cannon, 2002). However, it is not necessary that proteins be large or complex in order to generate strong, stable fibrils and intermediate filament proteins. The conserved domains are now known to be arranged in specific juxtapositions, sometimes controlled by highly regulated alternative splicing (Hynes, 2009), indicating thereby that the specific domains and architectures of ECM proteins contain information of biological importance and evolutionary value. In plants, abundant wall proteins include those rich in hydroxyproline or proline (HRGPs, PRPs), glycine (GRPs) and arabinogalactan (AGPs). Expansins, which relax the linkages of the wall during cell elongation, play a crucial role in development. Peroxidases, methyltransferases, galactosidases, glycanases and proteases have also been identified as the cell wall resident proteins having an N-terminal targeting sequence. Perhaps the protein most expected to be similar to their metazoan counterparts in the plant cell wall is aggrecan, which binds hyaluronan orthologs. At least three classes of hydroxyproline-rich glycoproteins exist in higher plants, namely extensins, arabinogalactans and solanaceae lectins (Hall & Cannon, 2002,). Extensins, which comprise 5-10% of wall proteins, are assumed to play a role in the structure of plant cell walls and may, therefore, be important in controlling growth. Increasing evidence suggests that the level of extension is developmentally regulated. It also accumulates upon wounding and pathogen attack, suggesting its involvement in plant defence (Cassab, 1998). The fact that extensins and collagens are hydroxyproline containing glycoproteins means that they may have common evolutionary precursors (Chen & Verner, 1985). In addition, the primary cell wall includes numerous enzymes, viz. endoglucanases, xyloglucan endotransglycosylases and a number of other glycosyl transferases that alter carbohydrate linkages and modify secreted cell wall components. Tetraspanin - one of the important classes of ECM protein in higher plants but absent from unicellular eukaryotes - is known as the secretory carrier membrane protein, important for synaptic vesicle recycling in stigma-pollen interaction. Other cell wall proteins, some of which are heavily glycosylated, have been proposed as structural cell wall components and have been implicated in mediating multiple aspects of plant development (Irshad et al*.*, 2008). Germin is another ECM protein that signals the onset of growth and determines plant immunity. A chronic theme proverbial to the class of ECMcytoskeleton linker proteins of plant cells is that these mechano-transducing transmembrane molecules communicate and interact preferentially with the actin cytoskeleton on the cytoplasmic side of the plasma membrane. Generally, the actin cytoskeleton has been optimised during eukaryotic evolution for acting as a structural scaffold for diverse signalling complexes (Baluska et al., 2003). Bruce Kohorn classified putative plant-specific linker molecules in four categories, focusing on the four most appealing candidates: cell wall-associated kinases (WAKs), arabinogalactan proteins (AGPs), pectins and cellulose synthases. Progress made during the last three years has resulted in additional candidates, including formins, plant-specific class VIII myosins, phospholipase D and callose synthases. Unexpectedly, formins represent a new candidate for a putative ECM-cytoskeleton linker in plant cells. Current bioinformatic analyses show that there is one plant-specific group of formins not only abundant in cell wall but also moonlighting in cytosol.

Earlier it was believed that ECM proteins were large and complex, with multiple distinct domains, and were highly conserved among the different taxa (Hall & Cannon, 2002). However, it is not necessary that proteins be large or complex in order to generate strong, stable fibrils and intermediate filament proteins. The conserved domains are now known to be arranged in specific juxtapositions, sometimes controlled by highly regulated alternative splicing (Hynes, 2009), indicating thereby that the specific domains and architectures of ECM proteins contain information of biological importance and evolutionary value. In plants, abundant wall proteins include those rich in hydroxyproline or proline (HRGPs, PRPs), glycine (GRPs) and arabinogalactan (AGPs). Expansins, which relax the linkages of the wall during cell elongation, play a crucial role in development. Peroxidases, methyltransferases, galactosidases, glycanases and proteases have also been identified as the cell wall resident proteins having an N-terminal targeting sequence. Perhaps the protein most expected to be similar to their metazoan counterparts in the plant cell wall is aggrecan, which binds hyaluronan orthologs. At least three classes of hydroxyproline-rich glycoproteins exist in higher plants, namely extensins, arabinogalactans and solanaceae lectins (Hall & Cannon, 2002,). Extensins, which comprise 5-10% of wall proteins, are assumed to play a role in the structure of plant cell walls and may, therefore, be important in controlling growth. Increasing evidence suggests that the level of extension is developmentally regulated. It also accumulates upon wounding and pathogen attack, suggesting its involvement in plant defence (Cassab, 1998). The fact that extensins and collagens are hydroxyproline containing glycoproteins means that they may have common evolutionary precursors (Chen & Verner, 1985). In addition, the primary cell wall includes numerous enzymes, viz. endoglucanases, xyloglucan endotransglycosylases and a number of other glycosyl transferases that alter carbohydrate linkages and modify secreted cell wall components. Tetraspanin - one of the important classes of ECM protein in higher plants but absent from unicellular eukaryotes - is known as the secretory carrier membrane protein, important for synaptic vesicle recycling in stigma-pollen interaction. Other cell wall proteins, some of which are heavily glycosylated, have been proposed as structural cell wall components and have been implicated in mediating multiple aspects of plant development (Irshad et al*.*, 2008). Germin is another ECM protein that signals the onset of growth and determines plant immunity. A chronic theme proverbial to the class of ECMcytoskeleton linker proteins of plant cells is that these mechano-transducing transmembrane molecules communicate and interact preferentially with the actin cytoskeleton on the cytoplasmic side of the plasma membrane. Generally, the actin cytoskeleton has been optimised during eukaryotic evolution for acting as a structural scaffold for diverse signalling complexes (Baluska et al., 2003). Bruce Kohorn classified putative plant-specific linker molecules in four categories, focusing on the four most appealing candidates: cell wall-associated kinases (WAKs), arabinogalactan proteins (AGPs), pectins and cellulose synthases. Progress made during the last three years has resulted in additional candidates, including formins, plant-specific class VIII myosins, phospholipase D and callose synthases. Unexpectedly, formins represent a new candidate for a putative ECM-cytoskeleton linker in plant cells. Current bioinformatic analyses show that there is one plant-specific group of formins not only abundant in cell wall but also

**1.1.1 The ECM protein sink: A dynamic framework for multiple functions** 

moonlighting in cytosol.

#### **1.1.2 ECM proteins: Cross talk in signalling and stress**

ECM senses and physiologically responds to environmental stress via signalling pathways. Signalling events are clearly not linear and induce many different reactions, including stressrelated processes that crosstalk with hormone signalling pathways. It is known that cell wall stress provokes a transient depolarised distribution of the cell wall biosynthetic enzyme glucan synthase and its regulatory subunit RHO1, possibly as a mechanism to repair general damage to the wall. Both environmental and patho-stress are thought to cause wall weakening which in turn transduces a signal to the interior of the cell as a homeostatic mechanism to repair the wall. Various kinases mediate the stress-induced synthesis of ECM proteins to combat cell wall interfering factors, such as pathogens, osmotic stress, dehydration and other environmental stresses. Recently, it has been found that wall-associated kinase (*WAK)* expression was induced when *Arabidopsis* plants were infected with a pathogen or stimulated by exogenous SA or its analogue INA. *WAK1* mRNA induction requires the positive regulator NPR1/NIM1 (Cheng et al., 2009). It provides a direct link between a protein kinase that could mediate signals from the ECM to the events that are precipitated by pathogen infection. It also suggests that while pathogen infection induces protective hangs in cells, these changes can be detrimental if certain cellular components, such as WAK1, are not present in sufficient amounts (Jones et al., 2004). In osmotic, salinity and dehydration stress, the expansion ability of the cell wall decreases. Correlated with this weakening was a substantial decrease in the proportion of crystalline cellulose in the primary cell wall while the amount of insoluble proteins (such as HRGPs) associated with the wall was increased relative to other wall components (Sakurai et al., 1998).

#### **2. Methodology and strategy**

We have compared the ECM proteome of six plants, namely *Arabidopsis thaliana*, *Cicer arietinum Medicago sp*, *Oryza sativa*, *Zea mays* and *Brassica napus*. The *modus operandi* in investigating the cell wall proteomes of available crops was the extensive literature and availability of relevant databases (wallprotDB, Phosida, UniPro, ProtAnnDB pep2proandSwissprot) search. The CWPs identified in these works were classified into functional categories. This classification is only tentative, since the biological role of many of the proteins identified has not been established experimentally. Furthermore, we applied a cross-species comparison on the available datasets. When analysing proteomes within the specified group of plants, a logical strategy was used to maximise efficiency and the overall comparative results. Thus, it was imperative to first evaluate the available proteomes, followed by an analysis of organ-specific proteomes of the model plant *Arabidopsis*. Once the organspecific differential proteomes of the model plant were analysed, we then tentatively evaluated the developmental proteomics of rice at various leaf stages so as to understand the acquisition of major pathways involved in the development of the cereal. We then moved on to assessing the stress-responsive plant proteomes in order to understand the overlap and specificity amongst different environmental and patho-stress. These comparative studies were customised for specific protein families. For example, when the environmental stressresponsive proteomes were compared, the parallel analysis of the proteomes of different clades of vascular plants were performed, viz. *Arabidopsis* vs. maize for osmotic stress, and chick pea vs. rice for dehydration. Similarly, in case of patho-stress, *Arabidopsis* and *Brassica* proteomes were compared. It is to be noted that protein consensus can be obtained across any combination of proteomes based on the type of extraction procedure.

Comparative Analyses of Extracellular

proteome.

Matrix Proteome: An Under-Explored Area in Plant Research 151

Fig. 1. A flowchart illustrating the overall experimental strategy for the analysis of the ECM

ECM/cell wall design and protein composition has been shown to differ between two major clades, viz. the monocots and dicots of vascular plants. Results have mainly been obtained with the model plants *Arabidopsis thaliana* (Liepman et al*.*, 2010; Basu et al*.*, 2006; Bayer et al*.,* 2006; Borderies et al*.*, 2003; Chivasa et al*.*, 2002; Feiz et al*.*, 2006; Jamet et al. 2008a), *Medicago sativa* (Soares et al*.*, 2007; Watson et al*.*, 2004)*,* and crop plants for, e.g., *Oryza sativa* (Choudhary et al*.,* 2010), *Brassica napus* (Basu et al*.,* 2006) *Zea mays* (Zhu et al*.,* 2006) and *Cicer arietinum*  (Bhushan et al*.,* 2006). Around 500 CWPs of *Arabidopsis*, representing about one third of its estimated cell wall proteome, have been described (Liepman et al*.*, 2010) while 219, 143, 102, 58 CWPs were identified in rice, chickpea, maize and Brassica, respectively. Our comparative analysis of different species in relation to their function showed that a high percentage of proteins were found to be unique to each proteome: 87% in *A. thaliana*, 82% in *B. napus*, 84% in *C. arietinum*, 76% in *M. sativa*, 80% in *O. sativa* and 71% in *Z. mays*, with only peroxidase and

**3.1 Analysis of ECM proteome dynamics in plants: social class vs. diversity** 

glycosyl hydrolase being the social class of proteins present ubiquitously in all (Fig. 2).

#### **2.1 Description of tools**

An outline of the procedure and an illustration of the data that can be generated with the methodology are shown in Figure 1. Each proteomic study is described through a simplified flowchart showing its different steps, from plant material to protein identification. As illustrated in Figure 1, two types of methods can be used to prepare a CWP fraction. Non-destructive methods leave the cells alive and allow the elution of CWPs from cell walls using different buffered solutions, while destructive methods start with tissue grinding, thus mixing CWPs and intracellular proteins (Boudart et al., 2005; Bayers et al., 2006). The CWP fraction needs to be fractionated in order to allow for the identification of proteins by mass spectrometry (MS). Proteins can be directly submitted to enzymatic digestion with the appropriate proteases, such as trypsin, or to chemical treatment to get peptides of the appropriate mass (usually between 750 and 4000 Da). Alternatively, proteins are separated prior to cleavage into peptides. Since most CWPs are basic glycoproteins which are poorly resolved by bi-dimensional electrophoresis (2D-E), the most efficient means to separate them are either mono-dimensional electrophoresis (1D-E) (Boudart et al., 2005) or else cationic exchange chromatography followed by 1D-E of protein fractions eluted with a salt gradient (Irshad et al*.*, 2008). The identification of proteins can then be done either by peptide sequencing through liquid chromatography (LC) coupled to MS (LC-MS/MS) or by peptide mass mapping using the matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) MS followed by *in silico* analyses. At the bioinformatics end, custom ECM protein databases markedly increase the identification of extensively modified peptides. New generations of mass spectrometers will help meet the demand for high-throughput identification and the localisation of biologically significant peptide modifications.

#### **3. Results and discussion**

Proteomics has turned out to be an imperative benefactor for studying the acquaintance of plants' ECM structure and functions by allowing the identification of proteins present in this cellular compartment. It is a well known fact that the field of proteomics is evolving from the cataloguing proteins under static conditions to comparative analyses. Defining proteins that change in abundance, form, location or other activities may indicate the presence and functional significance of a protein. Whereas comparative ECM proteome research is quite advanced in animals (Zhu et al., 2007) and yeast (Kim et al., 2007)*,* there is less information as to plants. The identification and cataloguing of plant ECM proteomes in recent years raises the following important questions: What are the essential plant ECM proteins? Do ECM proteins show clade specificity in vascular plants? What are those organ-specific cell wall proteins, if any? Does the cell wall developmental proteomics of one of the clades yield any astonishing or prolific results? How does ECM protein remodelling during environmental and/or patho-stress provide new perspectives? Are some of the ECM proteins unexpected? And, last but not the least, what sort of post-translational modifications have so far been characterised in CWP? Here we analyse and compare the experimental results of the thus far available proteomes so as to elucidate the dynamics of plant ECM /cell wall proteins.

An outline of the procedure and an illustration of the data that can be generated with the methodology are shown in Figure 1. Each proteomic study is described through a simplified flowchart showing its different steps, from plant material to protein identification. As illustrated in Figure 1, two types of methods can be used to prepare a CWP fraction. Non-destructive methods leave the cells alive and allow the elution of CWPs from cell walls using different buffered solutions, while destructive methods start with tissue grinding, thus mixing CWPs and intracellular proteins (Boudart et al., 2005; Bayers et al., 2006). The CWP fraction needs to be fractionated in order to allow for the identification of proteins by mass spectrometry (MS). Proteins can be directly submitted to enzymatic digestion with the appropriate proteases, such as trypsin, or to chemical treatment to get peptides of the appropriate mass (usually between 750 and 4000 Da). Alternatively, proteins are separated prior to cleavage into peptides. Since most CWPs are basic glycoproteins which are poorly resolved by bi-dimensional electrophoresis (2D-E), the most efficient means to separate them are either mono-dimensional electrophoresis (1D-E) (Boudart et al., 2005) or else cationic exchange chromatography followed by 1D-E of protein fractions eluted with a salt gradient (Irshad et al*.*, 2008). The identification of proteins can then be done either by peptide sequencing through liquid chromatography (LC) coupled to MS (LC-MS/MS) or by peptide mass mapping using the matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) MS followed by *in silico* analyses. At the bioinformatics end, custom ECM protein databases markedly increase the identification of extensively modified peptides. New generations of mass spectrometers will help meet the demand for high-throughput identification and the localisation of

Proteomics has turned out to be an imperative benefactor for studying the acquaintance of plants' ECM structure and functions by allowing the identification of proteins present in this cellular compartment. It is a well known fact that the field of proteomics is evolving from the cataloguing proteins under static conditions to comparative analyses. Defining proteins that change in abundance, form, location or other activities may indicate the presence and functional significance of a protein. Whereas comparative ECM proteome research is quite advanced in animals (Zhu et al., 2007) and yeast (Kim et al., 2007)*,* there is less information as to plants. The identification and cataloguing of plant ECM proteomes in recent years raises the following important questions: What are the essential plant ECM proteins? Do ECM proteins show clade specificity in vascular plants? What are those organ-specific cell wall proteins, if any? Does the cell wall developmental proteomics of one of the clades yield any astonishing or prolific results? How does ECM protein remodelling during environmental and/or patho-stress provide new perspectives? Are some of the ECM proteins unexpected? And, last but not the least, what sort of post-translational modifications have so far been characterised in CWP? Here we analyse and compare the experimental results of the thus far available proteomes so as to elucidate the dynamics of

**2.1 Description of tools** 

biologically significant peptide modifications.

**3. Results and discussion** 

plant ECM /cell wall proteins.

Fig. 1. A flowchart illustrating the overall experimental strategy for the analysis of the ECM proteome.

#### **3.1 Analysis of ECM proteome dynamics in plants: social class vs. diversity**

ECM/cell wall design and protein composition has been shown to differ between two major clades, viz. the monocots and dicots of vascular plants. Results have mainly been obtained with the model plants *Arabidopsis thaliana* (Liepman et al*.*, 2010; Basu et al*.*, 2006; Bayer et al*.,* 2006; Borderies et al*.*, 2003; Chivasa et al*.*, 2002; Feiz et al*.*, 2006; Jamet et al. 2008a), *Medicago sativa* (Soares et al*.*, 2007; Watson et al*.*, 2004)*,* and crop plants for, e.g., *Oryza sativa* (Choudhary et al*.,* 2010), *Brassica napus* (Basu et al*.,* 2006) *Zea mays* (Zhu et al*.,* 2006) and *Cicer arietinum*  (Bhushan et al*.,* 2006). Around 500 CWPs of *Arabidopsis*, representing about one third of its estimated cell wall proteome, have been described (Liepman et al*.*, 2010) while 219, 143, 102, 58 CWPs were identified in rice, chickpea, maize and Brassica, respectively. Our comparative analysis of different species in relation to their function showed that a high percentage of proteins were found to be unique to each proteome: 87% in *A. thaliana*, 82% in *B. napus*, 84% in *C. arietinum*, 76% in *M. sativa*, 80% in *O. sativa* and 71% in *Z. mays*, with only peroxidase and glycosyl hydrolase being the social class of proteins present ubiquitously in all (Fig. 2).

Comparative Analyses of Extracellular

than in *Cicer* (28 proteins).

Matrix Proteome: An Under-Explored Area in Plant Research 153

The available ECM proteome of the six plants compared in Figure 2 varied in molecular weight from 8.9 to 133.8 kDa and had a spread of p*I* values from 5.2 to 10.1. Seventy-six percent of the ECM proteins were basic in nature, concordant with the acidic environment of the wall. Between monocots and dicots, it was found that rice and *Arabidopsis* have a similar number of cell wall-related gene families and members within each family, even though rice has a far greater number of genes than Arabidopsis. This implies that similar numbers of genes are required for wall construction and maintenance, at least among Angiosperms (Yokoyama et al 2004). However, the cell wall proteome data of *Arabidopsis* is better explored than rice and therefore comparison of their proteome may not yield the postulated results as defined by genome analysis. When the maize cell wall proteome was compared with that of *Arabidopsis*, the results revealed an evolutionary divergence as well as tissue specificity, with few conserved proteins (Fig. 2). The protein network of maize (Zhu et al., 2006) revealed the predominance of the inhibitors of hemicellulose-degrading enzymes from monocots, such as endoxylanase inhibitors, and the *Arabidopsis* protein network (Slabas et al., 2004; Peck, 2005) was found to be rich in xyloglucan endoglucanase-inhibiting proteins and glycine rich protein as cell wall remodelling or biosynthetic enzymes. Comparison of the functional classes of cell wall proteins amongst dicot species like *Arabidopsis, Brassica, Medicago* and *Cicer* confirms the dynamic nature of the cell wall, as exemplified by the presence of cellulose synthase and peroxidase in all dicots. However, surprisingly the protein turnover rate of these enzymes are greater in *Medicago*. A more comprehensive investigation of the studied legume proteomes revealed that the proportion of proteins involved in cell wall modification is three times greater in *Medicago (*99 proteins*)*

This may be due to the fact that ionically bound and soluble ECM proteins can be separated with ease from *Medicago* as compared with *Cicer*. Additional variation of cell wall proteomes in *Cicer* and *Medicago* is provided by the presence of ferritin in the former and Polypolyglutamatesynthase in the latter, illustrating that nature invented vastly different solutions to a common problem, viz. transport and storage. When the studies on the legumes like *Cicer* and *Medicago* were compared to *Arabidopsis* belonging to the Brassicaceae family (Fig. 2), it can be readily observed that the protein machinery of the wall for activating the wall-modifying enzymes is diverse between the two families as well as between the members of the same family, leguminosae. Investigation between *Arabidopsis* and *Brassica* proteomes by MudPIT, using a homology-based search, unambiguously identified 16 proteins which were common to the 52 proteins of *Arabidopsis*. When the cell wall proteomes of *Oryza* (145) and *Zea* (128) were compared, less diversity was observed in Poaceae (Pandey et al., 2010; Zhu et al., 2006) except for the fact that one of the CWP expansins - HPT - is expressed by a moderate amount in maize, whereas in rice PRP is represented by a moderate number (Fig. 2). It may be assumed that the divergence in the resulting proteomes of the vascular plants is due to the presence of the different design of their wall based on their carbohydrate composition. It is known that type I carbohydrates which typically contains xyloglucan and/or glucomannan and 20–35% pectin - are found in all dicotyledons whereas type II carbohydrate rich in arabinoglycan are only characteristic of the Poaceae family in the monocot, suggesting the occurrence of clade-specific ECM proteins that would bind to their cognitive carbohydrate molecules. Most intriguing are the remaining 10% of ECM proteins that do not have any similarity to the known proteins in other organisms. The challenge is to elucidate their biological role within the cell wall.

Fig. 2. Cross-species comparison of ECM proteomes. The functional classification of the identified proteins was made according to the biological processes in which they are involved. The length of the bar indicates the number of proteins present in a particular species, such as *Arabidopsis thaliana*, *Cicer arientum*, *Medicago sativa*, *Oryza sativa*, *Zea mays*  and *Brassica napus.* The pie chart inset represents the fraction of unknown protein classes in each of these plants.

Fig. 2. Cross-species comparison of ECM proteomes. The functional classification of the identified proteins was made according to the biological processes in which they are involved. The length of the bar indicates the number of proteins present in a particular species, such as *Arabidopsis thaliana*, *Cicer arientum*, *Medicago sativa*, *Oryza sativa*, *Zea mays*  and *Brassica napus.* The pie chart inset represents the fraction of unknown protein classes in

each of these plants.

The available ECM proteome of the six plants compared in Figure 2 varied in molecular weight from 8.9 to 133.8 kDa and had a spread of p*I* values from 5.2 to 10.1. Seventy-six percent of the ECM proteins were basic in nature, concordant with the acidic environment of the wall. Between monocots and dicots, it was found that rice and *Arabidopsis* have a similar number of cell wall-related gene families and members within each family, even though rice has a far greater number of genes than Arabidopsis. This implies that similar numbers of genes are required for wall construction and maintenance, at least among Angiosperms (Yokoyama et al 2004). However, the cell wall proteome data of *Arabidopsis* is better explored than rice and therefore comparison of their proteome may not yield the postulated results as defined by genome analysis. When the maize cell wall proteome was compared with that of *Arabidopsis*, the results revealed an evolutionary divergence as well as tissue specificity, with few conserved proteins (Fig. 2). The protein network of maize (Zhu et al., 2006) revealed the predominance of the inhibitors of hemicellulose-degrading enzymes from monocots, such as endoxylanase inhibitors, and the *Arabidopsis* protein network (Slabas et al., 2004; Peck, 2005) was found to be rich in xyloglucan endoglucanase-inhibiting proteins and glycine rich protein as cell wall remodelling or biosynthetic enzymes. Comparison of the functional classes of cell wall proteins amongst dicot species like *Arabidopsis, Brassica, Medicago* and *Cicer* confirms the dynamic nature of the cell wall, as exemplified by the presence of cellulose synthase and peroxidase in all dicots. However, surprisingly the protein turnover rate of these enzymes are greater in *Medicago*. A more comprehensive investigation of the studied legume proteomes revealed that the proportion of proteins involved in cell wall modification is three times greater in *Medicago (*99 proteins*)* than in *Cicer* (28 proteins).

This may be due to the fact that ionically bound and soluble ECM proteins can be separated with ease from *Medicago* as compared with *Cicer*. Additional variation of cell wall proteomes in *Cicer* and *Medicago* is provided by the presence of ferritin in the former and Polypolyglutamatesynthase in the latter, illustrating that nature invented vastly different solutions to a common problem, viz. transport and storage. When the studies on the legumes like *Cicer* and *Medicago* were compared to *Arabidopsis* belonging to the Brassicaceae family (Fig. 2), it can be readily observed that the protein machinery of the wall for activating the wall-modifying enzymes is diverse between the two families as well as between the members of the same family, leguminosae. Investigation between *Arabidopsis* and *Brassica* proteomes by MudPIT, using a homology-based search, unambiguously identified 16 proteins which were common to the 52 proteins of *Arabidopsis*. When the cell wall proteomes of *Oryza* (145) and *Zea* (128) were compared, less diversity was observed in Poaceae (Pandey et al., 2010; Zhu et al., 2006) except for the fact that one of the CWP expansins - HPT - is expressed by a moderate amount in maize, whereas in rice PRP is represented by a moderate number (Fig. 2). It may be assumed that the divergence in the resulting proteomes of the vascular plants is due to the presence of the different design of their wall based on their carbohydrate composition. It is known that type I carbohydrates which typically contains xyloglucan and/or glucomannan and 20–35% pectin - are found in all dicotyledons whereas type II carbohydrate rich in arabinoglycan are only characteristic of the Poaceae family in the monocot, suggesting the occurrence of clade-specific ECM proteins that would bind to their cognitive carbohydrate molecules. Most intriguing are the remaining 10% of ECM proteins that do not have any similarity to the known proteins in other organisms. The challenge is to elucidate their biological role within the cell wall.

Comparative Analyses of Extracellular

Matrix Proteome: An Under-Explored Area in Plant Research 155

Fig. 3. The organ-specific comparative ECM proteome in *Arabidopsis*. The functional classification of the identified proteins was according to the biological processes in which they are involved. The length of the bars indicates the number of proteins present in a particular organ or culture, such as roots, the stem, the cell suspension culture, the culture medium of the cell suspension culture, etiolated hypocotyls, etiolated seedlings grown in the liquid medium, the culture medium of the etiolated seedlings, protoplast and leaves.

#### **3.2 Discerning organ-specific ECM proteomes in** *Arabidopsis thaliana*

We further analysed the organ-specific proteome of *Arabidopsis thaliana*, namely root, stem, leaves, etiolated hypocotyl, etiolated seedlings from liquid and cultured media, and protoplast- and leaf-derived cell suspension (Miller & Fry, 1992; Feiz et al., 2006; Minic et al., 2007, Irshad et al., 2008; Minic et al., 2008). Comparative analysis (Fig. 3) revealed that cell wall modifying proteins, structural proteins and proteins involved in signalling and development constitute 58% of the ECM's proteins in mature stems (71) and dark-grown hypocotyls (147) with high and moderate expression. However, it was intriguing to note that most of these CWPs identified by the proteomics study originate from genes whose level of transcripts was low (between 37% and 58%) or below the background (between 18% and 25%) as reported in Minic et al., 2007 indicating thereby the importance of the post-transcriptional regulation of organ-specific ECM proteomes. A further 29 and 54 cell wall modifying proteins were identified in the roots and leaves respectively, in which members of the hydroxyproline-rich glycoprotein family and other major structural proteins were not detected. For a few protein sequences within a particular organ, there also exists a certain degree of heterogeneity in terms of the occasional amino acid substitution as well as their appearance at different molecular weights. The former may be explained due to the origin of these protein species from different genes and the latter by post-translational modifications, such as glycosylation. Expansin, a cell wall modifying component, was the most dominant class in all the major organs, while wellknown cell wall enzymes like glycoside hydrolase, pectin methylesterases, peroxidases and glycosyl transferases were represented by several members of the same family (Fig. 3). The analysis of protoplast and suspension-cultured cell derived proteomes in *Arabidopsis* and rice showed the relative abundance of the GH family of ECM proteins. They might be involved in the modification of mixed glycan polymers, only found in monocot cell walls during the regeneration of the cell wall in the protoplast. However, the role of GH family of proteins has not yet been elucidated in *Arabidopsis*. A moderate number of carbohydrate esterases were identified in the ECM proteome of the cell suspension culture, etiolated hypocotyl and leaves while a novel family of HRGP, called LRR-extensin proteins (LRX), has only been found in the case of cell suspension cultures. The only organ in which a few salt-extractable structural proteins were identified is etiolated hypocotyl, possibly because such proteins are not yet completely insolubilised from other organs. Proteins having domains of interaction with proteins or polysaccharides are well-represented in all organs, and especially in rosettes. As expected from the fact that GH represents almost 20% of the identified CWPs (Fig 2 and 3), proteins acting on cell wall polysaccharides are also the category with the highest diversity within each organ. Oxidoreductases are particularly numerous in cell suspension cultures, probably due to the mechanical stress produced by continuous spinning and the oxidative stress that occurs in the liquid media culture. At least 20% of the identified CWPs represent a social class in one organ not found in the others. This may be partially linked to the high redundancy in the number of genes encoding each CWP family, presumably differentiallyregulated during organ development (Fig. 3).

#### **3.3 Exploring the variability of the developmental stage specific ECM proteome**

A cornerstone of evolution is associated with the diversity of individuals within a population. This diversity is generally understood to arise at the genetic level and leads to characteristics that may be advantageous or disadvantageous within the context of the

We further analysed the organ-specific proteome of *Arabidopsis thaliana*, namely root, stem, leaves, etiolated hypocotyl, etiolated seedlings from liquid and cultured media, and protoplast- and leaf-derived cell suspension (Miller & Fry, 1992; Feiz et al., 2006; Minic et al., 2007, Irshad et al., 2008; Minic et al., 2008). Comparative analysis (Fig. 3) revealed that cell wall modifying proteins, structural proteins and proteins involved in signalling and development constitute 58% of the ECM's proteins in mature stems (71) and dark-grown hypocotyls (147) with high and moderate expression. However, it was intriguing to note that most of these CWPs identified by the proteomics study originate from genes whose level of transcripts was low (between 37% and 58%) or below the background (between 18% and 25%) as reported in Minic et al., 2007 indicating thereby the importance of the post-transcriptional regulation of organ-specific ECM proteomes. A further 29 and 54 cell wall modifying proteins were identified in the roots and leaves respectively, in which members of the hydroxyproline-rich glycoprotein family and other major structural proteins were not detected. For a few protein sequences within a particular organ, there also exists a certain degree of heterogeneity in terms of the occasional amino acid substitution as well as their appearance at different molecular weights. The former may be explained due to the origin of these protein species from different genes and the latter by post-translational modifications, such as glycosylation. Expansin, a cell wall modifying component, was the most dominant class in all the major organs, while wellknown cell wall enzymes like glycoside hydrolase, pectin methylesterases, peroxidases and glycosyl transferases were represented by several members of the same family (Fig. 3). The analysis of protoplast and suspension-cultured cell derived proteomes in *Arabidopsis* and rice showed the relative abundance of the GH family of ECM proteins. They might be involved in the modification of mixed glycan polymers, only found in monocot cell walls during the regeneration of the cell wall in the protoplast. However, the role of GH family of proteins has not yet been elucidated in *Arabidopsis*. A moderate number of carbohydrate esterases were identified in the ECM proteome of the cell suspension culture, etiolated hypocotyl and leaves while a novel family of HRGP, called LRR-extensin proteins (LRX), has only been found in the case of cell suspension cultures. The only organ in which a few salt-extractable structural proteins were identified is etiolated hypocotyl, possibly because such proteins are not yet completely insolubilised from other organs. Proteins having domains of interaction with proteins or polysaccharides are well-represented in all organs, and especially in rosettes. As expected from the fact that GH represents almost 20% of the identified CWPs (Fig 2 and 3), proteins acting on cell wall polysaccharides are also the category with the highest diversity within each organ. Oxidoreductases are particularly numerous in cell suspension cultures, probably due to the mechanical stress produced by continuous spinning and the oxidative stress that occurs in the liquid media culture. At least 20% of the identified CWPs represent a social class in one organ not found in the others. This may be partially linked to the high redundancy in the number of genes encoding each CWP family, presumably differentially-

**3.2 Discerning organ-specific ECM proteomes in** *Arabidopsis thaliana*

regulated during organ development (Fig. 3).

**3.3 Exploring the variability of the developmental stage specific ECM proteome** 

A cornerstone of evolution is associated with the diversity of individuals within a population. This diversity is generally understood to arise at the genetic level and leads to characteristics that may be advantageous or disadvantageous within the context of the

Fig. 3. The organ-specific comparative ECM proteome in *Arabidopsis*. The functional classification of the identified proteins was according to the biological processes in which they are involved. The length of the bars indicates the number of proteins present in a particular organ or culture, such as roots, the stem, the cell suspension culture, the culture medium of the cell suspension culture, etiolated hypocotyls, etiolated seedlings grown in the liquid medium, the culture medium of the etiolated seedlings, protoplast and leaves.

Comparative Analyses of Extracellular

Matrix Proteome: An Under-Explored Area in Plant Research 157

Fig. 4. The developmental stage specific comparative ECM proteome in rice. The functional classification of the identified proteins was according to the biological processes in which they are involved. The length of the bars indicates the number of proteins present in a

particular leaf stage/day of suspension culture.

environment (Taraszka et al., 2005). The emerging field of developmental proteomics, in which large mixtures of proteins are characterised in a single experimental sequence, may allow for the assessment of variability or similarity in an individual at the level of the proteome (Hunter et al., 2002). The developmental proteomics of rice is perhaps the least studied, but its importance was realised when the proteome of rice at 5 days and the third and fourth leaf stages were analysed (Jung et al 2008; Chen et al., 2009). When we compared the existing dataset, even though the proteomes were found to be similar, some of the CWPs which were unknown earlier were uniquely present at a particular developmental stage. For example, COBRA and Leucine rich repeat extensins were found only in the third leaf stage while the polysaccharide lyase appeared in the fourth leaf stage (Fig. 4). Although ECM proteins which regulate development and expansion form the major class, very few have been functionally characterised so far. Such a protein, COBRA (COB), anchored to the extracellular surface of the plasma membrane by a glycosyl phosphatidylinositol (GPI) moiety is thought to regulate and link oriented-cell expansion in root cells (Brady, 2007). Another protein, LRX1, a chimeric leucine-rich repeat/extensin is also expressed in root hair cells. The interaction between the cell wall and the LRX1 protein is important for proper root hair development and expansion (Diet et al., 2006). A family of secreted proteins called SCAs (stigma/stylar cysteine-rich adhesion) was identified as a pollen tube adhesion molecule for the wall material of the style found in the lily (Baumberger et al., 2001). One of the ECM protein family Arabinogalactan-proteins (AGPs) belonging to the category of HRGP consists of a rather small and highly glycosylated protein moiety which has been found to play vital role in cell wall development (Gillmor et al., 2005). THESEUS1 (THE1), which is a member of the subfamily of the *Catharanthus roseus* protein kinase1-like receptor kinases also has efficacy in cell wall integrity, sensing and development (Hematy et al., 2007). Thus, the resulting cell wall proteomes were different, showing in another way that the cell wall structure and composition are regulated during development. However, the biological functions of most CWPs involved in development have not yet been experimentally studied.

#### **3.4** *In silico* **protein profiling of comparative ECM stress proteomes**

The plant cell wall or the extracellular matrix (ECM) is the first compartment that senses stress signals, transmits them to the cell interior and eventually influences the cell fate decision (Ellis et al., 2002), and thus it can be envisaged that ECM proteomes primarily regulate the environmental and patho-stress response in plants. We analysed the cell wall proteomes of *Arabidopsis* and maize in response to osmotic stress (Kachroo et al., 2001; Amaya et al., 1999), and the dehydration responsive ECM proteomes of chickpea and rice (Bhushan et al., 2007; Pandey et al., 2008; Choudhary et al., 2009; Pandey, et al., 2010; Bhushan et al., 2011). Interestingly, a great deal of divergence in the protein classes amongst these organisms was observed (Fig. 5A).

To our surprise, except for peroxidase, serine protease and subtilisin none of the ECM proteins was found to be common in all the organisms under both kinds of the stresses studied. The families of antimicrobial peptides such as thionins, defensins and knottin-like peptides have been found in the dehydration-responsive proteome of chickpea, while it was found that rice DRPs comprised of antimicrobial peptides such as oryzacystatin, thioredoxin and oligopeptidase. The *Cicer* dehydration-responsive protein network showed the exclusive presence of glycine-rich protein, methionine synthase, ferritin, tubby-like protein

environment (Taraszka et al., 2005). The emerging field of developmental proteomics, in which large mixtures of proteins are characterised in a single experimental sequence, may allow for the assessment of variability or similarity in an individual at the level of the proteome (Hunter et al., 2002). The developmental proteomics of rice is perhaps the least studied, but its importance was realised when the proteome of rice at 5 days and the third and fourth leaf stages were analysed (Jung et al 2008; Chen et al., 2009). When we compared the existing dataset, even though the proteomes were found to be similar, some of the CWPs which were unknown earlier were uniquely present at a particular developmental stage. For example, COBRA and Leucine rich repeat extensins were found only in the third leaf stage while the polysaccharide lyase appeared in the fourth leaf stage (Fig. 4). Although ECM proteins which regulate development and expansion form the major class, very few have been functionally characterised so far. Such a protein, COBRA (COB), anchored to the extracellular surface of the plasma membrane by a glycosyl phosphatidylinositol (GPI) moiety is thought to regulate and link oriented-cell expansion in root cells (Brady, 2007). Another protein, LRX1, a chimeric leucine-rich repeat/extensin is also expressed in root hair cells. The interaction between the cell wall and the LRX1 protein is important for proper root hair development and expansion (Diet et al., 2006). A family of secreted proteins called SCAs (stigma/stylar cysteine-rich adhesion) was identified as a pollen tube adhesion molecule for the wall material of the style found in the lily (Baumberger et al., 2001). One of the ECM protein family Arabinogalactan-proteins (AGPs) belonging to the category of HRGP consists of a rather small and highly glycosylated protein moiety which has been found to play vital role in cell wall development (Gillmor et al., 2005). THESEUS1 (THE1), which is a member of the subfamily of the *Catharanthus roseus* protein kinase1-like receptor kinases also has efficacy in cell wall integrity, sensing and development (Hematy et al., 2007). Thus, the resulting cell wall proteomes were different, showing in another way that the cell wall structure and composition are regulated during development. However, the biological functions of most CWPs involved in development have not yet been

experimentally studied.

these organisms was observed (Fig. 5A).

**3.4** *In silico* **protein profiling of comparative ECM stress proteomes** 

The plant cell wall or the extracellular matrix (ECM) is the first compartment that senses stress signals, transmits them to the cell interior and eventually influences the cell fate decision (Ellis et al., 2002), and thus it can be envisaged that ECM proteomes primarily regulate the environmental and patho-stress response in plants. We analysed the cell wall proteomes of *Arabidopsis* and maize in response to osmotic stress (Kachroo et al., 2001; Amaya et al., 1999), and the dehydration responsive ECM proteomes of chickpea and rice (Bhushan et al., 2007; Pandey et al., 2008; Choudhary et al., 2009; Pandey, et al., 2010; Bhushan et al., 2011). Interestingly, a great deal of divergence in the protein classes amongst

To our surprise, except for peroxidase, serine protease and subtilisin none of the ECM proteins was found to be common in all the organisms under both kinds of the stresses studied. The families of antimicrobial peptides such as thionins, defensins and knottin-like peptides have been found in the dehydration-responsive proteome of chickpea, while it was found that rice DRPs comprised of antimicrobial peptides such as oryzacystatin, thioredoxin and oligopeptidase. The *Cicer* dehydration-responsive protein network showed the exclusive presence of glycine-rich protein, methionine synthase, ferritin, tubby-like protein

Fig. 4. The developmental stage specific comparative ECM proteome in rice. The functional classification of the identified proteins was according to the biological processes in which they are involved. The length of the bars indicates the number of proteins present in a particular leaf stage/day of suspension culture.

Comparative Analyses of Extracellular

orthologs vs. paralogs.

et al., 2001; Tran & Plaxton, 2008).

Matrix Proteome: An Under-Explored Area in Plant Research 159

and leucine aminopeptidases. Another important finding was the presence of falacinin-like AGPs during osmotic stress in *Arabidopsis*, but not in other cases. Extensin, hydroxyproline transferase and carbohydrate esterase were predominantly found during the dehydration response but were absent in response to osmotic stress. Interestingly, rice as well as maize cell wall proteomes under both types of abiotic stresses revealed the presence of class III chitinase, plastocyanin, S-adenosylmethionine transferase and cyclosporine, suggesting their clade-specific expression. Our analysis revealed the presence of monocot and dicot peroxidases having specific protein sequences that clearly demonstrate the diversity of the identical CWPs in two divisions of angiosperm. This may be attributed to the evolution of

Moreover, plant cell walls constitute the first stage of defence against invading pathogens. The endogenous wall metabolism might facilitate pathogen infection, either because wall substrates are made more physically accessible to pathogens or because the plant enzymes convert wall polymers into appropriate nutritional substrates for the invading microorganism. In addition to the crucial role of CWPs in growth and development, these proteins or peptides are also involved in plant defence mechanisms in response to pathostress. Earlier, a number of ECM proteins have been shown to play a crucial role in plant defence against microbes (Sakurai, 1998), including pathogenesis-related (PR) proteins, chitinases and endo-b-1,3-glucanases, that are known to directly interact with pathogens (Jung et al., 2004; Jones et al., 2006). However, plants also deploy a repertoire of proteins in the wall that act as a surveillance system to allow the early detection of an impending pathogen assault. We analysed the cell wall proteomes of *Arabidopsis* and *Brassica napus* in response to fungal stresses (Ndimba et al., 2003; Floerl et al., 2008), and elicitor-induced ECM proteome of *Zea mays* (Chivasa et al., 2005). The common ECM proteins identified in fungal stress were jacalin-related, LRR-containing proteins, chitinase, thaumatin-like proteins, esterase/lipase thioesterase and the GLIP1 lipase. On contrary the, S-AMT, COBRA, FLA, BBE, CE and GRPs were found to be exclusive in the case of *Arabidopsis-Fusarium* interactions, suggesting that the cell wall is a dynamic milieu and responds differently in response to different pathogen within the divisions or in between the divisions of the angiosperm (Fig. 5B). Likewise, in order to assess the generality of the cell wall proteome of *A. thaliana* (Oh et al., 2005) and *B. napus* (Floerl et al., 2008) under pathostress were compared. The results indicate that the fungal stress-induced changes in CWPs were diverse in both of the plants except for the oxidoreductases, stress- and adaptation-related proteins and structural proteins. Meanwhile signalling-, transport- and development-related proteins were induced mostly in *A. thaliana*, except for the AGPs which were commonly present in both of the proteomes (Fig. 5B). Thus, *B. napus* may depend exclusively on AGPs-mediated stress signalling responses, whereas diverse signalling pathways operate in *A. thaliana.* Pathogen elicitor-induced changes in maize ECM proteomes revealed the involvement of lipases, esterases and thiols similar to the response of *Arabidopsis* to pathogen invasion**.** However, how monocot cell wall proteins respond to pathogens still needs further investigation in order to comprehend the monocot-dicot difference in response to pathogen invasion. In addition, a number of extracellular proteins and peptides have been identified that contribute to signalling and the recognition of not only pathogens but also other cell-type responses, such as in pollen–pistil interactions and the phosphate deficiency proteome of *Arabidopsis* (Kachroo

Fig. 5. Comparative stress proteome: A comparison of various functional classes of the extracellular matrix protein in environmental stress (A) and in patho-stress (B). The functional classification of the identified proteins was according to the biological processes in which they are involved. The length of the bars indicates the number of proteins present in a particular stress.

Fig. 5. Comparative stress proteome: A comparison of various functional classes of the extracellular matrix protein in environmental stress (A) and in patho-stress (B). The functional classification of the identified proteins was according to the biological processes in which they are involved. The length of the bars indicates the number of proteins present

in a particular stress.

and leucine aminopeptidases. Another important finding was the presence of falacinin-like AGPs during osmotic stress in *Arabidopsis*, but not in other cases. Extensin, hydroxyproline transferase and carbohydrate esterase were predominantly found during the dehydration response but were absent in response to osmotic stress. Interestingly, rice as well as maize cell wall proteomes under both types of abiotic stresses revealed the presence of class III chitinase, plastocyanin, S-adenosylmethionine transferase and cyclosporine, suggesting their clade-specific expression. Our analysis revealed the presence of monocot and dicot peroxidases having specific protein sequences that clearly demonstrate the diversity of the identical CWPs in two divisions of angiosperm. This may be attributed to the evolution of orthologs vs. paralogs.

Moreover, plant cell walls constitute the first stage of defence against invading pathogens. The endogenous wall metabolism might facilitate pathogen infection, either because wall substrates are made more physically accessible to pathogens or because the plant enzymes convert wall polymers into appropriate nutritional substrates for the invading microorganism. In addition to the crucial role of CWPs in growth and development, these proteins or peptides are also involved in plant defence mechanisms in response to pathostress. Earlier, a number of ECM proteins have been shown to play a crucial role in plant defence against microbes (Sakurai, 1998), including pathogenesis-related (PR) proteins, chitinases and endo-b-1,3-glucanases, that are known to directly interact with pathogens (Jung et al., 2004; Jones et al., 2006). However, plants also deploy a repertoire of proteins in the wall that act as a surveillance system to allow the early detection of an impending pathogen assault. We analysed the cell wall proteomes of *Arabidopsis* and *Brassica napus* in response to fungal stresses (Ndimba et al., 2003; Floerl et al., 2008), and elicitor-induced ECM proteome of *Zea mays* (Chivasa et al., 2005). The common ECM proteins identified in fungal stress were jacalin-related, LRR-containing proteins, chitinase, thaumatin-like proteins, esterase/lipase thioesterase and the GLIP1 lipase. On contrary the, S-AMT, COBRA, FLA, BBE, CE and GRPs were found to be exclusive in the case of *Arabidopsis-Fusarium* interactions, suggesting that the cell wall is a dynamic milieu and responds differently in response to different pathogen within the divisions or in between the divisions of the angiosperm (Fig. 5B). Likewise, in order to assess the generality of the cell wall proteome of *A. thaliana* (Oh et al., 2005) and *B. napus* (Floerl et al., 2008) under pathostress were compared. The results indicate that the fungal stress-induced changes in CWPs were diverse in both of the plants except for the oxidoreductases, stress- and adaptation-related proteins and structural proteins. Meanwhile signalling-, transport- and development-related proteins were induced mostly in *A. thaliana*, except for the AGPs which were commonly present in both of the proteomes (Fig. 5B). Thus, *B. napus* may depend exclusively on AGPs-mediated stress signalling responses, whereas diverse signalling pathways operate in *A. thaliana.* Pathogen elicitor-induced changes in maize ECM proteomes revealed the involvement of lipases, esterases and thiols similar to the response of *Arabidopsis* to pathogen invasion**.** However, how monocot cell wall proteins respond to pathogens still needs further investigation in order to comprehend the monocot-dicot difference in response to pathogen invasion. In addition, a number of extracellular proteins and peptides have been identified that contribute to signalling and the recognition of not only pathogens but also other cell-type responses, such as in pollen–pistil interactions and the phosphate deficiency proteome of *Arabidopsis* (Kachroo et al., 2001; Tran & Plaxton, 2008).

Comparative Analyses of Extracellular

hydroxyproline-rich glycoprotein, THRGP;

*Lett.*, 457: 80-84.

*Physiol.,* 133: 482-491.

**6. Abbreviations** 

**7. References** 

Matrix Proteome: An Under-Explored Area in Plant Research 161

Polysaccharide lyase, PL; gibberellin acid-stimulated Arabidopsis (GASA) protein, GASA; pectin methyl esterase inhibitor, PMEI; Expansin, Exp; carbohydrate esterase, CE; glycoside hydrolase, GH; Laccase, lacc; blue copper binding protein, BCuBP; berberine-bridge oxidoreductase, B-BOxRe; multicopper oxidase, MCuOx; Peroxidase, Pox; glycine-rich protein, GRP; proline-rich protein, PRP; leucine-rich repeat extensin, LRX; LRR protein, LRR;signal eptidase, SP; COBRA-like family, COBRA; fasciclin-like arabinogalactan protein, FLA; arabinogalactan protein, AGP; lipid transfer protein, LTP Ser carboxypeptidase, SerCP; Cys protease inhibitor, CysPI; Asp protease, AspP; purple acid phosphatase, PAP; glycosyl transferases, GT; a-D-mannosidases, a-D-Mann; b-D-galactosidases, b-D-Gac; b21,3 Glucanase, b21,3 Gluc; glycerophosphodiesterases, GPD; Mannose-1 phosphateguanyltranferase, MPT; berberine-bridge enzyme, BBE; Superoxide dismutase, SD; Putative cyclosporin, PC; Proline-rich proteins (PRPs), PRPs; Hydroxyl proline transferase, HPT; Putative protease inhibitor, PPI; LRR-receptor protein kinases; LRR-rPK; S-adenosylmethioninetransferaae, S-AMT; Wall- associated kinase, WAK; Arabinogalactanproteins, AGPs; Thaumatin-like protein, ThP; Class II chitinase, CIIChit; Pathogenesisrelated protein 5, PRP5; methionine synthase, MS; Class III chitinase, CIIIChit; threonine-

Amaya, I., Botella, M.A., de la Calle, M., Medina, M.I., Heredia, A., Bressan, R.A.,

Baluska, F., Samaj, J., Wojtaszek, P., Volkmann, D. and Menzel, D. (2003) Cytoskeleton-

Basu, U., Francis, J.L., Whittal, R.M., Stephens, J.L., Wang, Y., Zaiane, O.R., Goebel, R.,

Baumberger, N., Ringli, C. and Keller, B. (2001). The chimeric leucine-rich repeat/extensin

Bayer, E.M., Bottrill, A.R., Walshaw, J., Vigouroux, M., Naldrett, M.J., Thomas, C.L. and

Bhushan, D., Jaiswal, D.K., Ray, D., Basu, D., Datta, A., Chakraborty, S. and Chakraborty, N.

Bhushan, D., Pandey, A., Chattopadhyay, A., Choudhary, M.K., Chakraborty, S., Datta, A.

MudPIT and LC-MS/MS. *Plant Soil*, 286: 357-376.

protein identification technology. *Proteomics*, 6 : 301-11.

contrasting tolerance. *J. Proteome Res.*, 10: 2027-2046.

evolutionary perspect. *J. Proteome Res.* 5: 1711-1720.

*thaliana*. *Genes Dev.,* 15: 1128-1139.

Hasegawa, P.M., Quesada, M.A. and Valpuesta,V. (1999). Improved germination under osmotic stress of tobacco plants overexpressing a cell wall peroxidase. *FEBS* 

plasma membrane-cell wall continuum in plants. Emerging links revisited. *Plant* 

Muench, D.G., Good, A.G. and Taylor, G.J. (2006). Extracellular proteomes of Arabidopsis thaliana and Brassica napus roots: analysis and comparison by

cell wall protein LRX1 is required for root hair morphogenesis in *Arabidopsis* 

Maule, A.J. (2006). Arabidopsis cell wall proteome defined using multidimensional

(2011). Dehydration-responsive reversible and irreversible changes in the extracellular matrix: comparative proteomics of chickpea genotypes with

and Chakraborty, N. (2006) Extracellular matrix proteome of chickpea (Cicer arietinum L.) illustrates pathway abundance, novel protein functions and

The comparative analysis of clade and organ-specific, developmentally-regulated, stressresponsive plant ECM proteomes revealed the presence of certain proteins that were unexpected, either in their abundance, form, number or else localisation. These unexpected or non-canonical proteins suggest the constant remodelling of cell wall proteomes. The exact function and specificity of these candidates can only be comprehended once they are functionally characterised.

#### **4. Conclusion and perspectives**

In this study, cross-species as well as cross-condition comparisons of ECM proteomes in vascular plants illustrates the divergence in protein profiles within only a few social classes. Across species, cell wall modifying proteins (23%) represent the largest category, followed by oxidoreductase (19%) and cell wall structural protein (18.5%). In total, 213 and 110 glycoside hydrolase were found in the organ specific proteome of Arabidopsis and development specific proteomes of rice, respectively. Oxidoreductase constitutes the second largest category in both these cases. Furthermore, the dehydration responsive comparative proteome in legumes, chickpeas and cereals, rice showed both genotypic- and crop-specific adaptation. As expected, the proteins involved in cell-wall remodelling were found to be the most predominant across all conditions. Nonetheless, a large number of proteins were unique or novel to each of the plant species, organs, stages of development and different stresses. It may be thought that the ubiquitously present classes of proteins are the essential proteins for sustenance while the unique classes bring out the condition-specific special function. The differences in terms of protein pattern and protein function appear to encompass both genetic and physiological information. It may be speculated that the differential proteome is shaped by the cellular environment and the ecological niche of the corresponding organism. The divergence may arise due to codon bias, amino acid composition and protein length. However, a much more comprehensive survey of the ECM proteomes in several plants will ultimately draw a more complete picture of the social class vs. protein diversity in this organelle. We are witnessing a significant but inadequate progress in the understanding the ECM proteomes of various crops of agricultural importance. Our understanding of ECM composition, organisation and homeostasis has been greatly enhanced through targeted biochemical and genetic approaches. Unbiased 'discovery' methods, such as proteomics, have only recently gained traction in the field of matrix biology. To date, a key word search using "ECM proteome" retrieves only 43 results in a pubmed search, emphasising the need for in-depth study in the field of the plant ECM proteome. Our future efforts will focus on the development and analysis of comparative ECM proteomes towards an understanding of crop- and genotype-specific adaptation as an important amendment for the determination of protein networks influenced by the internal and external cues associated with the complex cellular biochemical and physiological process that bring about phenome variation.

#### **5. Acknowledgements**

This work was supported by a grant from the Department of Biotechnology (DBT), Govt. of India and a grant from National Institute of Plant Genome research. KN and EE are the recipients of pre-doctoral fellowships from the Council of Scientific and Industrial Research and DBT-TWAS, Govt. of India. Authors thank Mr. Jasbeer Singh for illustrations in the manuscript.

### **6. Abbreviations**

160 Crop Plant

The comparative analysis of clade and organ-specific, developmentally-regulated, stressresponsive plant ECM proteomes revealed the presence of certain proteins that were unexpected, either in their abundance, form, number or else localisation. These unexpected or non-canonical proteins suggest the constant remodelling of cell wall proteomes. The exact function and specificity of these candidates can only be comprehended once they are

In this study, cross-species as well as cross-condition comparisons of ECM proteomes in vascular plants illustrates the divergence in protein profiles within only a few social classes. Across species, cell wall modifying proteins (23%) represent the largest category, followed by oxidoreductase (19%) and cell wall structural protein (18.5%). In total, 213 and 110 glycoside hydrolase were found in the organ specific proteome of Arabidopsis and development specific proteomes of rice, respectively. Oxidoreductase constitutes the second largest category in both these cases. Furthermore, the dehydration responsive comparative proteome in legumes, chickpeas and cereals, rice showed both genotypic- and crop-specific adaptation. As expected, the proteins involved in cell-wall remodelling were found to be the most predominant across all conditions. Nonetheless, a large number of proteins were unique or novel to each of the plant species, organs, stages of development and different stresses. It may be thought that the ubiquitously present classes of proteins are the essential proteins for sustenance while the unique classes bring out the condition-specific special function. The differences in terms of protein pattern and protein function appear to encompass both genetic and physiological information. It may be speculated that the differential proteome is shaped by the cellular environment and the ecological niche of the corresponding organism. The divergence may arise due to codon bias, amino acid composition and protein length. However, a much more comprehensive survey of the ECM proteomes in several plants will ultimately draw a more complete picture of the social class vs. protein diversity in this organelle. We are witnessing a significant but inadequate progress in the understanding the ECM proteomes of various crops of agricultural importance. Our understanding of ECM composition, organisation and homeostasis has been greatly enhanced through targeted biochemical and genetic approaches. Unbiased 'discovery' methods, such as proteomics, have only recently gained traction in the field of matrix biology. To date, a key word search using "ECM proteome" retrieves only 43 results in a pubmed search, emphasising the need for in-depth study in the field of the plant ECM proteome. Our future efforts will focus on the development and analysis of comparative ECM proteomes towards an understanding of crop- and genotype-specific adaptation as an important amendment for the determination of protein networks influenced by the internal and external cues associated with the complex cellular biochemical and physiological

This work was supported by a grant from the Department of Biotechnology (DBT), Govt. of India and a grant from National Institute of Plant Genome research. KN and EE are the recipients of pre-doctoral fellowships from the Council of Scientific and Industrial Research and DBT-TWAS, Govt. of India. Authors thank Mr. Jasbeer Singh for illustrations in the manuscript.

functionally characterised.

**4. Conclusion and perspectives** 

process that bring about phenome variation.

**5. Acknowledgements** 

Polysaccharide lyase, PL; gibberellin acid-stimulated Arabidopsis (GASA) protein, GASA; pectin methyl esterase inhibitor, PMEI; Expansin, Exp; carbohydrate esterase, CE; glycoside hydrolase, GH; Laccase, lacc; blue copper binding protein, BCuBP; berberine-bridge oxidoreductase, B-BOxRe; multicopper oxidase, MCuOx; Peroxidase, Pox; glycine-rich protein, GRP; proline-rich protein, PRP; leucine-rich repeat extensin, LRX; LRR protein, LRR;signal eptidase, SP; COBRA-like family, COBRA; fasciclin-like arabinogalactan protein, FLA; arabinogalactan protein, AGP; lipid transfer protein, LTP Ser carboxypeptidase, SerCP; Cys protease inhibitor, CysPI; Asp protease, AspP; purple acid phosphatase, PAP; glycosyl transferases, GT; a-D-mannosidases, a-D-Mann; b-D-galactosidases, b-D-Gac; b21,3 Glucanase, b21,3 Gluc; glycerophosphodiesterases, GPD; Mannose-1 phosphateguanyltranferase, MPT; berberine-bridge enzyme, BBE; Superoxide dismutase, SD; Putative cyclosporin, PC; Proline-rich proteins (PRPs), PRPs; Hydroxyl proline transferase, HPT; Putative protease inhibitor, PPI; LRR-receptor protein kinases; LRR-rPK; S-adenosylmethioninetransferaae, S-AMT; Wall- associated kinase, WAK; Arabinogalactanproteins, AGPs; Thaumatin-like protein, ThP; Class II chitinase, CIIChit; Pathogenesisrelated protein 5, PRP5; methionine synthase, MS; Class III chitinase, CIIIChit; threoninehydroxyproline-rich glycoprotein, THRGP;

#### **7. References**


Comparative Analyses of Extracellular

*Plant Biol.*, 8, 129.

1172.

62.

312-329.

1566.

synthase. Plant Cell, 18: 1630-1641.

*Mol. Cell. Proteomics*, 2: 1261-1270.

Arabidopsis hypocotyls. *Plant Methods*, 2: 10.

wall. *Curr. Opin. Plant Biol.*, 13: 693-699.

responses. *Plant Cell,* 8: 1773-1791.

actors and newcomers. *BMC Plant Biol.*, 8: 94.

remodeling in the grasses. *Plant Physiol.*, 149: 27-37.

Matrix Proteome: An Under-Explored Area in Plant Research 163

Ellis, C., Karafyllidis, I., Wasternack, C. and Turner, J.G. (2002). The arabidopsis mutant cev1

Elortza, F., Nuhse, T.S., Foster, L.J., Stensballe, A., Peck, S.C. and Jensen, O.N. (2003).

Feiz, L., Irshad, M., Pont-Lezica, R.F., Canut, H. and Jamet, E. (2006). Evaluation of cell wall

Fincher, G. (2009). Revolutionary times in our understanding of cell wall biosynthesis and

Floerl, S., Druebert, C., Majcherczyk, A., Karlovsky, P., Kües, U. and Polle, A. (2008).

Gail McLean, B., Hempel, F.D. and Zambryski, P.C. (1997). Plant intercellular

Geitmann, A. (2010). Mechanical modeling and structural analysis of the primary plant cell

Hammond-Kosack, K.E. and Jones, J.D.G. (1996). Resistance gene–dependent plant defense

Hematy, K., Sado, P.E., VanTuinen, A., Rochange, S., Desnos, T., Balzergue, S., Pelletier, S.,

Irshad, M., Canut, H., Borderies, G., Pont-Lezica, R. and Jamet, E. (2008). A new picture of

Jamet, E., Albenne, C., Boudart, G., Irshad, M., Canut, H. and Pont-Lezica, R. (2008a) Recent

Jones, D.A. and Takemoto, D. (2004) Plant innate immunity—direct and indirect recognition

Jones, G., Jonathan, D. and Dangl, J.L. (2006). The plant immune system. *Nat. Biotechnol.* 16:

Hynes, R.O. (2009). The extracellular matrix: not just pretty fibrils. *Science*, 326: 1216.

advances in plant cell wall proteomics. *Proteomics*, 8: 893-908.

Gillmor, C.S., Lukowitz, W., Brininstool, G., Sedbrook, J.C., Hamann, T., Poindexter, P.,

communication via plasmodesmata. *Plant Cell.,* 9: 1043-1054.

suppressed by mutations in the RHM1 gene encoding a UDP-L-Rhamnose

links cell wall signaling to jasmonate and ethylene responses. *Plant Cell*, 14L 1557-

Proteomic analysis of glycosylphosphatidylinositolanchored membrane proteins.

preparations for proteomics: a new procedure for purifying cell walls from

Defence reactions in the apoplastic proteome of oilseed rape (Brassica napus var. napus) attenuate Verticillium longisporum growth but not disease symptoms. *BMC* 

Somerville, C. *(*2005*)* Glycosylphosphatidyl inositol-anchored proteins are required for cell wall synthesis and morphogenesis in *Arabidopsis. Plant Cell* 17*:*1128*–*1140 Hall, Q. and Cannon, M.C. (2002). The Cell Wall Hydroxyproline-Rich Glycoprotein RSH Is

Essential for Normal Embryo Development in Arabidopsis. Plant Cell, 14: 1161-

Renou, J.P. and Hofte, H. (2007). A receptor-like kinase mediates the response of Arabidopsis cells to the inhibition of cellulose synthesis*. Curr. Biol.,* 17*:* 922-931. Hunter, T.C., Andon, N.<., Koller, A., Yates, J.R. and Haynes, P.A. (2002). The functional proteomics toolbox: methods and applications. *J. Chromatogr,* B 782: 161-181.

cell wall protein dynamics in elongating cells of Arabidopsis thaliana : confirmed

of general and specific pathogen-associated molecules. *Curr. Opin. Immunol.* 16: 48-


Bhushan, D., Pandey, A., Choudhary, M.K., Datta, A., Chakraborty, S. and Chakraborty, N.

Borderies, G., Jamet, E., Lafitte, C., Rossignol, M., Jauneau, A., Boudart, G., Monsarrat, B.,

Boudart, G., Jamet, E., Rossignol, M., Lafitte, C., Borderies, G., Jauneau, A., Esquerré-

Brady, S.M., Song, S., Dhugga, K.S., Rafalski, J.A. and Benfey, P.N. (2007). Combining

Brownlee, C. (2002). Role of the extracellular matrix in cell-cell signaling: paracrine

Brownlee, C. and Berger, F. (1995). Extracellular matrix and pattern in plant embryos: on the

Cassab, G.I. (1998). Plant cell wall proteins. *Annu. Rev. Plant Physiol. Plant Mol. Biol.*, 49: 281-

Chaudhary, M.K., Basu, D., Datta, A., Chakraborty, N. and Chakraborty, S. (2009)

Chen, J. and Varner, J.E. (1985). An extraceliular matrLx protein in plants: characterization

Chen, X.Y., Kim, S.T., Cho, W.K., Rim, Y., Kim, S., Kim, S.W., Kang, K.Y., Park, Z.Y. and

Cheng, F.-Y., Blackburn, K., Lin, Y.-M., Goshe, M.B. and Wiliamson, J.D. (2009). Absolute

Chivasa, S., Ndimba, B., Simon, W., Robertson, D., Yu, X.-L., Knox, J., Bolwell, P. and Slabas,

Chivasa, S., Simon, W.J., Yu, X.-L., Yalpani, N. and Slabas, A.R. (2005). Pathogen elicitor-

Degenhardt, B. and Gimmer, H. (2000). Cell wall adaptation to multiple environmental

Diet, A., Link, B., Seifert, G.J., Schellenberg, B., Wagner, U., Pauly, M., Reiter, W.-D. and

Cosgrove, D. J. (2005). Growth of the plant cell wall. *Nat. Rev. Mol. Cell. Biol.*, 6: 850-61. Cosgrove, D.J. (1997). Assembly and enlargement of the primary cell wall in plants. *Annu.* 

Dehydration-responsive nuclear proteome of rice (*Oryza sativa* L.) illustrates protein network, novel regulators of cellular adaptation and evolutionary

Kim, J.Y. (2009) Proteomics of weakly bound cell wall proteins in rice calli. *J. Plant* 

protein quantification by LC/MS for global analysis of salicylic acid-induced plant

A. (2002). Proteomic analysis of the Arabidopsis thaliana cell wall. *Electrophoresis*,

induced changes in the maize extracellular matrix proteome. *Protgeomics*, 5: 4894-

Ringli, C. ( 2006) The arabidopsis root hair cell wall formation mutant lrx1 Is

lookout for developmental information. *Trends Genet.,* 11: 344-348.

of a genomic clone for carrot extension. *EMBO J.*, 4: 2145-2151.

critical analysis. *Electrophoresis,* 24:3421-3432.

paradigms. *Curr. Opin. Plant Biol*, 5: 396-401.

perspective. *Mol. Cell. Proteomics*, 8: 1579-1598.

protein secretion responses. *J. Proteome Res.*, 8: 82-93.

bioinformatics. *Proteomics*, 5: 212-221.

*Physiol.*, 143: 172-187.

*Physiol.*, 166: 675-685.

*Rev. Cell Dev. Biol*. 13, 171–201.

stresses in maize roots. *J. Exp. Bot.*, 51: 595-603.

23: 1754-1765.

4904.

309.

1868 -1884.

(2007) Comparative proteomics analysis of differentially expressed proteins in chickpea extracellular matrix during dehydration stress. *Mol. Cell. Proteomics*, 6:

Esquerré-Tugayé, M.-T., Boudet, A. and Pont-Lezica, R. (2003). Proteomics of loosely bound cell wall proteins of *Arabidopsis thaliana* cell suspension cultures: A

Tugayé, M.-T. and Pont-Lezica, R. (2005). Cell wall proteins in apoplastic fluids of *Arabidopsis thaliana* rosettes: Identification by mass spectrometry and

expression and comparative evolutionary analysis. The COBRA gene family. *Plant* 

suppressed by mutations in the RHM1 gene encoding a UDP-L-Rhamnose synthase. Plant Cell, 18: 1630-1641.


Comparative Analyses of Extracellular

*Physiol*, 138: 591-599.

Physiol. 152: 151-167.

*Res.* 111: 133-148.

7: 2070-2082.

1466-1476.

*Cell*, 19: 3339-3346.

5: 1111-1121.

deficiency. *Proteomics*, 8: 4317-4326.

*Expert Rev. Prtoemics*, 7: 803-806.

*Annu. Rev. Plant Biol.*, 63: 162-181.

cell walls. *Phytochemistry*, 65:1709-1720.

*Biochem. Soc. Trans.*, 32: 524-528.

Mobility-MS J. of Proteome Res. 4: 1238-1247.

Matrix Proteome: An Under-Explored Area in Plant Research 165

Peck, S. (2005). Update on proteomics in Arabidopsis. Where do we go from here? *Plant* 

Peters, W.S., Hagemann, W. and Tomos, A.D. (2000) What makes plants different?

Rose, J.K., Saladié, M. and Catalá, C. (2004). The plot thickens: New perspectives of primary

Sakurai, N. (1998). Dynamic function and regulation of apoplast in the plant body. *J. Plant* 

Šamaj, J., Bobák, M. and Volkmann, D. (1999). Extracellular matrix surface network of

Soares, N.C., Francisco, R., Ricardo, C.P., and Jackson, P.A. (2007). Proteomics of ionically

Taraszka, T.A., Gao, X., ValentineRena, S.J., Sowell, A., Koeniger, S.L., Miller, D.F.,

Telewski, F.W. (2006). A unified hypothesis of mechanoperception in plants. *Ame. J. Bot.*, 93:

The Arabidopsis Genome Initiative. (2000). Analysis of the genome sequence of the

Thelen, J. and Peck, S. (2007). Quantitative proteomics in plants: choices in abundance. *Plant* 

Tran, H.T. and Plaxton, W.C. (2008) Proteomic analysis of alterations in the secretome of

Watson, B.S., Lei, Z., Dixon, R.A. and Sumner, L.W. (2004). Proteomics of Medicago sativa

Wilson, R. (2010). The extracellular matrix: an underrexplored but important proteome.

Wolf, S., Hematy, K. and Hoft, H. (2009). Growth control and cell wall signaling in plants.

Yokoyama, R. and Nishitani, K. (2004). Genomic basis for cell-wall diversity in plants. A

Zhu, J., Chen, S., Alvarez, S., Asirvatham, V. S. (2006). Cell wall proteome in the maize

lightly ionically bound proteins. *Plant Physiol*., 140: 311-325.

comparative approach to gene families in Rice and Arabidopsis. *Plant Cell Physiol.*,

primary root elongation zone. Extraction and identification of water-soluble and

Arabidopsis thaliana suspension cells subjected to nutritional phosphate

flowering plant Arabidopsis thaliana. *Nature*, 408: 796-815.

recognized by monoclonal antibody JIM4. *Plant Cell Rep.*, 18: 369-374. Schröder, F., Lisso, J., Lange, P. and Müssig, C. (2009). The extracellular EXO protein mediates cell expansion in Arabidopsis leaves. *BMC Plant Biol.*, 13: 9-20. Slabas, A.R., Ndimba, B., Simon, W.J. and Chivasa, S. (2004). Proteomic analysis of the

cell wall modification. *Curr. Opin. Plant Biol.*, 7: 296-301. Rose, J.K.C. (2003). The plant cell wall. *Annu. Plant Rev.*, 8: 190-513.

Principles of extracellular matrix function in 'soft' plant tissues. Comp. Biochem.

embryogenic units of friable maize callus contains arabinogalactan-proteins

Arabidopsis cell wall reveals unexpected proteins with new cellular locations.

bound and soluble extracellular proteins in Medicago truncatula leaves. *Proteomics*,

Kaufman, T.C. and Clemmer, D.E. (2005). Proteome Profiling for Assessing Diversity: Analysis of Individual Heads of Drosophila melanogaster Using LC-Ion


Jung, E.H., Jung, H.W., Lee, S.C., Han, S.W., Heu, S. and Hwang, B.K. (2004) Identification of

phloem cells of Capsicum annuum. *Biochim. Biophys. Acta*, 1676: 211-222. Jung, Y.-H., Jeong, S.-H., Kim, S.H., Singh, R., Lee, J.-E., Cho, Y.-S., Agrawal, G.K., Rakwal,

density two-dimensional gel reference maps. *J. Proteome Res.*, 7: 5187-5210. Kachroo, A., Schopfer, C.R., Nasrallah, M.E. and Nasrallah, J.B. (2001) Allelespecific

Kim, I., Yun, H. and Jin, I. (2007)Comparative proteomic analyses of the yeast

Kumar, S., Maxwell, I.Z., Heisterkamp, A., Polte, T.R., Lele, T.P., Mazur, M.S.E. and Ingber,

Kwon, H.-K., Yokoyama, R. and Nishitani, K. (2005). A proteomic approach to apoplastic

Liepman, A.H., Wightman, R., Geshi, N., Turner, S.R. and Scheller, H.V. (2010). Arabidopsis – a powerful model system for plant cell wall research. *Plant J.*, 61: 1107-1121. Mann, M. and Jensen, O.N. (2003). Proteomic analysis of post-translational modifications.

Miller, J.G. and Fry, S.C. (1992). Production and harvesting of ionically wall-bound extensin from living cell suspension cultures. *Plant Cell, Tissue Organ Culture*, 31: 61-66.

Ndimba, B.K., Chivasa, S., Hamilton, J.M., Simon, W.J., and Slabas, A.R. (2003). Proteomic

Oh, I.S., Park, A.R., Bae, M.S., Kwon, S.J., Kim, Y.S., Lee, J.E., Kang, N.Y., Lee, S., Cheong, H.

Pandey, A., Rajamani U., Verma, J., Subba, P., Chakraborty, N., Datta, A., Chakraborty, S.

Pandey, A., Chakraborty, S., Datta, A. and Chakraborty, N. (2008). Proteomics approach to

Pandey, A., Rajamani, U., Verma, J., Subba, P., Chakraborty, N., Datta, A., Chakraborty, S.

Minic, Z. (2008). Physiological roles of plant glycoside hydrolases. *Planta*, 227: 723- 740. Minic, Z., Jamet, E., Negroni, L., der Garabedian, P.A., Zivy, M. and Jouanin, L. (2007). A

suspension-cultured cells. *Plant Cell Physiol.*, 46: 843-857.

cell wall glycoside hydrolases. *J. Exp. Bot.*, 58: 2503-2512.

culture induced by fungal elicitors *Proteomics*, 3: 1047-1059.

approach. *J. Proteome Res.,* 9: 3443-3464.

approach. *J. Proteome Res.*, 9: 3443-3464.

*Mol. Cell. Proteomics*, 7: 88-107.

defense against Alternaria brassicicola. *Plant Cell*, 17: 2832-2847.

stress. *J. Microbiol. Biotechnol.*, 17: 207-217.

*Nat. Biotechnol.*, 21: 255-261.

3773.

a novel pathogen-induced gene encoding a leucinerich repeat protein expressed in

R. and Jwa, N.-S. (2008) Systematic secretome analyses of rice leaf and seed callus suspension-cultured cells: Workflow development and establishment of high-

receptor–ligand interactions in Brassica self-incompatibility. *Science*, 293: 1824-1826.

Saccharomyces cerevisiae KNU5377 strain against menadione-induced oxidative

D.E. (2006). Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix. *Mech. Biophy. J.*, 90: 3762-

proteins involved in cell wall regeneration in protoplasts of Arabidopsis

sub-proteome of Arabidopsis thaliana trapped on Concanavalin A is enriched in

analysis of changes in the extracellular matrix of Arabidopsis cell suspension

and Park, O.K. (2005) Secretome analysis reveals an Arabidopsis lipase involved in

and Chakraborty, N., (2010). Identification of extracellular matrix proteins of rice (Oryza sativa L.) involved in dehydration-responsive network: a proteomic

identify dehydration responsive nuclear proteins from chickpea (*Cicer arietinum L.*).

and Chakraborty, N. (2010). Identification of extracellular matrix proteins of rice (*Oryza sativa* L.) involved in dehydration-responsive network: a proteomic


**1. Introduction** 

**8** 

*Venezuela* 

**The Shade Avoidance Syndrome Under** 

*Universidad Central de Venezuela, Facultad de Agronomía, Maracay* 

Sugarcane is grown mainly for sugar and ethanol production, belongs to the Poaceae family, genus *Saccharum* native to Southest Asia and India and cultivated intensively in tropical and subtropical areas throughout the world, and it plays a significant role in the world economy and the area cultivated yields observed to have progressively increased to remarkable levels in the last 10 years (Azevedo et al., 2011). Commercial sugarcane, mainly the interspecific hybrids of *S*. *officinarum* and *S. spontaneum* would greatly benefit from biotechnological improvements due to the long duration (10-15 years) required to breed elite cultivars, more importantly there is an ongoing need to provide durable and disease and pest resistance in

There is an increasing pressure worldwide to enhance the productivity of sugarcane cultivation in order to sustain profitable sugar industries (Hanlon et al., 2000), for example, improvement of industrial processes along with strong sugarcane breeding programs in Brazil, brought technologies that currently support a cropland of 7 million hectares of sugarcane with an average yield of 75 tons/ha (Matsuoka et al., 2009). Besides, biomass has gained prominence in the last years as new technologies for energy production from crushed sugarcane stalks developed a sugarcane industry that currently is one the most efficient systems for the conversion of photosynthate into different forms of energy, for

The crop is vegetatively propagated by stalk cuttings, having one to three buds, known as seed pieces or setts, is a perennial crop regrowing from these vegetative buds after the crop has been harvested giving subsequent regrowth or crop cycles known as rattoning. The germinating bud develops its own root system, and several shoots arise by heavy tillering which produces a canopy of leaves during closing-in stages of crop growth; the term "closed crop" defines a community of plants, of uniform height, which extends indefinitely in the horizontal plane. Within a "closed crop" canopy, we might expect the leaves in any particular horizon to experience a uniform environment, and we might further expect the only significant source of environmental variation to be found in the vertical plane (Charles-Edwards, 1981), thus for the sugarcane crop the production of stalks, to quickly achieving a closed canopy, is important as a means of increasing competition with the weeds growing

combination with superior agronomic performance (Lakshman et al., 2005).

example, the production of ethanol as a liquid fuel.

underneath and for crop protection against adverse conditions.

**the Sugarcane Crop** 

Jocelyne Ascencio and Jose Vicente Lazo

Zhu, Q., Zheng, X., Luo, J., Gaut, B.S. and Ge, S*.* (2007). Multilocus analysis of nucleotide variation of *Oryza sativa* and its wild relatives: severe bottleneck during domestication of rice*. Mol. Biol. Evol.,* 24*:* 875*-*888.
