**Plant Phosphate Nutrition and Environmental Challenges**

Mohammad Ali Malboobi, Ali Samaeian, Mohammad Sadegh Sabet and Tahmineh Lohrasebi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53424

## **1. Introduction**

Since the ancient times, food production was exercised in farms where animals and plants were grown together (Figure 1). However, as a part of Green Revolution, the use of synthetic fertilizers turned out to be an integrated part of industrial agriculture which has progressively encouraged disintegration between cropping and animal husbandry. As a result, the consumptions of fertilizers have remarkably increased since half a century ago [1]. Indeed, disruptions in the cycles of nutrients have brought about environmental challenge that caused irreversible damages to natural ecosystems while reasonably justified by the real needs for food security for growing human population. As one of the main challenges in the world of agriculture, provision of phosphorus (P) for plant nutrition requires a closer look from several points of views.

**Figure 1.** Painted grain and livestock growing together in ancient Egypt.

© 2012 Malboobi et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Malboobi et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In this chapter, we firstly explain the importance of P in living organisms and the evolved adaptive mechanisms, particularly from the molecular and genomic aspects. Subsequently, the cycle of exchanging P between physical and biological worlds will be described to show the extent of disturbance by current agricultural practices. Then, possible solutions to the experienced problems in industrialized agriculture will be discussed. The needs for introducing less-energy demanding production and consumption methods for P provision and the use of new generation of fertilizers, particularly organic and biological ones in combination with chemical P fertilizers will be described in details to address integrative measures for sustainable agriculture.

Plant Phosphate Nutrition and Environmental Challenges 5

its deficiency in plants [4, 16-18]. It is also influenced by biological processes such as the

The uptake of Pi from soil by plants depends on both the rate of diffusion of Pi towards roots and the growth of the root system to access unexploited soil [19]. Roots rapidly deplete Pi in the soil solution so that its concentration at the root surface is estimated around 0.05– 0.2 mM [19]. Although this establishes a Pi-diffusion gradient from the rhizosphere to bulk soil [19-21], low Pi diffusion rate effectively limits its uptake [22]. It is believed that proper application of the fertilizers not only provides Pi, but also promotes root growth into

Under experimental conditions, both high and low affinity Pi uptake mechanisms have been recognized in plants [23,24,25]. Nevertheless, it is generally accepted that if Pi level is within the micromolar range (1–10 µm), which corresponds to Pi concentrations in most cultivated soils, the high-affinity transporters handles Pi uptake. The Km for high-affinity transporters varies from 1.8 to 9.9 µM [25]. It is an energy mediated co-transport process, driven by protons generated by a plasma membrane H+-ATPase [23, 26]. Additional evidence for the involvement of protons in Pi uptake comes from the use of inhibitors that disrupts proton

Both experimental data and genome sequence analyses indicate that plants possess families of Pi transporter genes [24, 28-31]. Current data suggest that members of the PHT1 Pi transporter family mediate transfer of Pi into cells, whereas members of the PHT2, PHT3, PHT4, and pPT families are involved in Pi transfer across internal cellular and organelle

Members of the PHT1 Pi transporter gene family have been identified in a wide range of plant species including Arabidopsis, rice, medicago, tomato and soybean [28, 36-41]. Analysis of Arabidopsis whole genome sequences revealed a set of nine PHT1 transporters. Eight of them expressed in roots from which four are expressed in the epidermal cells. In contrast, there is less redundancy in the aerial tissues [42]. In rice, at least 10 of the PHT1

Overlapping expression patterns have also been reported for the PHT1 Pi transporters in other plant species [38,41,43-46]. The function of some PHT1 transporters have been analyzed either by expression in yeast Pi transport mutants or in plant cells [27,30,36,43,47]. In Arabidopsis two Pi transporters, PHt1;1 and PHt1;4, mediate 75% of the Pi uptake

After uptake into the roots, Pi moves symplastically from root surface to xylem at a rate of about 2 mm/h and to the other organs afterwards [2]. Entering into the xylem for long-distance translocation to the shoot is facilitated by another set of transporter-like proteins [49-50]. Most of the absorbed Pi by the roots is transported through xylem to growing leaves of Pi-fed plants. In Pi-starved plants. Stored Pi in older leaves is retranslocated to both younger leaves

capacity of the roots system in a wide range of environmental conditions [48].

hydrolysis of ester bonds in organic Pi compounds by phosphatase enzymes.

gradient across membranes causing the suppression of Pi uptake [25,27].

unexploited soil [18].

membranes [32-35].

transporters are expressed in roots [40].

**4. Pi uptake and reallocation in plants** 

## **2. Pi importance**

P, in the form of phosphate ion (Pi), is the most vital element for all living organisms playing major roles in the structures of essential biomolecules such as nucleic acids, phospholipids and phosphosugars, in almost all metabolic reactions including photosynthesis and respiration, in energy delivering molecules such as ATP, ADP or NADPH and in transduction of signals within the cells. To ensure functional metabolic reactions, Pi homeostasis must be kept between 5 to 20 mM in the cytoplasm. Plants absorb P only in its soluble inorganic form of Pi, H2PO4- or HPO42-, which occur in the soil between 0.1 to 1 µM [2-4]. Therefore, it is one of the most needed nutrients for plant growth and development and considered as a major limiting factor in crop yield.

### **3. Soil Pi and plants uptake**

Most soils contain a significant amount of P compounds, ranging from 200 to 3000 mg/kg, averaged at 1200 mg/kg [5]. P compounds in soil comprise a wide variety of organic and inorganic forms [6]. However, only a small proportion (generally less than 1%) is immediately available to plants as free Pi. The majority of inorganic compounds are predominantly associated with calcium (Ca) in alkaline soils or with iron (Fe) and aluminum (Al) in acidic soils [6]. Organic Pi accounts for 30 to 80 percent of soil P, among them monoester P occurs predominantly as cation derivatives of inositol hexakisphosphates (mainly as phytate), whereas sugar phosphates and diester phosphates (e.g. nucleic acids and phospholipids) constitute only a small proportions (~5%)[7, 8]. Factors that contribute to the accumulation and turnover of different forms of in/organic P in soil are complex and controlled by various competing processes that have been the subject of several reviews [6, 9-13].

High concentrations of Pi are generally found in the surface layer of soil profiles or in nutrientrich patches. Plants usually produce more roots in the surface soil than the subsoil. For instance, an analysis of traits associated with the rate of Pi uptake in wheat showed that root length density in the surface soil was the most important trait for Pi acquisition [14]. Because of that, drying the surface soil can cause ceased Pi uptake or 'nutritional drought' [15].

Low solubility of Pi in water (0.5 mg/lit), slow diffusion rates of Pi in soil (10-12 to 10-15 m/s) and limited capacity for replenishment of soil Pi-solution are major factors that contribute to its deficiency in plants [4, 16-18]. It is also influenced by biological processes such as the hydrolysis of ester bonds in organic Pi compounds by phosphatase enzymes.

The uptake of Pi from soil by plants depends on both the rate of diffusion of Pi towards roots and the growth of the root system to access unexploited soil [19]. Roots rapidly deplete Pi in the soil solution so that its concentration at the root surface is estimated around 0.05– 0.2 mM [19]. Although this establishes a Pi-diffusion gradient from the rhizosphere to bulk soil [19-21], low Pi diffusion rate effectively limits its uptake [22]. It is believed that proper application of the fertilizers not only provides Pi, but also promotes root growth into unexploited soil [18].

#### **4. Pi uptake and reallocation in plants**

4 Plant Science

measures for sustainable agriculture.

**3. Soil Pi and plants uptake** 

**2. Pi importance** 

In this chapter, we firstly explain the importance of P in living organisms and the evolved adaptive mechanisms, particularly from the molecular and genomic aspects. Subsequently, the cycle of exchanging P between physical and biological worlds will be described to show the extent of disturbance by current agricultural practices. Then, possible solutions to the experienced problems in industrialized agriculture will be discussed. The needs for introducing less-energy demanding production and consumption methods for P provision and the use of new generation of fertilizers, particularly organic and biological ones in combination with chemical P fertilizers will be described in details to address integrative

P, in the form of phosphate ion (Pi), is the most vital element for all living organisms playing major roles in the structures of essential biomolecules such as nucleic acids, phospholipids and phosphosugars, in almost all metabolic reactions including photosynthesis and respiration, in energy delivering molecules such as ATP, ADP or NADPH and in transduction of signals within the cells. To ensure functional metabolic reactions, Pi homeostasis must be kept between 5 to 20 mM in the cytoplasm. Plants absorb P only in its soluble inorganic form of Pi, H2PO4- or HPO42-, which occur in the soil between 0.1 to 1 µM [2-4]. Therefore, it is one of the most needed nutrients for plant growth and

Most soils contain a significant amount of P compounds, ranging from 200 to 3000 mg/kg, averaged at 1200 mg/kg [5]. P compounds in soil comprise a wide variety of organic and inorganic forms [6]. However, only a small proportion (generally less than 1%) is immediately available to plants as free Pi. The majority of inorganic compounds are predominantly associated with calcium (Ca) in alkaline soils or with iron (Fe) and aluminum (Al) in acidic soils [6]. Organic Pi accounts for 30 to 80 percent of soil P, among them monoester P occurs predominantly as cation derivatives of inositol hexakisphosphates (mainly as phytate), whereas sugar phosphates and diester phosphates (e.g. nucleic acids and phospholipids) constitute only a small proportions (~5%)[7, 8]. Factors that contribute to the accumulation and turnover of different forms of in/organic P in soil are complex and controlled by various

High concentrations of Pi are generally found in the surface layer of soil profiles or in nutrientrich patches. Plants usually produce more roots in the surface soil than the subsoil. For instance, an analysis of traits associated with the rate of Pi uptake in wheat showed that root length density in the surface soil was the most important trait for Pi acquisition [14]. Because of

Low solubility of Pi in water (0.5 mg/lit), slow diffusion rates of Pi in soil (10-12 to 10-15 m/s) and limited capacity for replenishment of soil Pi-solution are major factors that contribute to

development and considered as a major limiting factor in crop yield.

competing processes that have been the subject of several reviews [6, 9-13].

that, drying the surface soil can cause ceased Pi uptake or 'nutritional drought' [15].

Under experimental conditions, both high and low affinity Pi uptake mechanisms have been recognized in plants [23,24,25]. Nevertheless, it is generally accepted that if Pi level is within the micromolar range (1–10 µm), which corresponds to Pi concentrations in most cultivated soils, the high-affinity transporters handles Pi uptake. The Km for high-affinity transporters varies from 1.8 to 9.9 µM [25]. It is an energy mediated co-transport process, driven by protons generated by a plasma membrane H+-ATPase [23, 26]. Additional evidence for the involvement of protons in Pi uptake comes from the use of inhibitors that disrupts proton gradient across membranes causing the suppression of Pi uptake [25,27].

Both experimental data and genome sequence analyses indicate that plants possess families of Pi transporter genes [24, 28-31]. Current data suggest that members of the PHT1 Pi transporter family mediate transfer of Pi into cells, whereas members of the PHT2, PHT3, PHT4, and pPT families are involved in Pi transfer across internal cellular and organelle membranes [32-35].

Members of the PHT1 Pi transporter gene family have been identified in a wide range of plant species including Arabidopsis, rice, medicago, tomato and soybean [28, 36-41]. Analysis of Arabidopsis whole genome sequences revealed a set of nine PHT1 transporters. Eight of them expressed in roots from which four are expressed in the epidermal cells. In contrast, there is less redundancy in the aerial tissues [42]. In rice, at least 10 of the PHT1 transporters are expressed in roots [40].

Overlapping expression patterns have also been reported for the PHT1 Pi transporters in other plant species [38,41,43-46]. The function of some PHT1 transporters have been analyzed either by expression in yeast Pi transport mutants or in plant cells [27,30,36,43,47]. In Arabidopsis two Pi transporters, PHt1;1 and PHt1;4, mediate 75% of the Pi uptake capacity of the roots system in a wide range of environmental conditions [48].

After uptake into the roots, Pi moves symplastically from root surface to xylem at a rate of about 2 mm/h and to the other organs afterwards [2]. Entering into the xylem for long-distance translocation to the shoot is facilitated by another set of transporter-like proteins [49-50]. Most of the absorbed Pi by the roots is transported through xylem to growing leaves of Pi-fed plants. In Pi-starved plants. Stored Pi in older leaves is retranslocated to both younger leaves

and growing roots, from where Pi can again be recycled to the shoot [51]. Consequently, the uptake and allocation of Pi in plants requires multiple transport systems that must function in concert to maintain homeostatic level of it throughout the plant tissues [52].

Plant Phosphate Nutrition and Environmental Challenges 7

about by Pi-induced biosynthetic enzymes in each step of the pathways leading to the

A reduction in the phospholipid content of Pi-starved plant membranes coincided with increased sulfolipid sulfoquinovosyldiacylglycerol and galactolipid digalactosyldiacylglycerol membrane lipids. SQD1 and SQD2 are Pi starvation inducible enzymes required for sulfolipid biosynthesis in Arabidopsis [74,77]. Consistently, an Arabidopsis *sqd2* T-DNA insertional mutant showed reduced growth under Pi starvation conditions [78]. Galactolipid digalactosyldiacylglycerol accumulation was reduced in the roots of Pi-starved *pldz1* single and *pldz1/pldz2* double mutants [79]. PLDz generates phosphatidic acid that can be dephosphorylated by an APase to release Pi and diacylglycerol serving as a second messenger. The latter activates a protein kinase-mediated protein phosphorylation cascade which controls root growth. In contrast to PLDz function in roots, a non-specific phospholipase C5 is responsible for phospholipid degradation in leaves during Pi starvation

As a consequence of harsh Pi stress, large (up to 80%) reductions in intracellular levels of ATP, ADP, and related nucleoside phosphates also occur [81-82]. A noticeable feature of plant metabolism alterations is that some step in metabolic pathways could be bypassed to reduce dependence on Pi or ATP. This was confirmed by silencing of genes encoding enzymes traditionally considered to be essential. The growth and development of resulting transgenic plants were more or less normal [82] while it was expected to have inhibition of C flux. Protein phosphorylation and glycosylation were found responsible for controlling the activity and/or subcellular targeting of some enzymes involved in bypassed metabolic

A key plant response to Pi deprivation is the up-regulation of a large number of intracellular and secreted APase enzymes that hydrolyze Pi from a broad range of Pi compounds. Secreted APases are believed to function in scavenging nutritional Pi from many exogenous organic Pi substrates, including phytate, RNA, DNA, ATP, 3-phosphoglycerate, and various hexose phosphates that typically constitutes 20-85% of P compounds in soil [17,73-75,86-88]. Similarly, intracellular APases scavenge and remobilize Pi from expendable intracellular Pi monoesters and anhydrides. This is accompanied by marked reductions in levels of the

It is noteworthy that APase activity in rhizosphere or soil solution may also originate from fungi such as Aspergillus [89] and mycorriza [90] or from bacteria [89,90]. Microorganisms may produce both acid and alkaline phosphatase [89] while plants secrete APases only

An adaptive strategy for Pi acquisition is the excretion of proton and organic acids from roots which results in acidification of rhizosphere. The importance of this mechanism was

cytoplasmic Pi-containing metabolites during extended Pi deprivation [75,81].

synthesis of cyanidin, pelargonidin, flavonoids and anthocyanin [74-76].

reactions in response to Pi deprivation [83-85].

**5.4. Organic acid biosynthesis and secretion** 

**5.3. Acid Phosphatases and Pi recycle** 

[80].

[89,92].

## **5. Plant adaptive strategies toward low Pi**

### **5.1. Morphological changes in root architecture**

Factors that affect the initiation and activity of the meristems have a large effect on the three dimensional patterns of roots in space, the so-called Root System Architecture (RSA) [53] which is greatly influenced by surroundings soil and particularly the availability and distribution of nutrients, including Pi [54]. Several studies demonstrated that *Arabidopsis thaliana* exposed to low available Pi have reduced primary root growth and at the same time increased lateral root formation and growth and, also, root hair production and elongation [55]. Under sever Pi starvation, root hairs disappeared entirely in tomato [56]. Modification of RSA enable plant roots to explore the upper parts of the soil, a strategy described as 'topsoil foraging' [57]. Symbiotic associations with fungi (Vesicular-Arbuscular Mycorrhizae; see below) and formation of cluster roots are adaptive responses to increase Pi uptake in many plants which allow competent exploration of soils for fixed Pi [58-63].

Detailed analysis demonstrated some differences in RSA responses among ecotypes [64]. Among 73 Arabidopsis ecotypes, half showed reduced primary root growth on low Pi suggesting that root growth inhibition is determined genetically rather than being controlled metabolically only [65].

The first visible event upon Pi starvation is reduction of primary root cell elongation followed by a reduction of cell division as traced by rapid repressonof the cell cycle marker CYCB1;1. This is accompanied by a loss of quiescent center identity as detected by the QC46 marker [66].

Transcriptomics approach as well as mutations analyses have revealed an inventory of genes which are repressed or induced during Pi starvation. For example, PRD (Pi root development) gene is rapidly repressed in roots under low Pi conditions [67-70]. In this context, PRD repression mediated primary root growth arrest [66].

#### **5.2. Metabolic adaptations to Pi deficiency conditions**

As mentioned above, Pi is an essential macronutrient that plays a central role in virtually all metabolic processes in plants. This was clearly illustrated for Pi-induced inhibition of a major regulatory enzyme of starch biosynthesis, ADPGlc pyrophosphorylase. Similarly, a vacuolar acid phosphatase (APase) displayed strong activity inhibition by sufficient Pi [71- 73]. Conversely, the depletion of vacuolar Pi pools by extended Pi deprivation effectively relieved the inhibition of some APase expressions [74].

A common feature of the plant response to long-term Pi starvation conditions is the development of dark-green or purple shoots due to anthocyanin accumulation. It is brought about by Pi-induced biosynthetic enzymes in each step of the pathways leading to the synthesis of cyanidin, pelargonidin, flavonoids and anthocyanin [74-76].

A reduction in the phospholipid content of Pi-starved plant membranes coincided with increased sulfolipid sulfoquinovosyldiacylglycerol and galactolipid digalactosyldiacylglycerol membrane lipids. SQD1 and SQD2 are Pi starvation inducible enzymes required for sulfolipid biosynthesis in Arabidopsis [74,77]. Consistently, an Arabidopsis *sqd2* T-DNA insertional mutant showed reduced growth under Pi starvation conditions [78]. Galactolipid digalactosyldiacylglycerol accumulation was reduced in the roots of Pi-starved *pldz1* single and *pldz1/pldz2* double mutants [79]. PLDz generates phosphatidic acid that can be dephosphorylated by an APase to release Pi and diacylglycerol serving as a second messenger. The latter activates a protein kinase-mediated protein phosphorylation cascade which controls root growth. In contrast to PLDz function in roots, a non-specific phospholipase C5 is responsible for phospholipid degradation in leaves during Pi starvation [80].

As a consequence of harsh Pi stress, large (up to 80%) reductions in intracellular levels of ATP, ADP, and related nucleoside phosphates also occur [81-82]. A noticeable feature of plant metabolism alterations is that some step in metabolic pathways could be bypassed to reduce dependence on Pi or ATP. This was confirmed by silencing of genes encoding enzymes traditionally considered to be essential. The growth and development of resulting transgenic plants were more or less normal [82] while it was expected to have inhibition of C flux. Protein phosphorylation and glycosylation were found responsible for controlling the activity and/or subcellular targeting of some enzymes involved in bypassed metabolic reactions in response to Pi deprivation [83-85].

#### **5.3. Acid Phosphatases and Pi recycle**

6 Plant Science

and growing roots, from where Pi can again be recycled to the shoot [51]. Consequently, the uptake and allocation of Pi in plants requires multiple transport systems that must function in

Factors that affect the initiation and activity of the meristems have a large effect on the three dimensional patterns of roots in space, the so-called Root System Architecture (RSA) [53] which is greatly influenced by surroundings soil and particularly the availability and distribution of nutrients, including Pi [54]. Several studies demonstrated that *Arabidopsis thaliana* exposed to low available Pi have reduced primary root growth and at the same time increased lateral root formation and growth and, also, root hair production and elongation [55]. Under sever Pi starvation, root hairs disappeared entirely in tomato [56]. Modification of RSA enable plant roots to explore the upper parts of the soil, a strategy described as 'topsoil foraging' [57]. Symbiotic associations with fungi (Vesicular-Arbuscular Mycorrhizae; see below) and formation of cluster roots are adaptive responses to increase Pi uptake in many plants which allow competent exploration of soils for fixed Pi [58-63].

Detailed analysis demonstrated some differences in RSA responses among ecotypes [64]. Among 73 Arabidopsis ecotypes, half showed reduced primary root growth on low Pi suggesting that root growth inhibition is determined genetically rather than being

The first visible event upon Pi starvation is reduction of primary root cell elongation followed by a reduction of cell division as traced by rapid repressonof the cell cycle marker CYCB1;1. This is accompanied by a loss of quiescent center identity as detected by the QC46

Transcriptomics approach as well as mutations analyses have revealed an inventory of genes which are repressed or induced during Pi starvation. For example, PRD (Pi root development) gene is rapidly repressed in roots under low Pi conditions [67-70]. In this

As mentioned above, Pi is an essential macronutrient that plays a central role in virtually all metabolic processes in plants. This was clearly illustrated for Pi-induced inhibition of a major regulatory enzyme of starch biosynthesis, ADPGlc pyrophosphorylase. Similarly, a vacuolar acid phosphatase (APase) displayed strong activity inhibition by sufficient Pi [71- 73]. Conversely, the depletion of vacuolar Pi pools by extended Pi deprivation effectively

A common feature of the plant response to long-term Pi starvation conditions is the development of dark-green or purple shoots due to anthocyanin accumulation. It is brought

context, PRD repression mediated primary root growth arrest [66].

**5.2. Metabolic adaptations to Pi deficiency conditions** 

relieved the inhibition of some APase expressions [74].

concert to maintain homeostatic level of it throughout the plant tissues [52].

**5. Plant adaptive strategies toward low Pi** 

**5.1. Morphological changes in root architecture** 

controlled metabolically only [65].

marker [66].

A key plant response to Pi deprivation is the up-regulation of a large number of intracellular and secreted APase enzymes that hydrolyze Pi from a broad range of Pi compounds. Secreted APases are believed to function in scavenging nutritional Pi from many exogenous organic Pi substrates, including phytate, RNA, DNA, ATP, 3-phosphoglycerate, and various hexose phosphates that typically constitutes 20-85% of P compounds in soil [17,73-75,86-88]. Similarly, intracellular APases scavenge and remobilize Pi from expendable intracellular Pi monoesters and anhydrides. This is accompanied by marked reductions in levels of the cytoplasmic Pi-containing metabolites during extended Pi deprivation [75,81].

It is noteworthy that APase activity in rhizosphere or soil solution may also originate from fungi such as Aspergillus [89] and mycorriza [90] or from bacteria [89,90]. Microorganisms may produce both acid and alkaline phosphatase [89] while plants secrete APases only [89,92].

#### **5.4. Organic acid biosynthesis and secretion**

An adaptive strategy for Pi acquisition is the excretion of proton and organic acids from roots which results in acidification of rhizosphere. The importance of this mechanism was unknown until plasma membrane H+-pumping ATPases were shown to be involved in plant adaptation to Pi starvation [93]. Acidification was also correlated with the up-regulation of novel membrane channels needed to transport anions such as citrate and malate from root cells into the rhizosphere [94]. Organic acid excretion results in the chelating of metal cations that immobilize Pi (e.g. Ca2+, Al3+, Fe2/3+), thus, increasing free Pi concentrations in soil upto 1000-fold. Acidification of rhizosphere also enhances the hydrolysis of organic Pi by secreted APases. As well, organic acids could function as carbon source for symbiotic rhizobacteria that facilitate root Pi acquisition [17,74,75,86].

Plant Phosphate Nutrition and Environmental Challenges 9

responses, signal transduction, transcriptional regulation, protein degradation, cell wall metabolism and so on. The suppressed genes are involved in lipid synthesis, reactive oxygen controlling and protein synthesis. In another research, Morcuende et al. [69] showed P deprivation led to transcriptional alterations in over 1000 genes involved in Pi uptake, the mobilization of organic Pi, the conversion of phosphorylated glycolytic intermediates to carbohydrates and organic acids, the replacement of phospholipids with galactolipids and the repression of gene implicated in nucleotide/nucleic acid synthesis which were reversed within 3 h after Pi re-supply. In addition, analysis of metabolites confirmed that P deprivation leads to a shift towards the accumulations of carbohydrates, organic acids and amino acids. Pi deprived plants also showed large changes in the expression of many genes involved in secondary metabolism and photosynthesis. Hammond et al. [101]used an oligonucleotide potato microarray to investigate the transcriptional profile of potato leaves under Pi deficiency and compare their data with previously described transcriptional profiles for the leaves of Arabidopsis and rice. They identified novel components to these profiles, including the increased expression of potato patatin genes- with potential phospholipase A2 activity- in the leaves of Pi deficient potatoes. A set of 200 genes were identified that show differential expression patterns between fertilized and unfertilized

Müller et al. [102] investigated the effect of interaction of Pi and sucrose signals on the gene expression pattern in Arabidopsis. They found several genes that were previously identified to be either sugar-responsive or Pi-responsive genes. In addition, 150 genes were

In a comprehensive analysis, Lin et al. [103] conducted time course microarray experiments and co-expression-based clustering of Pi-responsive genes by pair wise comparison of genes against a customized data base. Three major clusters enriched in genes functioning in transcriptional regulation, root hair formation and developmental adaptations were distinguished in this analysis. The genome-wide transcriptional approach may be used to infer inclusive scenarios for involved mechanisms in the signaling and adaptation of plants

Metabolic adjustments to Pi limitation are largely cellular responses to sensed by internal Pi status which trigger a systemically integrated regulating mechanisms involving microRNAs, non-coding RNAs and PHO2 downstream of PHR as revealed in recent studies. PHR1, a MYB transcription factor, binds to the promoters of most of the Pi-responsive genes that are positively or negatively affected by Pi starvation [104]. Despite its central role in controlling the expression of numerous Pi-responsive genes, the phr1 mutant showed no major phenotypic defects except for a slight difference in the root-to-shoot ratio and root hair induction [105]. Indeed, PHR1 works with another MYB factor, PHL1, to control most transcriptional activations and repressions in responses to Pi deficiency [104]. Furthermore, about two thirds of the genes repressed in Pi-deprived wild type seedlings were markedly

synergistically or antagonistically regulated by the two signals.

potato plants.

to Pi deficiency.

**7. Pi sensing and gene expression** 

de-repressed in Pi-starved *phr1phl1* double mutants [104].

The amounts of exuded carbon as organic acids can be enormous, ranging from 10% to greater than 25% of the total plant dry weight [75]. Enhanced synthesis of organic acids in Pi-starved plants has been correlated with up-regulation of phosphoenolpyruvate carboxylase and its activation by reversible phosphorylation as well as malate dehydrogenase and citrate synthase and elevated rates of dark CO2 fixation [75,83].

## **6. Genomic analysis of Pi adaptation mechanisms**

As sessile organisms, plants stand on their own potential to retrieve Pi from their surrounding soil and utilize it as efficient as possible. Perhaps, this is why they carry numerous loci encoding APases and Pi transporters. Some representative genomes are shown in Table 1.


**Table 1.** Genome size and the number of APase and Pi transporter-encoding genes in four plant genomes with annotated sequenced.

Genome-wide profiling methods has been employed to compare the transcriptional profiles of Pi-starved and Pi-fed plants [67-69,95-99]. These studies have shown that the changes in gene expression can be detected within hours after exposure to Pi starvation [67,68,95]. Wu et al. [67] found that within 72 h from the onset of Pi starvation, the expression of 1800 of 6172 surveyed genes were changed over two-fold, which include more than 100 transcription factors and cell signaling proteins. Furthermore, differential expression patterns in leaves and roots demonstrated distinct responses to Pi starvation in those organs. A similar conclusion was obtained from the microarray analysis of the Pi-starved rice seedlings [100]. Using an Arabidopsis whole genome Affymetrix chip which includes 22,810 genes, Misson et al. [68] found that the expression of 612 genes were induced while 254 genes were suppressed under Pi-limiting conditions. In addition to the Pi transporters, RNase and APase genes, the induced genes include those that function in sulfate and iron transport and homeostasis, Pi salvaging from organic compounds, phospholipids degradation and galacto- and sulfolipid synthesis, anthocyanin synthesis, phytohormone responses, signal transduction, transcriptional regulation, protein degradation, cell wall metabolism and so on. The suppressed genes are involved in lipid synthesis, reactive oxygen controlling and protein synthesis. In another research, Morcuende et al. [69] showed P deprivation led to transcriptional alterations in over 1000 genes involved in Pi uptake, the mobilization of organic Pi, the conversion of phosphorylated glycolytic intermediates to carbohydrates and organic acids, the replacement of phospholipids with galactolipids and the repression of gene implicated in nucleotide/nucleic acid synthesis which were reversed within 3 h after Pi re-supply. In addition, analysis of metabolites confirmed that P deprivation leads to a shift towards the accumulations of carbohydrates, organic acids and amino acids. Pi deprived plants also showed large changes in the expression of many genes involved in secondary metabolism and photosynthesis. Hammond et al. [101]used an oligonucleotide potato microarray to investigate the transcriptional profile of potato leaves under Pi deficiency and compare their data with previously described transcriptional profiles for the leaves of Arabidopsis and rice. They identified novel components to these profiles, including the increased expression of potato patatin genes- with potential phospholipase A2 activity- in the leaves of Pi deficient potatoes. A set of 200 genes were identified that show differential expression patterns between fertilized and unfertilized potato plants.

Müller et al. [102] investigated the effect of interaction of Pi and sucrose signals on the gene expression pattern in Arabidopsis. They found several genes that were previously identified to be either sugar-responsive or Pi-responsive genes. In addition, 150 genes were synergistically or antagonistically regulated by the two signals.

In a comprehensive analysis, Lin et al. [103] conducted time course microarray experiments and co-expression-based clustering of Pi-responsive genes by pair wise comparison of genes against a customized data base. Three major clusters enriched in genes functioning in transcriptional regulation, root hair formation and developmental adaptations were distinguished in this analysis. The genome-wide transcriptional approach may be used to infer inclusive scenarios for involved mechanisms in the signaling and adaptation of plants to Pi deficiency.

#### **7. Pi sensing and gene expression**

8 Plant Science

shown in Table 1.

genomes with annotated sequenced.

unknown until plasma membrane H+-pumping ATPases were shown to be involved in plant adaptation to Pi starvation [93]. Acidification was also correlated with the up-regulation of novel membrane channels needed to transport anions such as citrate and malate from root cells into the rhizosphere [94]. Organic acid excretion results in the chelating of metal cations that immobilize Pi (e.g. Ca2+, Al3+, Fe2/3+), thus, increasing free Pi concentrations in soil upto 1000-fold. Acidification of rhizosphere also enhances the hydrolysis of organic Pi by secreted APases. As well, organic acids could function as carbon source for symbiotic

The amounts of exuded carbon as organic acids can be enormous, ranging from 10% to greater than 25% of the total plant dry weight [75]. Enhanced synthesis of organic acids in Pi-starved plants has been correlated with up-regulation of phosphoenolpyruvate carboxylase and its activation by reversible phosphorylation as well as malate

As sessile organisms, plants stand on their own potential to retrieve Pi from their surrounding soil and utilize it as efficient as possible. Perhaps, this is why they carry numerous loci encoding APases and Pi transporters. Some representative genomes are

Organism **Genome size (Mb) APases Pi transporters**  *Oryza sativa* 450 40 14 *Arabidopsis thaliana* 125 58 15 *Glycine max* 975 128 35 *Populus trichocarpa* 10 51 22 **Table 1.** Genome size and the number of APase and Pi transporter-encoding genes in four plant

Genome-wide profiling methods has been employed to compare the transcriptional profiles of Pi-starved and Pi-fed plants [67-69,95-99]. These studies have shown that the changes in gene expression can be detected within hours after exposure to Pi starvation [67,68,95]. Wu et al. [67] found that within 72 h from the onset of Pi starvation, the expression of 1800 of 6172 surveyed genes were changed over two-fold, which include more than 100 transcription factors and cell signaling proteins. Furthermore, differential expression patterns in leaves and roots demonstrated distinct responses to Pi starvation in those organs. A similar conclusion was obtained from the microarray analysis of the Pi-starved rice seedlings [100]. Using an Arabidopsis whole genome Affymetrix chip which includes 22,810 genes, Misson et al. [68] found that the expression of 612 genes were induced while 254 genes were suppressed under Pi-limiting conditions. In addition to the Pi transporters, RNase and APase genes, the induced genes include those that function in sulfate and iron transport and homeostasis, Pi salvaging from organic compounds, phospholipids degradation and galacto- and sulfolipid synthesis, anthocyanin synthesis, phytohormone

dehydrogenase and citrate synthase and elevated rates of dark CO2 fixation [75,83].

rhizobacteria that facilitate root Pi acquisition [17,74,75,86].

**6. Genomic analysis of Pi adaptation mechanisms** 

Metabolic adjustments to Pi limitation are largely cellular responses to sensed by internal Pi status which trigger a systemically integrated regulating mechanisms involving microRNAs, non-coding RNAs and PHO2 downstream of PHR as revealed in recent studies. PHR1, a MYB transcription factor, binds to the promoters of most of the Pi-responsive genes that are positively or negatively affected by Pi starvation [104]. Despite its central role in controlling the expression of numerous Pi-responsive genes, the phr1 mutant showed no major phenotypic defects except for a slight difference in the root-to-shoot ratio and root hair induction [105]. Indeed, PHR1 works with another MYB factor, PHL1, to control most transcriptional activations and repressions in responses to Pi deficiency [104]. Furthermore, about two thirds of the genes repressed in Pi-deprived wild type seedlings were markedly de-repressed in Pi-starved *phr1phl1* double mutants [104].

In Arabidopsis, a number of miRNA molecules have been shown to be specifically and strongly induced by Pi limitation, including miRNA399, miRNA778, miRNA827 and miRNA2111[106-108], though only the role of miRNA399 in the regulation of Pi homeostasis has been elucidated [77,109]. miRNA399 is a component of the shoot-to-root Pi-deficiency signaling pathway that moves from shoot to root via the phloem where it targets the transcripts of PHO2 [103,110]. The repression of PHO2 expression by miRNA399 causes upregulation of root Pi transporters (e.g. PHT1;8 and PHT1;9).

Plant Phosphate Nutrition and Environmental Challenges 11

Pi bearing minerals suitable for Pi production. The primary Pi minerals in PR are *phosphorites* that include fluor-apatite (Ca10(PO4)6F2), Hydroxy-apatite (Ca10(PO4)6(OH)2, carbonate-hydroxyl-apatites, and francolite (Ca10-x-y Nax Mgy(PO4)6-z(CO3)zF0.4zF2) which is a carbonated-substituted form of apatite mainly found in marine environments [116].

In PR industry the grade of the rocks are mostly reported as the percentage of P pentoxide (P2O5). Three major resources that can be profitably recovered today are as below [116-117]: 1. Sedimentary Pi deposits which are widespread throughout the world, occurring almost on all continental shelves. Francolite and apatites are deposited in layers that might cover thousands of square miles in several chemical compositions and a wide range of

2. Igneous Pi deposits which are mostly found in continental shelf and on seamounts in the Atlantic and Pacific oceans (Russia, Canada, Brazil and South Africa). Their exploitation is economically non-feasible so that they have mostly remained untouched

3. Biogenic deposits, also known as island Pi, which are mainly old bird and bat

About 80% of world Pi production comes from non-renewable sedimentary reserves, 15 to 19% from igneous and about 1-5% from biogenic and other deposits such as island Pi which

Today, PR is produced in over 40 countries, with 12 countries producing over 92 % of the world's total production [118]. US alone produce over 28% of the total production followed by China, Morocco and Russia. Among them, four major producers of PR (the United States of America, China, Morocco and Western Sahara, and the Russian Federation) produced about 72.0 percent of the world total. Twenty other countries produced the remaining 6-7

Production of fertilizers began in early 19th century when crushed bones were treated with acid and applied to soil. Since 1945 mining of PR was increased from 11.2 Mt to 145 Mt in 1999 [120]. This was translated to increasing production rate of Pi fertilizers (as P2O5) from 4 Mt by 1940's to over 42 Mt annually today [121]. Ever since new technologies has been evolving to maximize the production rate and purity of the fertilizers. Here a brief

The wet process or hemihydrate process is the newest and dominant production method used due to its ease, lesser investment size, energy efficient and higher yield [122]. Basically,

are near total depletion due to over exploitation during the past decade [116].

physical forms [116,118].

droppings built up.

percent of world production [119].

**11. Methods of Pi production** 

**11.1. Wet process** 

**10. Geographic distributions of Pi rocks** 

description of the major production methods is given.

[118].

The Pi-signaling network also involves IPS (Induced by Pi Starvation) genes that carry short open reading frames [111-112]. It is postulated that their transcripts operate by a mechanism called 'target mimicry' as they contain a conserved 23-bp region complementary to miRNA399 and to fine-tune the PHO2–miRNA399 pathway [113]. Since homologs of Arabidopsis IPS and PHO2 genes are present in numerous other plants such a mechanism is probably widespread in plants [114].

The role of miRNAs and non-coding RNAs appear to be extended to a possible role in its coordination with homeostasis of Pi and also other nutrients. For example, a hypothetical role was attributed to miRNA827, in the cross-talk between Pi-limitation and nitratelimitation signaling pathways that affect anthocyanin synthesis [108]. In a survey on small RNAs showing differential expression, miRNA169, miRNA395, and miRNA398 were found to be suppressed in response to Pi deficiency while they also responded to other nutritional stresses [106]. Furthermore, miRNA169 and miRNA398 target genes involved in drought tolerance and oxidative stress response [115]. Suppression of miRNA395 was suggested to up-regulate the expression of APS4 and SULTR2;1 leading to increased sulfate translocation and improved utilization of sulfolipid biosynthesis under Pi-deficient conditions [115].

Taken together, the genome-wide surveys affirm the participation of miRNAs in coordination of homeostatic pathways of Pi and possible links between them and metabolic adjustment [106].

#### **8. Limited Pi resources**

Despite the existence of high amounts of P in the soil [5], the concentration of available Pi in many soil solution averages at about 1 µM and seldom exceeds 10 µM [2] which is far below the cellular Pi concentrations (5–20 mM) required for optimal plant growth and development [73]. Such a limitation often lead to reduced productivity in natural ecosystems as well as cropping systems, unless it is supplied as fertilizer [74-75]. Whilst our limited global Pi reserves are non-renewable, P in many agricultural soils is being building up. This is because 80 to 90 percent of Pi applied as fertilizer is fixed by soil particles or compounds, rendering it unavailable for plants.

### **9. Types of Pi mines**

There are over 200 P minerals existing on the earth but only a few can be used for commercial extraction of Pi [61]. Phosphate Rocks (PR) is the commercial term applied to all Pi bearing minerals suitable for Pi production. The primary Pi minerals in PR are *phosphorites* that include fluor-apatite (Ca10(PO4)6F2), Hydroxy-apatite (Ca10(PO4)6(OH)2, carbonate-hydroxyl-apatites, and francolite (Ca10-x-y Nax Mgy(PO4)6-z(CO3)zF0.4zF2) which is a carbonated-substituted form of apatite mainly found in marine environments [116].

In PR industry the grade of the rocks are mostly reported as the percentage of P pentoxide (P2O5). Three major resources that can be profitably recovered today are as below [116-117]:


About 80% of world Pi production comes from non-renewable sedimentary reserves, 15 to 19% from igneous and about 1-5% from biogenic and other deposits such as island Pi which are near total depletion due to over exploitation during the past decade [116].

## **10. Geographic distributions of Pi rocks**

Today, PR is produced in over 40 countries, with 12 countries producing over 92 % of the world's total production [118]. US alone produce over 28% of the total production followed by China, Morocco and Russia. Among them, four major producers of PR (the United States of America, China, Morocco and Western Sahara, and the Russian Federation) produced about 72.0 percent of the world total. Twenty other countries produced the remaining 6-7 percent of world production [119].

## **11. Methods of Pi production**

Production of fertilizers began in early 19th century when crushed bones were treated with acid and applied to soil. Since 1945 mining of PR was increased from 11.2 Mt to 145 Mt in 1999 [120]. This was translated to increasing production rate of Pi fertilizers (as P2O5) from 4 Mt by 1940's to over 42 Mt annually today [121]. Ever since new technologies has been evolving to maximize the production rate and purity of the fertilizers. Here a brief description of the major production methods is given.

#### **11.1. Wet process**

10 Plant Science

In Arabidopsis, a number of miRNA molecules have been shown to be specifically and strongly induced by Pi limitation, including miRNA399, miRNA778, miRNA827 and miRNA2111[106-108], though only the role of miRNA399 in the regulation of Pi homeostasis has been elucidated [77,109]. miRNA399 is a component of the shoot-to-root Pi-deficiency signaling pathway that moves from shoot to root via the phloem where it targets the transcripts of PHO2 [103,110]. The repression of PHO2 expression by miRNA399 causes up-

The Pi-signaling network also involves IPS (Induced by Pi Starvation) genes that carry short open reading frames [111-112]. It is postulated that their transcripts operate by a mechanism called 'target mimicry' as they contain a conserved 23-bp region complementary to miRNA399 and to fine-tune the PHO2–miRNA399 pathway [113]. Since homologs of Arabidopsis IPS and PHO2 genes are present in numerous other plants such a mechanism is

The role of miRNAs and non-coding RNAs appear to be extended to a possible role in its coordination with homeostasis of Pi and also other nutrients. For example, a hypothetical role was attributed to miRNA827, in the cross-talk between Pi-limitation and nitratelimitation signaling pathways that affect anthocyanin synthesis [108]. In a survey on small RNAs showing differential expression, miRNA169, miRNA395, and miRNA398 were found to be suppressed in response to Pi deficiency while they also responded to other nutritional stresses [106]. Furthermore, miRNA169 and miRNA398 target genes involved in drought tolerance and oxidative stress response [115]. Suppression of miRNA395 was suggested to up-regulate the expression of APS4 and SULTR2;1 leading to increased sulfate translocation and improved utilization of sulfolipid biosynthesis under Pi-deficient conditions [115].

Taken together, the genome-wide surveys affirm the participation of miRNAs in coordination of homeostatic pathways of Pi and possible links between them and metabolic

Despite the existence of high amounts of P in the soil [5], the concentration of available Pi in many soil solution averages at about 1 µM and seldom exceeds 10 µM [2] which is far below the cellular Pi concentrations (5–20 mM) required for optimal plant growth and development [73]. Such a limitation often lead to reduced productivity in natural ecosystems as well as cropping systems, unless it is supplied as fertilizer [74-75]. Whilst our limited global Pi reserves are non-renewable, P in many agricultural soils is being building up. This is because 80 to 90 percent of Pi applied as fertilizer is fixed by soil particles or

There are over 200 P minerals existing on the earth but only a few can be used for commercial extraction of Pi [61]. Phosphate Rocks (PR) is the commercial term applied to all

regulation of root Pi transporters (e.g. PHT1;8 and PHT1;9).

probably widespread in plants [114].

adjustment [106].

**8. Limited Pi resources** 

**9. Types of Pi mines** 

compounds, rendering it unavailable for plants.

The wet process or hemihydrate process is the newest and dominant production method used due to its ease, lesser investment size, energy efficient and higher yield [122]. Basically,

this is the process where PR is treated with sulfuric acid to hydrolyze apatite minerals. Other acids can be used to harvest Pi depending on the costs and desired final product. For example, phosphoric acid addition yields TSP as the final product. Using of nitric acid and hydrochloric acid has also been reported but never industrialized. Phosphogypsium and silicon fluorine or HF are as the main side product in this method. Sulfuric acid added to PRs forms the soluble monocalcium Pi. This product can either go to the fertilizer production (i.e. MAP or DAP) or to concentrated forms for other applications [122-123]. Concentrating the product by evaporation can yield up to 53% phosphoric acid solution. The pollution of current technology wastes (4-5 tons phosphogypsium per one ton of P2O5) signifies the processing costs for the industry.

Plant Phosphate Nutrition and Environmental Challenges 13

crushed bones with sulfuric acid to produce superphosphate was followed by the same treatment of PRs to produce current Pi fertilizers which was the beginning of a new era in the world agriculture history [127]. Since then, Pi fertilizers production processes, molecular types, application methods and feasibility assessments have been the subject of a wide range of researches and analyses. Table 2 summarizes popular Pi fertilizers that are currently used

In 2006, 167 Mt of PR were mined in the world, while China alone was responsible for 56 Mt of it. 23% of the total PR production was directly used as fertilizers which equals 12 Mt P2O5 per year. Only 6% was used for production of elemental P, and almost the rest went through the wet process for production of phosphoric acid, which in turn is mostly (88-95%) used up for Pi fertilizers. World's total Pi supply (P2O5) has been estimated to rise from 42 Mt in 2010 to 45 Mt in 2015 (Table 3) [121]. It is expected that P fertilizer supply will have an increased trend of 3.2% annually with the major Pi balance (production minus consumption) surplus in North America and Africa and deficit in Latin America and Asia. With the current rate of consumption, various estimates showed that recoverable Pi mine will be vanished in 50 to

**Material P N K S P compound** 

**superphosphate (TSP)** 20 ----- ----- 1 to 1.5 Ca(H2PO4)2

18 to

11 to

**superphosphate** 7 to 10 ----- ----- 12 Ca(H2PO4)2

**phosphate** ----- 35 ----- KH2PO4

**phosphate** ----- 54 ----- K2HPO4

**Table 2.** Commonly used Pi fertilizers and the percentage of each elements (quoted from Havlin and

23 to

20 to 21

21 to 24

<sup>35</sup>----- ----- ----- H3PO4

<sup>24</sup>----- ----- ----- H3PO4

<sup>21</sup>----- 0-2 (NH4)2HPO4

<sup>13</sup>----- 0 to 2 NH4H2PO4

(CaCo3)x.(Ca(OH)2)x

18 ----- ----- ----- [Ca3(PO4)2]3.CaFx.

[128].

100 years [129].

**acid** 

**Concentrated** 

**Diammonium phosphate (DAP)** 

**Normal** 

**Monoammonium phosphate (MAP)** 

**Monopotassium** 

**Dipotassium** 

Beaton [128]).

**Superphosphoric acid** 30 to

**Phosphate Rock** 12 to

**Wet Process phosphric** 

**13. Trend of Pi usage in the world** 

#### **11.2. Thermal process**

Thermal process is an older technology that is used in only a few factories in the world. High electricity and water consumption as well as higher investment size and lower product yield makes such process less desirable for investors. This process can be performed in desired scales, but requires detailed financial feasibility estimation depending on the production site location [122]. Before heating, Pi rock is converted to 1-2 cm pellets by wet granulation and sintering to prevent blocking of furnace. Next step involves mixing with cokes (reducing agent) and SiO2 (for slag formation) before feeding into furnace. Heating at 1500C, Pi is reduced to P4 in gaseous state which is condensed afterward. Remaining CaO combines with SiO2 to form liquid slag that might be used for road construction [124].

#### **11.3. Bioprocess**

Bioprocessing of Pi rocks involves treatment of apatites with Pi solubilizing bacteria. This controlled fermentation process requires presence of a bacterial energy supply such as sugars which is consumed to produce organic acids to break down the PRs [125]. Optimization of this process is still under investigation of many scientific and financial institutes due to its promising outlook.

Bioprocessing of insoluble soil Pi compounds is also carried out directly in agricultural fields by the use of biofertilizers (see below) which converts a portion of precipitated fertilizers as well as natural occurring insoluble Pi. This method is extremely feasible with regards of purchasing, application and higher crop yields [126].

### **12. Emergence of Pi fertilizers**

As mentioned earlier in this chapter, Pi has been found to be the limiting macronutrient in most agricultural soils. The first surveys that revealed such deficiency was a simple study during early 19th century in England [127]. At that time the only source of artificially added Pi to the agricultural soils was farmyard manure, which resulted in higher crop yields near manure production sites. It was only a matter of time before it was realized that crushed bones might have a positive result on crop yield on some specific types of soils. Treating the crushed bones with sulfuric acid to produce superphosphate was followed by the same treatment of PRs to produce current Pi fertilizers which was the beginning of a new era in the world agriculture history [127]. Since then, Pi fertilizers production processes, molecular types, application methods and feasibility assessments have been the subject of a wide range of researches and analyses. Table 2 summarizes popular Pi fertilizers that are currently used [128].

#### **13. Trend of Pi usage in the world**

12 Plant Science

signifies the processing costs for the industry.

**11.2. Thermal process** 

**11.3. Bioprocess** 

institutes due to its promising outlook.

**12. Emergence of Pi fertilizers** 

regards of purchasing, application and higher crop yields [126].

this is the process where PR is treated with sulfuric acid to hydrolyze apatite minerals. Other acids can be used to harvest Pi depending on the costs and desired final product. For example, phosphoric acid addition yields TSP as the final product. Using of nitric acid and hydrochloric acid has also been reported but never industrialized. Phosphogypsium and silicon fluorine or HF are as the main side product in this method. Sulfuric acid added to PRs forms the soluble monocalcium Pi. This product can either go to the fertilizer production (i.e. MAP or DAP) or to concentrated forms for other applications [122-123]. Concentrating the product by evaporation can yield up to 53% phosphoric acid solution. The pollution of current technology wastes (4-5 tons phosphogypsium per one ton of P2O5)

Thermal process is an older technology that is used in only a few factories in the world. High electricity and water consumption as well as higher investment size and lower product yield makes such process less desirable for investors. This process can be performed in desired scales, but requires detailed financial feasibility estimation depending on the production site location [122]. Before heating, Pi rock is converted to 1-2 cm pellets by wet granulation and sintering to prevent blocking of furnace. Next step involves mixing with cokes (reducing agent) and SiO2 (for slag formation) before feeding into furnace. Heating at 1500C, Pi is reduced to P4 in gaseous state which is condensed afterward. Remaining CaO combines with SiO2 to form liquid slag that might be used for road construction [124].

Bioprocessing of Pi rocks involves treatment of apatites with Pi solubilizing bacteria. This controlled fermentation process requires presence of a bacterial energy supply such as sugars which is consumed to produce organic acids to break down the PRs [125]. Optimization of this process is still under investigation of many scientific and financial

Bioprocessing of insoluble soil Pi compounds is also carried out directly in agricultural fields by the use of biofertilizers (see below) which converts a portion of precipitated fertilizers as well as natural occurring insoluble Pi. This method is extremely feasible with

As mentioned earlier in this chapter, Pi has been found to be the limiting macronutrient in most agricultural soils. The first surveys that revealed such deficiency was a simple study during early 19th century in England [127]. At that time the only source of artificially added Pi to the agricultural soils was farmyard manure, which resulted in higher crop yields near manure production sites. It was only a matter of time before it was realized that crushed bones might have a positive result on crop yield on some specific types of soils. Treating the In 2006, 167 Mt of PR were mined in the world, while China alone was responsible for 56 Mt of it. 23% of the total PR production was directly used as fertilizers which equals 12 Mt P2O5 per year. Only 6% was used for production of elemental P, and almost the rest went through the wet process for production of phosphoric acid, which in turn is mostly (88-95%) used up for Pi fertilizers. World's total Pi supply (P2O5) has been estimated to rise from 42 Mt in 2010 to 45 Mt in 2015 (Table 3) [121]. It is expected that P fertilizer supply will have an increased trend of 3.2% annually with the major Pi balance (production minus consumption) surplus in North America and Africa and deficit in Latin America and Asia. With the current rate of consumption, various estimates showed that recoverable Pi mine will be vanished in 50 to 100 years [129].


**Table 2.** Commonly used Pi fertilizers and the percentage of each elements (quoted from Havlin and Beaton [128]).

An IFDC estimates shows that resources of unprocessed PR of varying grades could be as high as 290 Mt [130] that could become reserves once all high grade PR are depleted. Even if this takes place, the price of Pi would not be affordable for many poor farmers and still pollution because of Pi accumulation and associated wastes in the soil and water are of concerns.

Plant Phosphate Nutrition and Environmental Challenges 15

the granule, which in turn absorbs water, increasing the soil moisture. In these areas Pi uptake can take place efficiently by plants. Depending on type of soil and its cations composition, the released Pi can re-precipitate within a few days or weeks [127-128]. This behavior indicate the proper time of Pi fertilizer application in order to reduce the chance of Pi accumulation in the soil. The use of such information also helps calculating the correct amount of Pi that should be applied for maintaining the P-level of the soil after a harvesting season which is extremely important for balanced fertilization. It has been shown repeatedly

Historically, since mid-19th century, the use of local organic matters was replaced by other sources such as guano, deposited bird droppings in remote islands. Growing world consumption rapidly vanished guano resources by the end of 19th century shifting trades to PR. In recent years, the PR production has grown to over 140-160 Mt per year for extraction

**Figure 2.** Predicted peak of elemental P production curve based on actual data in previous years. It illustrates global P reserves are likely to peak around year 2033 when current P production will not be

Since PRs are non-renewable, it is estimated that the economically recoverable reserves will be depleted by the end of this century [129]. The point is that production rate will peak at a maximum when high quality and accessible Pi resources are depleted and mining and processing become quite expensive. As shown in Figure 2, the consumption of P since 19th century follows a Guassian distribution curve. Assuming the total earth reserve of elemental P is approximately 3240 Mt, then its production will peak at 29 Mt in 2033 as modeled by Cordell et al. [129] (Figure 2). Although the actual timing depends on several conditions to be met, however, this is really alarming if considering that the non-renewable Pi reserves are finite and there is no substitute for PRs. Besides, one might presume that the energy cost will be increased and the average grade of Pi will decrease in future as it was experienced to decline from 15% to 13% between 1970 to 1996 [132]. As this challenge silently threaten the food security, a global comprehensive effort is necessary. Web sites such as Pi Knowledge

no increase in yield is detected when Pi fertilizers are over-used [124,127,128].

**16. Need for Pi preservation and restoration** 

of about 25 Mt elemental P per year [132].

economical any more (Quoted from Cordell et al. [129]).


**Table 3.** World Pi supply, demand and balance according to FAO report (2008) [119].

It is noteworthy the extracted phosphoric acid, so-called green or wet process acid, contains upto 24% elemental P and is produced from the reaction of sulfuric acid and PR. It is used for agronomics means either by direct application to more alkaline soils or by reaction with PRs for partial or complete acidulation. Phosphoric acid is also produced by electric furnace to produce elemental P that in turn reacts with O2 and water to make phosphoric acid. This type of phosphoric acid, called white acid, is purified to make technical and food grades which is mostly used for non-agricultural purposes such as soft drinks due to its higher purity and expensive production costs [128].

## **14. Effect of P fertilizers on crop yields**

Soil scientists recognize three kinds of Pi: (1) deposited Pi as chemical compounds in the soil; (2) available Pi or free Pi ion in the soil; and (3) absorbable Pi or free Pi ion in vicinity of root. Even though the average use of P2O5 (19 kg/ha) has been less than recommended amounts in the literature (30Kg/ha) [131], it has resulted in significant increase in crop yields around the world. For example, an increase between 9 to 60% compared to control was observed in Middle East countries on barley, wheat, and chickpea. Productivity Indexes of cereal crops were as high as 11.1 when up to 90 kg/ha P2O5 were added. Data on oil crops shows productivity index upto 6 in groundnut and mustard. In Indonesia, upto 10 fold increase was observed after soluble Pi was added to soybeans [131]. Such examples are very easy to find in the literatures dated early 19th to 20th centuries when the application of Pi fertilizers were becoming dominant. The resultant high yield is accompanied by Pi removal of 6 kg/ha for cereals and 20 Kg/ha for other crops from soil [61].

## **15. The fate of Pi fertilizers in soil**

Two contrasting challenges of higher crop yield and environmental concerns necessitate tracing the fate of Pi fertilizer added to soil. As the Pi fertilizer granule or droplet is added to the soil, the moisture penetrates its way into the fertilizer. This results in the release of export of Pi into the soil which might expand as far as 3 to 5 cm from an average size granule [128]. As long as the original salt remains, a saturated solution is maintained around the granule, which in turn absorbs water, increasing the soil moisture. In these areas Pi uptake can take place efficiently by plants. Depending on type of soil and its cations composition, the released Pi can re-precipitate within a few days or weeks [127-128]. This behavior indicate the proper time of Pi fertilizer application in order to reduce the chance of Pi accumulation in the soil. The use of such information also helps calculating the correct amount of Pi that should be applied for maintaining the P-level of the soil after a harvesting season which is extremely important for balanced fertilization. It has been shown repeatedly no increase in yield is detected when Pi fertilizers are over-used [124,127,128].

#### **16. Need for Pi preservation and restoration**

14 Plant Science

concerns.

An IFDC estimates shows that resources of unprocessed PR of varying grades could be as high as 290 Mt [130] that could become reserves once all high grade PR are depleted. Even if this takes place, the price of Pi would not be affordable for many poor farmers and still pollution because of Pi accumulation and associated wastes in the soil and water are of

Year 2010 2011 2012 2013 2014 2015 Supply 39600 42094 43966 45011 46439 47788 Demand 41700 41679 42562 43435 44245 45015 Potential balance -2100 415 1404 1576 2194 2773

It is noteworthy the extracted phosphoric acid, so-called green or wet process acid, contains upto 24% elemental P and is produced from the reaction of sulfuric acid and PR. It is used for agronomics means either by direct application to more alkaline soils or by reaction with PRs for partial or complete acidulation. Phosphoric acid is also produced by electric furnace to produce elemental P that in turn reacts with O2 and water to make phosphoric acid. This type of phosphoric acid, called white acid, is purified to make technical and food grades which is mostly used for non-agricultural purposes such as soft drinks due to its higher

Soil scientists recognize three kinds of Pi: (1) deposited Pi as chemical compounds in the soil; (2) available Pi or free Pi ion in the soil; and (3) absorbable Pi or free Pi ion in vicinity of root. Even though the average use of P2O5 (19 kg/ha) has been less than recommended amounts in the literature (30Kg/ha) [131], it has resulted in significant increase in crop yields around the world. For example, an increase between 9 to 60% compared to control was observed in Middle East countries on barley, wheat, and chickpea. Productivity Indexes of cereal crops were as high as 11.1 when up to 90 kg/ha P2O5 were added. Data on oil crops shows productivity index upto 6 in groundnut and mustard. In Indonesia, upto 10 fold increase was observed after soluble Pi was added to soybeans [131]. Such examples are very easy to find in the literatures dated early 19th to 20th centuries when the application of Pi fertilizers were becoming dominant. The resultant high yield is accompanied by Pi removal

Two contrasting challenges of higher crop yield and environmental concerns necessitate tracing the fate of Pi fertilizer added to soil. As the Pi fertilizer granule or droplet is added to the soil, the moisture penetrates its way into the fertilizer. This results in the release of export of Pi into the soil which might expand as far as 3 to 5 cm from an average size granule [128]. As long as the original salt remains, a saturated solution is maintained around

**Table 3.** World Pi supply, demand and balance according to FAO report (2008) [119].

purity and expensive production costs [128].

**15. The fate of Pi fertilizers in soil** 

**14. Effect of P fertilizers on crop yields** 

of 6 kg/ha for cereals and 20 Kg/ha for other crops from soil [61].

Historically, since mid-19th century, the use of local organic matters was replaced by other sources such as guano, deposited bird droppings in remote islands. Growing world consumption rapidly vanished guano resources by the end of 19th century shifting trades to PR. In recent years, the PR production has grown to over 140-160 Mt per year for extraction of about 25 Mt elemental P per year [132].

**Figure 2.** Predicted peak of elemental P production curve based on actual data in previous years. It illustrates global P reserves are likely to peak around year 2033 when current P production will not be economical any more (Quoted from Cordell et al. [129]).

Since PRs are non-renewable, it is estimated that the economically recoverable reserves will be depleted by the end of this century [129]. The point is that production rate will peak at a maximum when high quality and accessible Pi resources are depleted and mining and processing become quite expensive. As shown in Figure 2, the consumption of P since 19th century follows a Guassian distribution curve. Assuming the total earth reserve of elemental P is approximately 3240 Mt, then its production will peak at 29 Mt in 2033 as modeled by Cordell et al. [129] (Figure 2). Although the actual timing depends on several conditions to be met, however, this is really alarming if considering that the non-renewable Pi reserves are finite and there is no substitute for PRs. Besides, one might presume that the energy cost will be increased and the average grade of Pi will decrease in future as it was experienced to decline from 15% to 13% between 1970 to 1996 [132]. As this challenge silently threaten the food security, a global comprehensive effort is necessary. Web sites such as Pi Knowledge

Center (http://www.GreenPi.info) or Global Phosphorus Research Initiative (http://www.phosphorusfutures.net) could greatly help worldwide data sharing and collaborations amongst scientists and other players to seek integrative solutions.

Plant Phosphate Nutrition and Environmental Challenges 17

underground waters [122]. Eutrophication and the consequent over-enrichment of aquatic ecosystems with nutrients, mostly Pi and nitrate lead to algal blooms and anoxic events that may initiate irreversible environmental damages. Although some lakes have recovered after sources of nutrient inputs were reduced, recycling of enriched Pi from sediments causes

Recycling of Pi from sewage systems, precision farming and agricultural and industrial treatment units has been exercised in developed countries such as Japan and Germany to reduce such effects [122,124]. Replacing Pi in detergents by aluminum silicate has also been considered as option, despite its much poorer performance. Crystallization of Pi in wastewater as struvite (ammonium magnesium phosphate), separation of urine and the use of sanitized faecal matters in municipalities have been attempted to recover or reuse wasted

Cordell et al. [129] warned about the peak time of Pi production (Figure 2) with assumption of PRs are the sole high-value Pi reserves. Taking into consideration the massive amounts of insoluble Pi compounds accumulated in the soil, a straightforward solution could be the utilization of PSMs in the form of biofertilizers [91,126]. Simplified application method and comparable cost of Pi biofertizers on one hand and increased quantity and quality of the

As shown in Figure 3, P like other mineral nutrients exists in soil, minerals, water as well as living organisms. Over time, geologic events bring ocean sediments to land and weathering

yields, on the other hand, have already attracted the attention of farmers.

**19. Cycle of P between the physical and biological worlds** 

**Figure 3.** Pi flow through food production and consumption.

lakes to remain eutrophic for years [122].

Pi [129].

## **17. Environmental concerns about Pi production**

Removal of massive amounts of soil and extensive washing with huge amounts of water are major changes in the environment of the mining sites and the surrounding regions by themselves. However, the most important issue is disposal of side products that pollute the ecosystem at the stacking sites massively.

Phosphogypsum, the side product of wet process, is deposited in large land areas called gypsum stacks. For the production of 50 Mt of P2O5 annually, about 250 Mt of gypsum is produced, out of which only 10 percent is used for other purposes (e.g. road construction, cement and housing) and the rest remains stacked [122].

Presence of radioactive materials in side products is another issue for Pi production industry. PRs contain mainly uranium (20-300 ppm) and thorium (1 to 5 ppm) and their decay products. This would mean 0.35 kg U2O8 per Mt P2O5 or 2100 tons of unused uranium annually [122,133]. About 80 percent of this amount and their decay products are concentrated within the gypsum waste materials in wet process. These could be as high as 7 to 100 pCi/g uranium, 11 to 35 pCi/g radium and 1.7 to 12 pCi/g radon, though radioactivity is different from stack to stack depend on the original PR. The fertilizers themselves might also exhibit radioactivity which might be as high as 33 pCi/g in US [133]. These concerns are reflected in a final rule of EPA issued in 1992 stating "phosphogypsum intended for agriculture use must have a certificated average concentration of radium-226 no greater than 10 pCi/g" [133].

## **18. Environmental impacts of Pi fertilizers consumption**

The ever growing population of the world has created an enormous pressure to cultivate larger agricultural lands and to increase production yields by improved mechanized methods and usage of fertilizers. Such practices not only alter chemical and physical properties of soils, but also affect macro and micro flora remarkably [122].

Application of Pi fertilizers to soil does not necessarily guarantee plant uptake due to their polycrystalline structure and their tendency to precipitate. An observation showed that if no crop is around, the addition of Pi fertilizer to soil increases the soluble P within the first couple of years, but it will decrease to a constant level later [127]. Studies in developed countries have also suggested that Pi application might not be as effective as it used to be in the past [125].

In addition to the effect of deposited Pi on the soil structure and microbial flora, leaching of Pi through the erosion of surface soil and organic matters to water streams and lakes cause vast eutrophication [17]. Discharged detergents sodium triphosphates as well as human or animal metabolic wastes must also be accounted for a portion of Pi inputs to surface and underground waters [122]. Eutrophication and the consequent over-enrichment of aquatic ecosystems with nutrients, mostly Pi and nitrate lead to algal blooms and anoxic events that may initiate irreversible environmental damages. Although some lakes have recovered after sources of nutrient inputs were reduced, recycling of enriched Pi from sediments causes lakes to remain eutrophic for years [122].

16 Plant Science

10 pCi/g" [133].

the past [125].

Center (http://www.GreenPi.info) or Global Phosphorus Research Initiative (http://www.phosphorusfutures.net) could greatly help worldwide data sharing and

Removal of massive amounts of soil and extensive washing with huge amounts of water are major changes in the environment of the mining sites and the surrounding regions by themselves. However, the most important issue is disposal of side products that pollute the

Phosphogypsum, the side product of wet process, is deposited in large land areas called gypsum stacks. For the production of 50 Mt of P2O5 annually, about 250 Mt of gypsum is produced, out of which only 10 percent is used for other purposes (e.g. road construction,

Presence of radioactive materials in side products is another issue for Pi production industry. PRs contain mainly uranium (20-300 ppm) and thorium (1 to 5 ppm) and their decay products. This would mean 0.35 kg U2O8 per Mt P2O5 or 2100 tons of unused uranium annually [122,133]. About 80 percent of this amount and their decay products are concentrated within the gypsum waste materials in wet process. These could be as high as 7 to 100 pCi/g uranium, 11 to 35 pCi/g radium and 1.7 to 12 pCi/g radon, though radioactivity is different from stack to stack depend on the original PR. The fertilizers themselves might also exhibit radioactivity which might be as high as 33 pCi/g in US [133]. These concerns are reflected in a final rule of EPA issued in 1992 stating "phosphogypsum intended for agriculture use must have a certificated average concentration of radium-226 no greater than

The ever growing population of the world has created an enormous pressure to cultivate larger agricultural lands and to increase production yields by improved mechanized methods and usage of fertilizers. Such practices not only alter chemical and physical

Application of Pi fertilizers to soil does not necessarily guarantee plant uptake due to their polycrystalline structure and their tendency to precipitate. An observation showed that if no crop is around, the addition of Pi fertilizer to soil increases the soluble P within the first couple of years, but it will decrease to a constant level later [127]. Studies in developed countries have also suggested that Pi application might not be as effective as it used to be in

In addition to the effect of deposited Pi on the soil structure and microbial flora, leaching of Pi through the erosion of surface soil and organic matters to water streams and lakes cause vast eutrophication [17]. Discharged detergents sodium triphosphates as well as human or animal metabolic wastes must also be accounted for a portion of Pi inputs to surface and

collaborations amongst scientists and other players to seek integrative solutions.

**17. Environmental concerns about Pi production** 

cement and housing) and the rest remains stacked [122].

**18. Environmental impacts of Pi fertilizers consumption** 

properties of soils, but also affect macro and micro flora remarkably [122].

ecosystem at the stacking sites massively.

Recycling of Pi from sewage systems, precision farming and agricultural and industrial treatment units has been exercised in developed countries such as Japan and Germany to reduce such effects [122,124]. Replacing Pi in detergents by aluminum silicate has also been considered as option, despite its much poorer performance. Crystallization of Pi in wastewater as struvite (ammonium magnesium phosphate), separation of urine and the use of sanitized faecal matters in municipalities have been attempted to recover or reuse wasted Pi [129].

Cordell et al. [129] warned about the peak time of Pi production (Figure 2) with assumption of PRs are the sole high-value Pi reserves. Taking into consideration the massive amounts of insoluble Pi compounds accumulated in the soil, a straightforward solution could be the utilization of PSMs in the form of biofertilizers [91,126]. Simplified application method and comparable cost of Pi biofertizers on one hand and increased quantity and quality of the yields, on the other hand, have already attracted the attention of farmers.

## **19. Cycle of P between the physical and biological worlds**

As shown in Figure 3, P like other mineral nutrients exists in soil, minerals, water as well as living organisms. Over time, geologic events bring ocean sediments to land and weathering

**Figure 3.** Pi flow through food production and consumption.

carry Pi compounds to terrestrial habitats where the land cycles occurs. Pi is absorbed by plants from soil, consumed by animals and returned to soil as organic matters that slowly released as inorganic Pi or incorporated in more stable molecules as soil organic compounds. Depending on temperature and moisture, soil microorganisms break down these compounds by secreting organic acids and APase enzymes. Pi leaching due to soil erosion or water run-off to surface or underground waters finally discharge in lakes, seas and oceans where another sub-cycle between marine organisms and Pi sediments occurs. Human action for the utilization of Pi in fertilizers, pesticides, detergents and food industries are the main sources of disturbance of these natural cycles [134].

Plant Phosphate Nutrition and Environmental Challenges 19

simply decomposed organic material that continuously occurs in nature, often without any assistance from mankind. The rate of decomposing depends on type of material, air,

Manures improve the texture of both clay and sandy soil; indeed, it is the best additive to make either clay or sandy soil into rich, moisture holding, and loamy soil. They are rich in nitrogen and potassium but have very little Pi. Yet, the availability of soil Pi is enhanced due to chelating polyvalent cations by organic acids and other decay products. In addition, organic materials are rich substrates for propagation of beneficial microorganisms (see below). However, poorly available, being costly and the unbalanced levels of elements in manures have hindered the effectiveness and wide usage. In addition, these still need

Pi solubilizing microorganisms (PSMs) are, as named, a group of soil fungi and bacteria involved in solubilization and mineralization of soil P from inorganic and organic pools as an integral part of the P cycle. In soil, Pi solubilizing bacteria out-number fungi with the same activity by 2–150 folds [135]. The majority of the PSMs solubilize Ca–P complexes in alkaline soils and only a few can solubilize Fe–P and Al–P in acidic soils. Nevertheless,

The principle mechanism for Pi solubilization is the secretion of organic acids and APases that play major role in the mineralization of both inorganic and organic Pi compounds in surrounding soil. Several mechanisms like lowering of pH by acidulation, ion chelating, or ion exchange reactions as well as enzymatic activities in the growth environment have also

Through receiving energetic carbon sources from plant, PSMs facilitate the uptake of Pi and also other nutrients such as zinc, molybdenum, copper and iron. Fungi have been reported to possess greater ability to solubilize rock Pi than bacteria, though they perform better in acidic soil conditions. Species of *Aspergillus*, *Penicillium*, *Curvularia* and yeast have been widely reported to solubiliz various forms of inorganic phosphates [137]. Another mechanism which indirectly leads to increased Pi acquisition by plants is the production of phytohormones (mainly auxins) by rhizobacteria that stimulate root growth [138-139]. Therefore, it makes more sense to name such bacteria as plant growth promoting

Among the soil Pi solubilizing bacteria communities, *Pseudomonas putida*, *P. strata*, *Bacillus sircalmous* and *Pantoa agglomeranse* could be referred to as the most important strains [91]. The positive effect of Pseudomonas inoculation on plant growth has been reported in many field trials [126]. The role of Pi solubilizing bacteria and their potential capacity to restore Pi

Symbiotic relationship between mycorrhizal fungi and plants exists in almost all ecosystems [141]. Fungi species spread around their mycelium acquiring Pi from longer distances which

PSMs can become effective in the latter soils when supplemented with PR.

been proposed to play a role in Pi solubilization by PSMs [91,126,136,137].

cycle processes in plant-soil system cannot be ignored [138].

moisture, and temperature and soil pH.

microbial activity to release the elements.

rhizobacteris (PGPR) [140].

**20.3. The use of Pi solubilizing microorganism** 

## **20. Sustainable Pi usage in agriculture**

The assessment of plant response to Pi level in a series of greenhouse pot trials over a wide range of soils with various characteristics established two values of soil Pi, (1) the critical value (Tc) below which it is recommended to apply Pi fertilizers in order to build up soil Pi fertility, and (2) the sufficient value (Ts) above which it is recommended not to apply it over a period of time. For Tc value, available soil Pi (measured by Olson method) ranged from 10 to15 ppm P2O5 whereas it ranged from 50 to 55 ppm for the Ts value [131]. Of course, it is crucial to provide a certain ratio of each nutrient to establish a proper balance. Legumes need N:P:K nutrients in a ratio of 0:1:1, 1:2:2 or 1:2:3, root crops, a ratio of 2:1:2, and other crops, a ratio of 2:1:1 or 1:1:1 [131].

An old statistics between year 1960 to 1981 shows that Pi fertilizer consumption has been well established as the share of developed countries in the world was decreased for from 75.1 % to 43.5% of the world consumption by practicing precision farming while it has increased from 6.5 to 21.3% in developing countries mainly because of the over usage [125]. The main alternative methods for Pi provision have been described below.

### **20.1. Direct application of PRs as Pi fertilizer**

Direct application of Pi rocks as a source of Pi fertilizer has been practiced since early 19th century. Except for grinding to increase surface area in contact with soil, no other modifications are required to be made which makes it a relatively inexpensive source of Pi. The catch is, however, the lower Pi content of the rocks and required specific soils conditions such as having acidic pH (below 5.5) and low exchangeable Ca2+ [116]. Besides, some plants have higher capabilities in mobilization of Pi into their systems. For example, legumes and some crops of *Cruciferae* family enhance Pi solubilization and uptake by excretion of organic acids that lowers the soil pH and increases the cation exchange capacity of the soil. Acidifying the rhizosphere, secretion of APases and morphological changes are prerequisite adaptations that enable different crops to salvage Pi from PRs.

### **20.2. The use of composted organic materials**

The increasing cost of chemical fertilizers has renewed interest of farmers across all sectors to utilize organic fertilizers as an alternative source of plant nutrients. Composted manure is simply decomposed organic material that continuously occurs in nature, often without any assistance from mankind. The rate of decomposing depends on type of material, air, moisture, and temperature and soil pH.

Manures improve the texture of both clay and sandy soil; indeed, it is the best additive to make either clay or sandy soil into rich, moisture holding, and loamy soil. They are rich in nitrogen and potassium but have very little Pi. Yet, the availability of soil Pi is enhanced due to chelating polyvalent cations by organic acids and other decay products. In addition, organic materials are rich substrates for propagation of beneficial microorganisms (see below). However, poorly available, being costly and the unbalanced levels of elements in manures have hindered the effectiveness and wide usage. In addition, these still need microbial activity to release the elements.

#### **20.3. The use of Pi solubilizing microorganism**

18 Plant Science

carry Pi compounds to terrestrial habitats where the land cycles occurs. Pi is absorbed by plants from soil, consumed by animals and returned to soil as organic matters that slowly released as inorganic Pi or incorporated in more stable molecules as soil organic compounds. Depending on temperature and moisture, soil microorganisms break down these compounds by secreting organic acids and APase enzymes. Pi leaching due to soil erosion or water run-off to surface or underground waters finally discharge in lakes, seas and oceans where another sub-cycle between marine organisms and Pi sediments occurs. Human action for the utilization of Pi in fertilizers, pesticides, detergents and food

The assessment of plant response to Pi level in a series of greenhouse pot trials over a wide range of soils with various characteristics established two values of soil Pi, (1) the critical value (Tc) below which it is recommended to apply Pi fertilizers in order to build up soil Pi fertility, and (2) the sufficient value (Ts) above which it is recommended not to apply it over a period of time. For Tc value, available soil Pi (measured by Olson method) ranged from 10 to15 ppm P2O5 whereas it ranged from 50 to 55 ppm for the Ts value [131]. Of course, it is crucial to provide a certain ratio of each nutrient to establish a proper balance. Legumes need N:P:K nutrients in a ratio of 0:1:1, 1:2:2 or 1:2:3, root crops, a ratio of 2:1:2, and other

An old statistics between year 1960 to 1981 shows that Pi fertilizer consumption has been well established as the share of developed countries in the world was decreased for from 75.1 % to 43.5% of the world consumption by practicing precision farming while it has increased from 6.5 to 21.3% in developing countries mainly because of the over usage [125].

Direct application of Pi rocks as a source of Pi fertilizer has been practiced since early 19th century. Except for grinding to increase surface area in contact with soil, no other modifications are required to be made which makes it a relatively inexpensive source of Pi. The catch is, however, the lower Pi content of the rocks and required specific soils conditions such as having acidic pH (below 5.5) and low exchangeable Ca2+ [116]. Besides, some plants have higher capabilities in mobilization of Pi into their systems. For example, legumes and some crops of *Cruciferae* family enhance Pi solubilization and uptake by excretion of organic acids that lowers the soil pH and increases the cation exchange capacity of the soil. Acidifying the rhizosphere, secretion of APases and morphological changes are

The increasing cost of chemical fertilizers has renewed interest of farmers across all sectors to utilize organic fertilizers as an alternative source of plant nutrients. Composted manure is

industries are the main sources of disturbance of these natural cycles [134].

The main alternative methods for Pi provision have been described below.

prerequisite adaptations that enable different crops to salvage Pi from PRs.

**20. Sustainable Pi usage in agriculture** 

**20.1. Direct application of PRs as Pi fertilizer** 

**20.2. The use of composted organic materials** 

crops, a ratio of 2:1:1 or 1:1:1 [131].

Pi solubilizing microorganisms (PSMs) are, as named, a group of soil fungi and bacteria involved in solubilization and mineralization of soil P from inorganic and organic pools as an integral part of the P cycle. In soil, Pi solubilizing bacteria out-number fungi with the same activity by 2–150 folds [135]. The majority of the PSMs solubilize Ca–P complexes in alkaline soils and only a few can solubilize Fe–P and Al–P in acidic soils. Nevertheless, PSMs can become effective in the latter soils when supplemented with PR.

The principle mechanism for Pi solubilization is the secretion of organic acids and APases that play major role in the mineralization of both inorganic and organic Pi compounds in surrounding soil. Several mechanisms like lowering of pH by acidulation, ion chelating, or ion exchange reactions as well as enzymatic activities in the growth environment have also been proposed to play a role in Pi solubilization by PSMs [91,126,136,137].

Through receiving energetic carbon sources from plant, PSMs facilitate the uptake of Pi and also other nutrients such as zinc, molybdenum, copper and iron. Fungi have been reported to possess greater ability to solubilize rock Pi than bacteria, though they perform better in acidic soil conditions. Species of *Aspergillus*, *Penicillium*, *Curvularia* and yeast have been widely reported to solubiliz various forms of inorganic phosphates [137]. Another mechanism which indirectly leads to increased Pi acquisition by plants is the production of phytohormones (mainly auxins) by rhizobacteria that stimulate root growth [138-139]. Therefore, it makes more sense to name such bacteria as plant growth promoting rhizobacteris (PGPR) [140].

Among the soil Pi solubilizing bacteria communities, *Pseudomonas putida*, *P. strata*, *Bacillus sircalmous* and *Pantoa agglomeranse* could be referred to as the most important strains [91]. The positive effect of Pseudomonas inoculation on plant growth has been reported in many field trials [126]. The role of Pi solubilizing bacteria and their potential capacity to restore Pi cycle processes in plant-soil system cannot be ignored [138].

Symbiotic relationship between mycorrhizal fungi and plants exists in almost all ecosystems [141]. Fungi species spread around their mycelium acquiring Pi from longer distances which

is then passed to plants in exchange for carbon compounds [142]. Nevertheless, limitations in mass production and restriction of this relationship to certain plant types have been unsolved issues for wide utilizations so far.

Plant Phosphate Nutrition and Environmental Challenges 21

**Main effect under P** 

biomass if phytate

phytate available

phytate available

activity and P content in root exudates

Increased P uptake, P content and biomass

Tobacco Increased P content and biomass

Clover Increased P content and biomass

growth

sterile media (phytate available)-no effect in

**Reference** 

[152]

[17]

[153]

[154]

[155]

[156]

[157]

[158]

[158]

**deficiency** 

available

Clover Increased P uptake if

soil

hydrolyze phytates which constitutes up to 50% of the total organic Pi in soil [8]. Overexpression of phytase in potato, clover, soybean and tobacco resulted in increased Pi acquisition and content. These results point to possible manipulation of biochemical pathways that increase the ability of the plant to release organic acids or enzymes that

Several mutant genes have been successfully introduced into commercial crop varieties that significantly enhance the nutritional value of crops. Mutants of barley, wheat, rice and soybean with low phytic acid have been released and can reduce both Pi pollution and increase bioavailability of Pi and micronutrient minerals in cereals and legumes [164]. A major QTL *pup1,* on chromosome 12 effective on Pi uptake from Pi‐deficient soils was fine‐mapped in rice. Liu and colleagues [165] demonstrated that chromosome 1R and 7R of rye might carry genes responsible for tolerance to Pi starvation stress, while 5R carry

**crop species** 

**Secretary phytase** synthetic Potato Increased P content and

**Phytase** *A. niger* Clover Increased P uptake in

**Phytase** *A. niger* Tobacco Increased P uptake if

**Phytase** *A. ficuum* Soybean Increased phytase

**Acid phosphatase** white lupin Tobacco Increased P uptake and

*A. thaliana* Arabidopsis

thaliana and tobacco

improve Pi acquisition from soils.

unfavorable genes regarding Pi starvation tolerance.

**Gene Name Gene source Transformed** 

*niger* 

*subtilis* 

*Medicago truncatula* 

**Phytase** *Aspergillus* 

**β-Propeller phytase** *Bacillus* 

**Phytase and acid phosphatase** 

**Purple acid Phosphatase 18** 

**20.6. Mutagenesis** 

#### **20.4. Plant species with high Pi efficiency**

Genotypic variations for tolerance to Pi deficiency has been demonstrated for many food crops [143] which provide sources for developing cultivars superior in Pi acquisition and higher yield particularly in deficient conditions through plant breeding [144]. Pi-efficient plants are defined as plants which could produce higher yields per unit of applied or absorbed nutrient compared to other plants grown under similar agroecological conditions [145]. Screening for plant species or genotypes with increased Pi absorption involves criteria such as: (i) greater root growth and, thus, extended soil exploration; (ii) the exuding of proton and organic acids that increase the solubility of Pi by decreasing pH and/or chelating; and (iii) the secretions of elevated levels of APase enzymes that break down organic Pi [146].

Most modern crop cultivars have been selected through conventional breeding approaches for better adaptation to deficient soil Pi by looking at root architectural traits which allow for more Pi acquisition from soil surface zone [147]. However, conventional plant breeding including phenotypic selection for improved root systems has proven to be difficult, prone to environmental effects, and time-consuming [148]. As tolerance to low Pi is a quantitative trait, an appropriate method to dissect its complex polygenic inheritance must be employed through quantitative trait loci (QTL) analysis. A number of QTL have been identified for tolerance mechanisms to low Pi in various food crops [149]. Molecular markers linked to the target traits are needed to be used for selection in the breeding process.

#### **20.5. Genetic engineering for Pi acquisition and utilization**

Researches on the molecular and physiological basis of Pi uptake, translocation, and internal utilization facilitate the design and generation of transgenic plants with enhanced Pi efficiency. For instance, over-expression of a Pi transporter gene in tobacco cell cultures resulted in an increased rate of Pi uptake when cells were grown under Pi limited conditions [47]. Several attempts have been made to improve soil Pi solubilization and acquisition processes in food crops through engineering of genes with bacterial, fungal or plant origins. Increasing knowledge on regulatory and signaling mechanisms involved in Pi acquisition might identify new useful genes. It is assumed that a combined overexpression of Pitransporter and dissolving encoding genes might result in synergistic effects [150].

An overview of reported transgenic crop plants is given in Table 4. Generating plants with an enhanced metabolical Pi uptake capacity were attempted by engineered tobacco plants to produce more organic acids, specifically citrate [150]. The promising result was that these transgenic plants yielded more leaf and fruit biomass than controls when grown under Pilimited conditions [151]. Many studies reported transgenic plants with overexpression of phytase in various food crops. Phytases are exuded into the rhizosphere and are able to hydrolyze phytates which constitutes up to 50% of the total organic Pi in soil [8]. Overexpression of phytase in potato, clover, soybean and tobacco resulted in increased Pi acquisition and content. These results point to possible manipulation of biochemical pathways that increase the ability of the plant to release organic acids or enzymes that improve Pi acquisition from soils.

#### **20.6. Mutagenesis**

20 Plant Science

organic Pi [146].

unsolved issues for wide utilizations so far.

**20.4. Plant species with high Pi efficiency** 

is then passed to plants in exchange for carbon compounds [142]. Nevertheless, limitations in mass production and restriction of this relationship to certain plant types have been

Genotypic variations for tolerance to Pi deficiency has been demonstrated for many food crops [143] which provide sources for developing cultivars superior in Pi acquisition and higher yield particularly in deficient conditions through plant breeding [144]. Pi-efficient plants are defined as plants which could produce higher yields per unit of applied or absorbed nutrient compared to other plants grown under similar agroecological conditions [145]. Screening for plant species or genotypes with increased Pi absorption involves criteria such as: (i) greater root growth and, thus, extended soil exploration; (ii) the exuding of proton and organic acids that increase the solubility of Pi by decreasing pH and/or chelating; and (iii) the secretions of elevated levels of APase enzymes that break down

Most modern crop cultivars have been selected through conventional breeding approaches for better adaptation to deficient soil Pi by looking at root architectural traits which allow for more Pi acquisition from soil surface zone [147]. However, conventional plant breeding including phenotypic selection for improved root systems has proven to be difficult, prone to environmental effects, and time-consuming [148]. As tolerance to low Pi is a quantitative trait, an appropriate method to dissect its complex polygenic inheritance must be employed through quantitative trait loci (QTL) analysis. A number of QTL have been identified for tolerance mechanisms to low Pi in various food crops [149]. Molecular markers linked to the

Researches on the molecular and physiological basis of Pi uptake, translocation, and internal utilization facilitate the design and generation of transgenic plants with enhanced Pi efficiency. For instance, over-expression of a Pi transporter gene in tobacco cell cultures resulted in an increased rate of Pi uptake when cells were grown under Pi limited conditions [47]. Several attempts have been made to improve soil Pi solubilization and acquisition processes in food crops through engineering of genes with bacterial, fungal or plant origins. Increasing knowledge on regulatory and signaling mechanisms involved in Pi acquisition might identify new useful genes. It is assumed that a combined overexpression of Pi-

An overview of reported transgenic crop plants is given in Table 4. Generating plants with an enhanced metabolical Pi uptake capacity were attempted by engineered tobacco plants to produce more organic acids, specifically citrate [150]. The promising result was that these transgenic plants yielded more leaf and fruit biomass than controls when grown under Pilimited conditions [151]. Many studies reported transgenic plants with overexpression of phytase in various food crops. Phytases are exuded into the rhizosphere and are able to

transporter and dissolving encoding genes might result in synergistic effects [150].

target traits are needed to be used for selection in the breeding process.

**20.5. Genetic engineering for Pi acquisition and utilization** 

Several mutant genes have been successfully introduced into commercial crop varieties that significantly enhance the nutritional value of crops. Mutants of barley, wheat, rice and soybean with low phytic acid have been released and can reduce both Pi pollution and increase bioavailability of Pi and micronutrient minerals in cereals and legumes [164]. A major QTL *pup1,* on chromosome 12 effective on Pi uptake from Pi‐deficient soils was fine‐mapped in rice. Liu and colleagues [165] demonstrated that chromosome 1R and 7R of rye might carry genes responsible for tolerance to Pi starvation stress, while 5R carry unfavorable genes regarding Pi starvation tolerance.



Plant Phosphate Nutrition and Environmental Challenges 23

the microorganisms. Such an elegant smart mechanism leads to natural growth and

To this end, years of researches on chemical, organic and biological Pi fertilizers have shown that none can replace the other. Practically, achieved higher yield and quality with combinations of all three in certain proportions address a call for precise farming with

, Ali Samaeian, and Tahmineh Lohrasebi *Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology,* 

We would like to express our appreciation to those who contributed to data compilation and sharing through Phosphate Knowledge Center (http://www. GreenPi.info), FAO, IFDC, IMPHOS and Global Phosphate Research Initiatives (http://www.phosphorusfutures.net).

[1] Vance CP. Symbiotic Nitrogen Fixation and Phosphorus Acquisition. Plant Nutrition in a World of Declining Renewable Resources. Plant Physiol; 2001. 127(2): 390-7. [2] Bieleski RL. Phosphate Pools, Phosphate Transport and Phosphate Availability. Annual Review of Plant Physiology; 1973. 24: 225–252. doi: 10.1146/annurev.pp.

[3] Hinsinger P. Bioavailability of Soil Inorganic P in the Rhizosphere as Affected by Root-

[4] Conyers MK., Moody PW. A Conceptual Framework for Improving the P Efficiency of Organic Farming without Inputs of Soluble P Fertiliser. Crop & Pasture Science; 2009.

[5] Harrison AF. Soil Organic Phosphorus: A Review of World Literature. CAB

[6] Sanyal SK., De Datta SK. Chemistry of Phosphorus Transformations in Soil. Advances

[7] DalalR C. Soil Organic Phosphorus. Advances in Agronomy; 1977. 29: 85–117.

Induced Chemical Changes: A Review. Plant Soil; 2001. 237: 173–195.

integrative plant nutrition programs prescribed based on crop and soil requirements.

*Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares* 

development of plants.

**Author details** 

*Tehran, I.R. Iran* 

Mohammad Ali Malboobi\*

Mohammad Sadegh Sabet

*University, Tehran, I.R. Iran* 

**Acknowledgement** 

**22. References** 

24.060173.001301

60: 100–104.

Corresponding Author

 \*

International: Wallingford, UK; 1987.

in Soil Science; 1991. 16: 1–120.

**Table 4.** An overview of some reported genetic engineering in crop species

## **21. Conclusion**

Chemical fertilizers have played a significant role in the Green Revolution to have improved yield and to combat hunger to some extent [166]. However, poorly managed consumption of resources and even poor applications of Pi fertilizers and side products have endangered our environment. These factors have encouraged researchers to seek bio-friendly, economically feasible, and replaceable sources of Pi before the world consumption rate will rich to a no-return point. In order to secure agricultural sustainability through reduction in Pi fertilizer overuse, plant and soil scientists have tried to address solutions for retrieval of Pi from P-compounds in the rhizosphere, soils in the a close proximity of root. Two major approaches in the current research and technology world are:


the microorganisms. Such an elegant smart mechanism leads to natural growth and development of plants.

To this end, years of researches on chemical, organic and biological Pi fertilizers have shown that none can replace the other. Practically, achieved higher yield and quality with combinations of all three in certain proportions address a call for precise farming with integrative plant nutrition programs prescribed based on crop and soil requirements.

## **Author details**

22 Plant Science

**Malate** 

**(MDH)** 

**Type I H+**

**AVP1** 

**gene** 

**High-affinity Ptransporter** 

**High-affinity Ptransporter** 

**dehydrogenase** 

**overexpression**

**Phosphoenolpyruvat e carboxylase (PFPC) overexpression** 

**-pyrophosphatase** 

**21. Conclusion** 

**Aluminum resistance** 

**Gene Name Gene source Transformed** 

**Table 4.** An overview of some reported genetic engineering in crop species

approaches in the current research and technology world are:

**crop species** 

*A. thaliana* Rice and tomato More robust roots,

Chemical fertilizers have played a significant role in the Green Revolution to have improved yield and to combat hunger to some extent [166]. However, poorly managed consumption of resources and even poor applications of Pi fertilizers and side products have endangered our environment. These factors have encouraged researchers to seek bio-friendly, economically feasible, and replaceable sources of Pi before the world consumption rate will rich to a no-return point. In order to secure agricultural sustainability through reduction in Pi fertilizer overuse, plant and soil scientists have tried to address solutions for retrieval of Pi from P-compounds in the rhizosphere, soils in the a close proximity of root. Two major

1. To bioengineer Pi efficient transgenic crops expressing enzymes such as phosphatases. This solution has been highly approached by many scientists (for a review see [84]). However, transgenic plants were found to be affected by the non-desired traits or epitasis among numerous APase-encoding genes. Even if a transgenic plant acquire release permission, still we are limited to use a lines variety of the transgenic crop. 2. To bioengineer rhizosphere by the use of beneficial microorganisms as biofertilizers involved in solubilization of Pi in the soil, namely *"Pi biofertilizers*". This approach is advantageous as Pi biofertilizers could be applied to wide range of crops and their varieties as desired. Besides, signaling of sufficient availability of Pi in the rhizosphere would regulate the expression and secretion of organic acids and/or APase enzymes by

wheat Barley Increased P uptake and

soils)

**Main effect under P** 

**Reference** 

[161]

[161]

[162]

[163]

**deficiency** 

Alfalfa Increased organic acid exudation

Alfalfa No increase in organic acid exudation

> higher shoot mass, higher yields

grain production (acid

Barley Barley No effect [43]

tobacco Rice Increased P uptake [160]

Mohammad Ali Malboobi\* , Ali Samaeian, and Tahmineh Lohrasebi *Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran, I.R. Iran* 

Mohammad Sadegh Sabet *Department of Plant Breeding and Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, I.R. Iran* 

## **Acknowledgement**

We would like to express our appreciation to those who contributed to data compilation and sharing through Phosphate Knowledge Center (http://www. GreenPi.info), FAO, IFDC, IMPHOS and Global Phosphate Research Initiatives (http://www.phosphorusfutures.net).

## **22. References**


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**Chapter 2** 

© 2012 Kamiński et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Kamiński et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Enzymatic Antioxidant Responses of Plants** 

Piotr Kamiński, Beata Koim-Puchowska, Piotr Puchowski, Leszek Jerzak,

Plants under natural conditions are frequently exposed to combined stressors including drought stress and desiccation, salt stress, chilling, heat shock, heavy metals, ultraviolet, radiation, air pollutants such as ozone and SO2, mechanical stress, nutrient deprivation, pathogen attack and high light stress [1]. A common result of most abiotic and biotic stresses is an increased production of reactive oxygen species (ROS), which frequently result in oxidative stress [2], [3]. The production of ROS results from pathways such as photorespiration, from the photosynthetic apparatus and from mitochondrial respiration [1]. The reduction of molecular oxygen to H2O yields to intermediates reactive oxygen species (superoxide anion (O2•‾), hydroxyl radical (OH•), hydroperoxyl radical (HO2•), hydrogen peroxide (H2O2), singlet oxygen (1O2•‾), which are potentially toxic because they are relatively reactive compared with O2 [4]. Reactive oxygen species may lead to the unspecific oxidations of proteins, membrane lipids or DNA injury. Malonodialdehyd (MDA) content, as a product of lipid peroxidation, has been considered as an indicator of oxidative damage in the cell membrane, resulting in disruption of metabolic function and loss of cellular integrity [5], [6]. Many reports confirm lipid peroxidation has been associated with damages provoked by a variety of environmental stresses [7], i.e. with salt stress [8], [9], [10] and heavy metals [11], [12].

However, there is evidence that ROS also plays important roles in the plant's defence system against pathogens and any pathogenic factors, mark certain development stages such as tracheary element formations, lignification and other cross linking process in the cell wall and act as intermediate signalling molecules to regulate the expression of genes [4]. Thus it is very important for cells to control of level of reactive oxygen species but not eliminate them completely. Defence mechanisms against free radical-induced oxidative stress involve: (1) preventive mechanisms, (2) repair mechanisms, (3) physical defences, and

**in Saline Anthropogenic Environments** 

Monika Wieloch and Karolina Bombolewska

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51149

**1. Introduction** 


**Chapter 2** 

## **Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments**

Piotr Kamiński, Beata Koim-Puchowska, Piotr Puchowski, Leszek Jerzak, Monika Wieloch and Karolina Bombolewska

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51149

## **1. Introduction**

34 Plant Science

[164] Mohan J., Suprasanna P. Induced Mutations for Enhancing Nutrition and Food

[165] Liu J., Li Y., Tong Y., Gao J., Li B., Li J., Li Z. Chromosomal Location of Genes Conferring the Tolerance to Pi Starvation Stress and Acid Phosphatase (APase)

[166] Ahmed S. Agriculture-Fertilizer Interface in Asia-Issues of Growth and Sustainability.

Secretion in the Genome of Rye (Secale L.) Plant and Soil; 2001. 237: 267–274.

Production. Geneconserve; 2011. 40: 201–215.

New Delhi. Oxford and IBH Publ. Co.; 1995

Plants under natural conditions are frequently exposed to combined stressors including drought stress and desiccation, salt stress, chilling, heat shock, heavy metals, ultraviolet, radiation, air pollutants such as ozone and SO2, mechanical stress, nutrient deprivation, pathogen attack and high light stress [1]. A common result of most abiotic and biotic stresses is an increased production of reactive oxygen species (ROS), which frequently result in oxidative stress [2], [3]. The production of ROS results from pathways such as photorespiration, from the photosynthetic apparatus and from mitochondrial respiration [1].

The reduction of molecular oxygen to H2O yields to intermediates reactive oxygen species (superoxide anion (O2•‾), hydroxyl radical (OH•), hydroperoxyl radical (HO2•), hydrogen peroxide (H2O2), singlet oxygen (1O2•‾), which are potentially toxic because they are relatively reactive compared with O2 [4]. Reactive oxygen species may lead to the unspecific oxidations of proteins, membrane lipids or DNA injury. Malonodialdehyd (MDA) content, as a product of lipid peroxidation, has been considered as an indicator of oxidative damage in the cell membrane, resulting in disruption of metabolic function and loss of cellular integrity [5], [6]. Many reports confirm lipid peroxidation has been associated with damages provoked by a variety of environmental stresses [7], i.e. with salt stress [8], [9], [10] and heavy metals [11], [12].

However, there is evidence that ROS also plays important roles in the plant's defence system against pathogens and any pathogenic factors, mark certain development stages such as tracheary element formations, lignification and other cross linking process in the cell wall and act as intermediate signalling molecules to regulate the expression of genes [4]. Thus it is very important for cells to control of level of reactive oxygen species but not eliminate them completely. Defence mechanisms against free radical-induced oxidative stress involve: (1) preventive mechanisms, (2) repair mechanisms, (3) physical defences, and

© 2012 Kamiński et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Kamiński et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(4) antioxidant defences [13]. The plants defend against these ROS by the induction of activities of certain antioxidative enzymes such as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and superoxide dismutase (SOD), which scavenge reactive oxygen species [14], [15].

Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 37

binding proteins such as calmodulins and calmodulin-binding proteins [2]. Reports showed close interaction between intracellular H2O2 and cytosolic calcium in response to biotic and abiotic stresses -increase in cytosolic calcium boosts the generation of H2O2. The protein calmodulin binds to activates plant catalases in the present of calcium. It indicated a dual

Metals are involved in the direct or indirect generation of free radicals (FR) and reactive oxygen species in the following ways: 1. direct transfer of electron in the single electron reduction; 2. disturbance of metabolic pathways resulting in an increase in the rate of FR and ROS formation; 3. inactivation and down regulation of the enzymes of the antioxidative defence system, and 4. depletion of low molecular weight of antioxidants [33]. Microelements such as Fe, Zn, Cu and Mn fulfil various roles in the metabolism of plant organism and are necessary for the regularity of physiological processes, however the excess and deficiency of these elements leads also to disturbance of ionic homeostasis [34]. Fe, Mn, Cu, and Zn as transition metals have frequently unpaired electrons and they are, therefore, very good catalysts of oxygen reduction. In aqueous solutions at neutral pH, O2•‾ can generate H2O2, which can subsequently decompose to produce •OH by the Haber-Weiss reaction in which Cu and Fe being involved: O2• + H2O2 → O2 + OH– + •OH. When iron is the

transition metal in the Haber-Weiss reaction, it is called the Fenton reaction [35], [33].

Toxic heavy metals Cd an Pb are considered as not essential metals for plant metabolism and stimulate formation of free radicals and reactive oxygen species [36], [37], [38]. The ROS formation by toxic metals is indirectly rather than directly [39]. The toxicity of Cd may result from its binding to sulfhydryl groups (S-H) of proteins leading to inhibition of activity and disruption of structure, due to perturbations in the nutrient balance and disturbance of cellular redox control 40]. Due to a Cd inhibitory effect on the Calvin cycle, there is a decrease in NADPH utilization, resulting in the one-electron reduction of a large number of oxygen molecules on the reducing side of PSI [39]. The reports of [41], [42], [43], [44], [45], [46], [47], and [48] have concluded that an oxidative stress could be involved in Cd toxicity, by either inducing oxygen free radical production, or by decreasing enzymatic and non-

The subject of numerous studies for at least the past four decades has been physiological responses of plants for salinity and heavy metals in the controlled laboratory conditions. However, many of these determinations are concentrated to one type of pollutant, whilst plants in the natural conditions are subjected to many stressful differentiated ecophysiological sources and factors. Therefore there still remains a need for research on the interdependencies of plants with multiple biotic and abiotic factors in their natural habitats, their adaptation mechanisms and responses. The aim of this paper was thus to investigate the enzymatic antioxidant mechanisms and responses in plants subjected to destabilization of chemical elements management in the natural conditions. We thus studied antioxidant enzymes SOD, CAT, and APOX, and the content of malondialdehyde (MDA) variations in different ecological groups of glycophytes Creeping thistle *Cirisium arvense*, Common nettle *Urtica dioica*, Yarrow *Achillea millefolium*, and Burdock *Arctium lappa* in various types of environments: salted and alkaline anthropogenic environments, agricultural environments

function of Ca in regulating H2O2 homeostasis [30].

enzymatic antioxidants.

Superoxide dismutase catalyses the reaction of dismutation of superoxide radicals: 2O2•‾ + 2H+ →H2O2 + O2. SOD is a metaloprotein with metals Cu, Zn, Mn, and Fe as co-factor [16], Stroiński 1999). Cu-Zn-SOD (homodimer; 31-33 kD) is present in cytosol and chloroplasts. Mn-SOD (tetramer 90kD) was found in the matrix mitochondrium [17], [18], whilst Fe-SOD, commonly occurring in *Procaryota*, was detected only in some plants [19], [18]. On the other hand catalase catalyses the reaction: 2 H2O2 →2H2O + O2. This enzyme contains three isoenzymatic forms (CAT-l, CAT-2, and CAT-3). CAT-1 and CAT-2 occur mainly in glyoxisomes, peroxysomes, and cytosol, while CAT-3 in mitochondria and cytosol [20], [18].

Another important factor decomposing H2O2 is peroxidase of ascorbic acid (APOX) (m.w. 28-34 kD) which contains protoheme as a prosthetic group and 4 cysteines responsible for sensitivity of the enzyme to compounds blocking a thiol group [21], [18]. It occurs in chloroplasts and cytosol [22], [18], vacuole [23], [18], and in apoplasts [24], [18]. APOX, in particular the chloroplast isoenzyme, becomes labile if the concentration of ascorbic acid drops significantly and it decomposes under the influence of its own radical product. Under such circumstances both monodehydroascorbate (MDHAA) and dehydroascorbate (DHAA) are easily reduced along the Halliwell-Asada path [18], [25]. The cell defensive mechanism also depends on small-cellular antioxidants such as glutathion (GSH), proline (Pro) or ascorbic acid (AA) which also react with radicals generated in the oxidative-stress [18].

The origin of oxidative stress under salinity conditions is well documented in leaves, where the inhibition of Calvin cycle results in over-reduction of oxygen and formation of superoxides [26]. Salt stress limits also gas exchange and thereby CO2 supply to the leaf. One consequence is the over-reduction of photosynthetic-chain electron transport. This induces the generation of ROS [15]. Little is known about the origin of oxidative stress in roots, although salt stress-related impairment of mitochondrial function is likely to be involved [27]. Recent studies have revealed that salt-induced oxidative stress occurs in root mitochondria in a wild salt-tolerant tomato species as indicated by increased concentrations of H2O2 and MDA [28], [27]. It is now widely accepted that superoxide radicals produced during respiration, in response to salt stress, are the main precursors of mitochondrial H2O2 and make an important contribution to the oxidative load experienced by the cell [26]. Root mitochondrial ROS production is increased as a result of high salinity as constraints are imposed on electron transport through mitochondrial complexes I and II [27].

Calcium as macroelement and important physiological chemical element reacts with heavy metals and is required for various roles in plant cell [29], [30]. It is implicated in the movement of cellular organelles such as the spindle apparatus and secretory vesicles, and may play a key role in integrating plant cell metabolism [31], [32]. However, the homeostasis of Ca is maintained principally by the action of extrusion proteins. The cytosol is strongly buffered against high concentrations of calcium by numerous ranges of calciumbinding proteins such as calmodulins and calmodulin-binding proteins [2]. Reports showed close interaction between intracellular H2O2 and cytosolic calcium in response to biotic and abiotic stresses -increase in cytosolic calcium boosts the generation of H2O2. The protein calmodulin binds to activates plant catalases in the present of calcium. It indicated a dual function of Ca in regulating H2O2 homeostasis [30].

36 Plant Science

reactive oxygen species [14], [15].

(4) antioxidant defences [13]. The plants defend against these ROS by the induction of activities of certain antioxidative enzymes such as catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and superoxide dismutase (SOD), which scavenge

Superoxide dismutase catalyses the reaction of dismutation of superoxide radicals: 2O2•‾ + 2H+ →H2O2 + O2. SOD is a metaloprotein with metals Cu, Zn, Mn, and Fe as co-factor [16], Stroiński 1999). Cu-Zn-SOD (homodimer; 31-33 kD) is present in cytosol and chloroplasts. Mn-SOD (tetramer 90kD) was found in the matrix mitochondrium [17], [18], whilst Fe-SOD, commonly occurring in *Procaryota*, was detected only in some plants [19], [18]. On the other hand catalase catalyses the reaction: 2 H2O2 →2H2O + O2. This enzyme contains three isoenzymatic forms (CAT-l, CAT-2, and CAT-3). CAT-1 and CAT-2 occur mainly in glyoxisomes, peroxysomes, and cytosol, while CAT-3 in mitochondria and cytosol [20], [18]. Another important factor decomposing H2O2 is peroxidase of ascorbic acid (APOX) (m.w. 28-34 kD) which contains protoheme as a prosthetic group and 4 cysteines responsible for sensitivity of the enzyme to compounds blocking a thiol group [21], [18]. It occurs in chloroplasts and cytosol [22], [18], vacuole [23], [18], and in apoplasts [24], [18]. APOX, in particular the chloroplast isoenzyme, becomes labile if the concentration of ascorbic acid drops significantly and it decomposes under the influence of its own radical product. Under such circumstances both monodehydroascorbate (MDHAA) and dehydroascorbate (DHAA) are easily reduced along the Halliwell-Asada path [18], [25]. The cell defensive mechanism also depends on small-cellular antioxidants such as glutathion (GSH), proline (Pro) or ascorbic acid (AA) which also react with radicals generated in the oxidative-stress [18].

The origin of oxidative stress under salinity conditions is well documented in leaves, where the inhibition of Calvin cycle results in over-reduction of oxygen and formation of superoxides [26]. Salt stress limits also gas exchange and thereby CO2 supply to the leaf. One consequence is the over-reduction of photosynthetic-chain electron transport. This induces the generation of ROS [15]. Little is known about the origin of oxidative stress in roots, although salt stress-related impairment of mitochondrial function is likely to be involved [27]. Recent studies have revealed that salt-induced oxidative stress occurs in root mitochondria in a wild salt-tolerant tomato species as indicated by increased concentrations of H2O2 and MDA [28], [27]. It is now widely accepted that superoxide radicals produced during respiration, in response to salt stress, are the main precursors of mitochondrial H2O2 and make an important contribution to the oxidative load experienced by the cell [26]. Root mitochondrial ROS production is increased as a result of high salinity as constraints are

Calcium as macroelement and important physiological chemical element reacts with heavy metals and is required for various roles in plant cell [29], [30]. It is implicated in the movement of cellular organelles such as the spindle apparatus and secretory vesicles, and may play a key role in integrating plant cell metabolism [31], [32]. However, the homeostasis of Ca is maintained principally by the action of extrusion proteins. The cytosol is strongly buffered against high concentrations of calcium by numerous ranges of calcium-

imposed on electron transport through mitochondrial complexes I and II [27].

Metals are involved in the direct or indirect generation of free radicals (FR) and reactive oxygen species in the following ways: 1. direct transfer of electron in the single electron reduction; 2. disturbance of metabolic pathways resulting in an increase in the rate of FR and ROS formation; 3. inactivation and down regulation of the enzymes of the antioxidative defence system, and 4. depletion of low molecular weight of antioxidants [33]. Microelements such as Fe, Zn, Cu and Mn fulfil various roles in the metabolism of plant organism and are necessary for the regularity of physiological processes, however the excess and deficiency of these elements leads also to disturbance of ionic homeostasis [34]. Fe, Mn, Cu, and Zn as transition metals have frequently unpaired electrons and they are, therefore, very good catalysts of oxygen reduction. In aqueous solutions at neutral pH, O2•‾ can generate H2O2, which can subsequently decompose to produce •OH by the Haber-Weiss reaction in which Cu and Fe being involved: O2• + H2O2 → O2 + OH– + •OH. When iron is the transition metal in the Haber-Weiss reaction, it is called the Fenton reaction [35], [33].

Toxic heavy metals Cd an Pb are considered as not essential metals for plant metabolism and stimulate formation of free radicals and reactive oxygen species [36], [37], [38]. The ROS formation by toxic metals is indirectly rather than directly [39]. The toxicity of Cd may result from its binding to sulfhydryl groups (S-H) of proteins leading to inhibition of activity and disruption of structure, due to perturbations in the nutrient balance and disturbance of cellular redox control 40]. Due to a Cd inhibitory effect on the Calvin cycle, there is a decrease in NADPH utilization, resulting in the one-electron reduction of a large number of oxygen molecules on the reducing side of PSI [39]. The reports of [41], [42], [43], [44], [45], [46], [47], and [48] have concluded that an oxidative stress could be involved in Cd toxicity, by either inducing oxygen free radical production, or by decreasing enzymatic and nonenzymatic antioxidants.

The subject of numerous studies for at least the past four decades has been physiological responses of plants for salinity and heavy metals in the controlled laboratory conditions. However, many of these determinations are concentrated to one type of pollutant, whilst plants in the natural conditions are subjected to many stressful differentiated ecophysiological sources and factors. Therefore there still remains a need for research on the interdependencies of plants with multiple biotic and abiotic factors in their natural habitats, their adaptation mechanisms and responses. The aim of this paper was thus to investigate the enzymatic antioxidant mechanisms and responses in plants subjected to destabilization of chemical elements management in the natural conditions. We thus studied antioxidant enzymes SOD, CAT, and APOX, and the content of malondialdehyde (MDA) variations in different ecological groups of glycophytes Creeping thistle *Cirisium arvense*, Common nettle *Urtica dioica*, Yarrow *Achillea millefolium*, and Burdock *Arctium lappa* in various types of environments: salted and alkaline anthropogenic environments, agricultural environments and also pollution free environments of the Pomeranian region of Poland. We also investigated the halophyte Common glasswort *Salicornia europaea* from only salted environments. Simultaneously, we examined the levels of chemical elements Na, Ca, Fe, Zn, Cu, Mn, Cd, and Pb in roots and green parts of plants as probably the factor generated reactive oxidant species and thus activated enzymatic antioxidant mechanisms. We compare environmental and ecophysiological determinations of plant groups under salinity and acidity and their adaptation strategies. These studies were planned in order to understand whether the consideration of particular relationships between destabilization of free radicals homeostasis are connected with the excess of Na and Ca and pro-antioxidant balance.

Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 39

**Figure 1.** Study area. Environments: C – control, AGRE – agricultural, WE – wetlands, AE – anthropogenic, SM – sodium manufactures (http://www.maps.google.pl/kujawsko-pomorskie,

The mainly human management caused secondary salinity and consequently alkalization of Kujawy areas as a result of inappropriate tightening of sediment traps causing wastes to infiltrate into the soil and grassland irrigation. Our research was done in selected habitats with a variety of anthropogenic disturb: 1) "Sodium manufactures" (SM) - Inowrocław and Janikowo district, closely by sodium factories; 2) "Anthropogenic" (AE) waste dumping sites near Giebnia and neighboured polluted areas; 3) "Wetlands" (WE) floodplains near Noteć Channel and Pakoskie Lakeland; 4) "Agriculture" (AGRE) – agriculture areas near Borkowo and 5) "Control" – in Tuchola Forestry named "Bory Tucholskie" (see Fig. 1).

Field studies were carried out in 2008-2010 during summer (May-June-July). Plant species were estimated on the basis of the work by [49] and were divided onto glycophytes - Creeping thistle *Cirisium arvense*, Common nettle *Urtica dioica*, Yarrow *Achillea millefolium*, Burdock *Arctium lappa* and obligatory halophyte: Common glasswort *Salicornia europea*. Common glasswort appears only in salted habitats and is known as succulent [50]. It is one of the most saline tolerant plants in general and is capable to grow under highly saline conditions in the lowest part of salt marshes [51], [52]. Samples of roots and green parts of

modified).

**3. Material and methods** 

**Fig. 1.**

#### **2. Study area**

The study area is situated in the Pomeranian Kujawy region (52-53°N, 18-20°E, central Poland) connected with Permian rock-salt uplifted in the form of salted domes and associated salt springs and saline ground waters, which had influence on the development of a sodium industry in districts of Janikowo and Inowrocław and nonpolluted and industrialized area of the Tuchola Forestry complex (54-53°N and 17°30'- 18°30' E); Fig. 1.

**Figure 1.** Study area. Environments: C – control, AGRE – agricultural, WE – wetlands, AE – anthropogenic, SM – sodium manufactures (http://www.maps.google.pl/kujawsko-pomorskie, modified).

The mainly human management caused secondary salinity and consequently alkalization of Kujawy areas as a result of inappropriate tightening of sediment traps causing wastes to infiltrate into the soil and grassland irrigation. Our research was done in selected habitats with a variety of anthropogenic disturb: 1) "Sodium manufactures" (SM) - Inowrocław and Janikowo district, closely by sodium factories; 2) "Anthropogenic" (AE) waste dumping sites near Giebnia and neighboured polluted areas; 3) "Wetlands" (WE) floodplains near Noteć Channel and Pakoskie Lakeland; 4) "Agriculture" (AGRE) – agriculture areas near Borkowo and 5) "Control" – in Tuchola Forestry named "Bory Tucholskie" (see Fig. 1).

### **3. Material and methods**

38 Plant Science

**2. Study area** 

18°30' E); Fig. 1.

and also pollution free environments of the Pomeranian region of Poland. We also investigated the halophyte Common glasswort *Salicornia europaea* from only salted environments. Simultaneously, we examined the levels of chemical elements Na, Ca, Fe, Zn, Cu, Mn, Cd, and Pb in roots and green parts of plants as probably the factor generated reactive oxidant species and thus activated enzymatic antioxidant mechanisms. We compare environmental and ecophysiological determinations of plant groups under salinity and acidity and their adaptation strategies. These studies were planned in order to understand whether the consideration of particular relationships between destabilization of free radicals homeostasis are connected with the excess of Na and Ca and pro-antioxidant balance.

The study area is situated in the Pomeranian Kujawy region (52-53°N, 18-20°E, central Poland) connected with Permian rock-salt uplifted in the form of salted domes and associated salt springs and saline ground waters, which had influence on the development of a sodium industry in districts of Janikowo and Inowrocław and nonpolluted and industrialized area of the Tuchola Forestry complex (54-53°N and 17°30'-

**SODIUM SODIUM**

1:220000

**CONTROL**

**CONTROL**

**ANTHRO POGENIC**

**ANTHRO POGENIC**

> Field studies were carried out in 2008-2010 during summer (May-June-July). Plant species were estimated on the basis of the work by [49] and were divided onto glycophytes - Creeping thistle *Cirisium arvense*, Common nettle *Urtica dioica*, Yarrow *Achillea millefolium*, Burdock *Arctium lappa* and obligatory halophyte: Common glasswort *Salicornia europea*. Common glasswort appears only in salted habitats and is known as succulent [50]. It is one of the most saline tolerant plants in general and is capable to grow under highly saline conditions in the lowest part of salt marshes [51], [52]. Samples of roots and green parts of

plants: leaves from glycophytes (No of samples N=220), and shoots of Common glasswort *Salicornia europea* (No of samples N=150), and samples of soils (No of samples N=300) from rhizosphera were collected from all studied environments (glycophytes) and only from anthropogenic salted areas (halophytes), because of their natural distribution.

Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 41

The activity of CAT was assayed according [57], [58] in reaction solution (3 ml) composed of 0.1 M phosphate buffer (pH 7.0) to which 30% (w/v) H2O2 was added until reaching an absorbance at 240 nm between 0.520 and 0.550. The reaction was initiated by the addition of 10 μl of plant extract and the activity of CAT was monitored by the decreasing absorbance at 240 nm at for 2 min. One unit of enzyme was the amount necessary to decompose 1 μmol of

The activity of APOX was measured according method of [59]. The soluble protein extract (25 μl) was added to 1.975 ml reaction mixture of 0.05 M phosphate buffer (pH 7.0), 0.5 mM sodium ascorbate, 0.1 mM H2O2 and the decrease absorbance of ascorbate at 290 nm for 2 min was used to calculate APOX activity. Enzyme activity was expressed in enzyme unit of mg-1 protein. One unit of enzyme was the amount necessary to decompose 1 μmol of

The intensity of lipid peroxidation was estimated following the method by [60]. Approximately 0.5 g of frozen plant tissue samples was cut into small pieces, homogenized with 2.5 ml of 5% trichloroacetic acid, and then centrifuged at 10000 g for 15 min. at room temperature. The equal volumes of supernatant and 0,5% thiobarbituric acid in 20% trichloroacetic acid were added in a new tube and incubated in 96°C for 25 minutes and then quickly cooled in an ice bath. After centrifugation at 8 000 g for 5 min, the absorbance of supernatant was recorded at 532 and 600 nm. The value for non-specific absorption at 600 nm was subtracted. The concentration of MDA was calculated using coefficient of

Arithmetic means and descriptive statistics of activation of antioxidant enzymes: SOD, CAT, APOX and concentrations of Na, Ca, Fe, Zn, Cu, Mn, Pb, and Cd in roots and green parts of plants were calculated. We also calculated arithmetic mean of concentrations of the same elements in soils from root zone of plants. The data did not show normal distribution, so non parametric tests were used. We used ANOVA Kruskal-Wallis test, followed by multiple Kruskal-Wallis comparison and U-Mann-Whitney test to estimate the significance of differences in the activation of antioxidant enzymes and concentrations of elements in roots and green parts from different environments, between organs and also groups of plants from the same environments (significance level at p<0.05), and also the differences between concentrations of metals in soil in particular environments. The dependence of activity of antioxidant enzymes and concentrations of elements in the roots and green parts of plants from different environments were calculated by correlation coefficient (r) according to the ranks of

This work required permits from General and Regional Nature Conservation Dpts. These were obtained and had the following respective numbers: DOPozgiz-4211/I-14/995/09/ep,

H2O2 per min at 25ºC (extinction coefficient 43.6 M-1\*cm-1).

substrate per min at 25ºC (extinction coefficient 2.8 mM-1\*cm-1).

absorbance 155 mM-1 cm-1 and was expressed as μmol g-1 FW.

Spearman test (significance level at p<0.05) [61].

RDOS.PN.6631/2/08/KLD, and RDOS.04.PN.6631/37/09/KLD.

**3.1. Statistical analysis** 

Samples of soil were air-dried to a constant mass up to 650C, homogenized and sieved through 1 mm mesh. The electrolythical conductivity (expressed as mS or μS) of soil (Ec) was measured by conductivity meter (Elmetron CC-401). Instead the soil acidity (pH) was determined in bidistilled water at soil solution ratio of 1:2.5 with a potentiometric glass electrode by used pH-meter (Elmetron C-501).

Samples were collected from randomly selected plants at midday, and were kept cool in freezer bags for transfer to the laboratory. Green parts samples were then washed three times with deionized water; roots were gently separated from soils, then abundantly washed with tap water to eliminate soil particles, and also rinsed three times with deionized water. Root and washed shoot samples were divided into two parts. One was immediately stocked at −80°C until further biochemical analysis, while a second was cut into small pieces and dried in oven for 24 h [53], [54]. Mineralized samples of soil and plants were analyzed for Na, Ca, Fe, Zn, Cu, Mn, Cr, Ni, Cd, and Pb using inductively coupled plasma mass spectrometry ICP-MS (AGILENT 7500 CE; plasma ICP-MS spectrophotometer from Agilent Technologies Inc. (Palo Alto, CA, USA). The results were interpreted comparatively with standard well-known concentration, i.e. in relation to the analysis of reference materials. Parallel measurements were taken in the blind trials. The results were given in mg\*kg-1 of dry weight.

To determine the levels of superoxide dismutase (SOD) and catalase (CAT) roots and green parts of plants (1g) were homogenized in a 0.05 M phosphate buffer (pH 7.0) containing 1mM EDTA (3 ml). During homogenization polyvinyl-pyrrolidone (PVP) (0.25 g) was added (modified by [40]). The homogenate was centrifugated twice for 10 min first at 10 000 x g and second at 20 000x g. For ascorbate peroxidase (APOX) assay the roots and green parts (1g) were homogenized according to [54], i.e. by adding 0.05 M phosphate buffer (pH 7.5) containing 1 mM EDTA, 1 mM sodium ascorbate, 1 mM DTT and 4.0% (w/v) polyvinyl-pyrrolidone (PVP), 1 mM EDTA (5 ml). The homogenate was centrifugated at 15 000x g for 20 min. Sodium ascorbate was added only in case of green parts. All assays were conducted at 4º C. The protein content in the supernatant was measured according to [55].The activity of all enzymes was expressed in units of mg-1 of protein.

The activity of SOD was assayed by measuring its ability to inhibit the photochemical reduction of NBT, adopting the method of [56]. The reaction mixture consist of 50 mM phosphate buffer (pH 7.8) containing 0.1 mM EDTA, 120 μM riboflavine, 97.5 mM methionine, 2.25 mM NBT. After the addition of plant homogenate switching on a UV lamp for one minute the start of reaction is begin. The absorbance at 560 nm of wave length was measured. Fifty percent reduction in color was considered as one unit of enzyme activity.

The activity of CAT was assayed according [57], [58] in reaction solution (3 ml) composed of 0.1 M phosphate buffer (pH 7.0) to which 30% (w/v) H2O2 was added until reaching an absorbance at 240 nm between 0.520 and 0.550. The reaction was initiated by the addition of 10 μl of plant extract and the activity of CAT was monitored by the decreasing absorbance at 240 nm at for 2 min. One unit of enzyme was the amount necessary to decompose 1 μmol of H2O2 per min at 25ºC (extinction coefficient 43.6 M-1\*cm-1).

The activity of APOX was measured according method of [59]. The soluble protein extract (25 μl) was added to 1.975 ml reaction mixture of 0.05 M phosphate buffer (pH 7.0), 0.5 mM sodium ascorbate, 0.1 mM H2O2 and the decrease absorbance of ascorbate at 290 nm for 2 min was used to calculate APOX activity. Enzyme activity was expressed in enzyme unit of mg-1 protein. One unit of enzyme was the amount necessary to decompose 1 μmol of substrate per min at 25ºC (extinction coefficient 2.8 mM-1\*cm-1).

The intensity of lipid peroxidation was estimated following the method by [60]. Approximately 0.5 g of frozen plant tissue samples was cut into small pieces, homogenized with 2.5 ml of 5% trichloroacetic acid, and then centrifuged at 10000 g for 15 min. at room temperature. The equal volumes of supernatant and 0,5% thiobarbituric acid in 20% trichloroacetic acid were added in a new tube and incubated in 96°C for 25 minutes and then quickly cooled in an ice bath. After centrifugation at 8 000 g for 5 min, the absorbance of supernatant was recorded at 532 and 600 nm. The value for non-specific absorption at 600 nm was subtracted. The concentration of MDA was calculated using coefficient of absorbance 155 mM-1 cm-1 and was expressed as μmol g-1 FW.

### **3.1. Statistical analysis**

40 Plant Science

dry weight.

protein.

activity.

plants: leaves from glycophytes (No of samples N=220), and shoots of Common glasswort *Salicornia europea* (No of samples N=150), and samples of soils (No of samples N=300) from rhizosphera were collected from all studied environments (glycophytes) and only from

Samples of soil were air-dried to a constant mass up to 650C, homogenized and sieved through 1 mm mesh. The electrolythical conductivity (expressed as mS or μS) of soil (Ec) was measured by conductivity meter (Elmetron CC-401). Instead the soil acidity (pH) was determined in bidistilled water at soil solution ratio of 1:2.5 with a potentiometric glass

Samples were collected from randomly selected plants at midday, and were kept cool in freezer bags for transfer to the laboratory. Green parts samples were then washed three times with deionized water; roots were gently separated from soils, then abundantly washed with tap water to eliminate soil particles, and also rinsed three times with deionized water. Root and washed shoot samples were divided into two parts. One was immediately stocked at −80°C until further biochemical analysis, while a second was cut into small pieces and dried in oven for 24 h [53], [54]. Mineralized samples of soil and plants were analyzed for Na, Ca, Fe, Zn, Cu, Mn, Cr, Ni, Cd, and Pb using inductively coupled plasma mass spectrometry ICP-MS (AGILENT 7500 CE; plasma ICP-MS spectrophotometer from Agilent Technologies Inc. (Palo Alto, CA, USA). The results were interpreted comparatively with standard well-known concentration, i.e. in relation to the analysis of reference materials. Parallel measurements were taken in the blind trials. The results were given in mg\*kg-1 of

To determine the levels of superoxide dismutase (SOD) and catalase (CAT) roots and green parts of plants (1g) were homogenized in a 0.05 M phosphate buffer (pH 7.0) containing 1mM EDTA (3 ml). During homogenization polyvinyl-pyrrolidone (PVP) (0.25 g) was added (modified by [40]). The homogenate was centrifugated twice for 10 min first at 10 000 x g and second at 20 000x g. For ascorbate peroxidase (APOX) assay the roots and green parts (1g) were homogenized according to [54], i.e. by adding 0.05 M phosphate buffer (pH 7.5) containing 1 mM EDTA, 1 mM sodium ascorbate, 1 mM DTT and 4.0% (w/v) polyvinyl-pyrrolidone (PVP), 1 mM EDTA (5 ml). The homogenate was centrifugated at 15 000x g for 20 min. Sodium ascorbate was added only in case of green

measured according to [55].The activity of all enzymes was expressed in units of mg-1 of

The activity of SOD was assayed by measuring its ability to inhibit the photochemical reduction of NBT, adopting the method of [56]. The reaction mixture consist of 50 mM phosphate buffer (pH 7.8) containing 0.1 mM EDTA, 120 μM riboflavine, 97.5 mM methionine, 2.25 mM NBT. After the addition of plant homogenate switching on a UV lamp for one minute the start of reaction is begin. The absorbance at 560 nm of wave length was measured. Fifty percent reduction in color was considered as one unit of enzyme

C. The protein content in the supernatant was

anthropogenic salted areas (halophytes), because of their natural distribution.

electrode by used pH-meter (Elmetron C-501).

parts. All assays were conducted at 4º

Arithmetic means and descriptive statistics of activation of antioxidant enzymes: SOD, CAT, APOX and concentrations of Na, Ca, Fe, Zn, Cu, Mn, Pb, and Cd in roots and green parts of plants were calculated. We also calculated arithmetic mean of concentrations of the same elements in soils from root zone of plants. The data did not show normal distribution, so non parametric tests were used. We used ANOVA Kruskal-Wallis test, followed by multiple Kruskal-Wallis comparison and U-Mann-Whitney test to estimate the significance of differences in the activation of antioxidant enzymes and concentrations of elements in roots and green parts from different environments, between organs and also groups of plants from the same environments (significance level at p<0.05), and also the differences between concentrations of metals in soil in particular environments. The dependence of activity of antioxidant enzymes and concentrations of elements in the roots and green parts of plants from different environments were calculated by correlation coefficient (r) according to the ranks of Spearman test (significance level at p<0.05) [61].

This work required permits from General and Regional Nature Conservation Dpts. These were obtained and had the following respective numbers: DOPozgiz-4211/I-14/995/09/ep, RDOS.PN.6631/2/08/KLD, and RDOS.04.PN.6631/37/09/KLD.

## **4. Results**

#### **4.1. Soil parameters**

As shown in Table 1, the soils at Kujawy region were in the various degree of alkalinity (sodium manufactures; strong alkaline), anthropogenic environments, wetlands, agriculture environment (moderate alkaline), whilst pH of soils from control was acid. The electrical conductivity of soils was higher in sodium manufactures and anthropogenic environments than in control (Table 1).

Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 43

**Table 1.** Soil characteristics of sodium manufacture (SM), anthropogenic environments (AE), wetlands (WE), agricultural environments (AGRE) and control (C); mean ± SD. Significant differences in the concentrations of Na, Ca, Fe, Zn, Cu, Mn, Pb, and Cd in soil among environments (p<0.05).

As soils collected in SM and AE environments mainly consisted of tailings from nearby sodium factory and AGRE environment near field used agriculturally, heavy metal contents in the sample of soils were high and varied greatly in comparison with control and wetlands. The concentration of Na, Ca, Fe, Zn, Cu, and Mn were higher in soils at disturbed than control environments but in case of the same elements also from wetlands. However, concentration of toxic metals Pb, and Cd were higher in Tuchola Forestry than in Wetlands and did not differ with concentrations in soils from remaining environments (Table 1).

## **4.2. Chemical elements in roots and green parts of glycophytes and Common glasswort** *Salicornia europaea*

#### *4.2.1. Glycophytes*

We found lower concentrations of Na, Ca, Fe, Cu, and Pb in roots in control in compare with environments in Kujawy region in opposite to level of Zn (higher in control than in agricultural and did not differ in the remaining environments). Similar to Zn concentration, Cd level was also higher in non-polluted environment (control) in comparison with factory disturbed sodium manufactures and anthropogenic (Table 2). Simultaneously, sodium level in glycophytes from anthropogenic environments was higher than in those from remaining areas.

Concentrations of Fe and Zn were lower in leaves from control but only in comparison with wetlands. Simultaneously, Pb level was lower in control areas than in wetlands and agriculture fields. It is interesting that the level of Cd, in opposite to Pb, was higher in the unpolluted environment (C) compared with all environments studied in Kujawy region, except anthropogenic areas. Similar concentration of Mn was also higher in the control (C) as compared with industrial disturbed SM and AE and also wetlands (Table 2). We did not find significant differences in the concentrations of Mn in roots and the level of Ca, and Cu in leaves among investigated environments (Table 2).

#### *4.2.2. Common glasswort Salicornia europaea*

We found only higher concentrations of Na in roots and Mn in leaves from anhropogenic than from sodium manufactures. Simultaneously, the roots and green parts of plants had higher concentrations of Pb in sodium manufactures than in the anthropogenic. Concentrations of Ca, Fe, Zn, Cu and Cd did not differ between both organs and amongst environments studied (Table 3).


**4. Results** 

**4.1. Soil parameters** 

than in control (Table 1).

**glasswort** *Salicornia europaea*

*4.2.1. Glycophytes* 

As shown in Table 1, the soils at Kujawy region were in the various degree of alkalinity (sodium manufactures; strong alkaline), anthropogenic environments, wetlands, agriculture environment (moderate alkaline), whilst pH of soils from control was acid. The electrical conductivity of soils was higher in sodium manufactures and anthropogenic environments

As soils collected in SM and AE environments mainly consisted of tailings from nearby sodium factory and AGRE environment near field used agriculturally, heavy metal contents in the sample of soils were high and varied greatly in comparison with control and wetlands. The concentration of Na, Ca, Fe, Zn, Cu, and Mn were higher in soils at disturbed than control environments but in case of the same elements also from wetlands. However, concentration of toxic metals Pb, and Cd were higher in Tuchola Forestry than in Wetlands and did not differ with concentrations in soils from remaining environments (Table 1).

**4.2. Chemical elements in roots and green parts of glycophytes and Common** 

We found lower concentrations of Na, Ca, Fe, Cu, and Pb in roots in control in compare with environments in Kujawy region in opposite to level of Zn (higher in control than in agricultural and did not differ in the remaining environments). Similar to Zn concentration, Cd level was also higher in non-polluted environment (control) in comparison with factory disturbed sodium manufactures and anthropogenic (Table 2). Simultaneously, sodium level in glycophytes from

Concentrations of Fe and Zn were lower in leaves from control but only in comparison with wetlands. Simultaneously, Pb level was lower in control areas than in wetlands and agriculture fields. It is interesting that the level of Cd, in opposite to Pb, was higher in the unpolluted environment (C) compared with all environments studied in Kujawy region, except anthropogenic areas. Similar concentration of Mn was also higher in the control (C) as compared with industrial disturbed SM and AE and also wetlands (Table 2). We did not find significant differences in the concentrations of Mn in roots and the level of Ca, and Cu

We found only higher concentrations of Na in roots and Mn in leaves from anhropogenic than from sodium manufactures. Simultaneously, the roots and green parts of plants had higher concentrations of Pb in sodium manufactures than in the anthropogenic. Concentrations of Ca, Fe, Zn, Cu and Cd did not differ between both organs and amongst

anthropogenic environments was higher than in those from remaining areas.

in leaves among investigated environments (Table 2).

*4.2.2. Common glasswort Salicornia europaea* 

environments studied (Table 3).

**Table 1.** Soil characteristics of sodium manufacture (SM), anthropogenic environments (AE), wetlands (WE), agricultural environments (AGRE) and control (C); mean ± SD. Significant differences in the concentrations of Na, Ca, Fe, Zn, Cu, Mn, Pb, and Cd in soil among environments (p<0.05).


Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 45

 **SM (N=70) AE (N=80) Mean SD Mean SD Z p** 

SOD 152,34 207,76 127,13 104,47 0,00 1,000 CAT 0,25 0,30 0,17 0,14 0,03 0,975 APOX 81,59 42,20 98,08 77,09 -0,03 0,975 MDA 5,19 1,73 5,08 0,91 -0,10 0,922 Na 10266,67 6637,63 18772,32 7713,88 -2,14 0,032 Ca 6112,00 5671,56 3900,34 1462,37 -0,09 0,926 Fe 467,42 391,22 470,88 274,67 -0,59 0,555 Zn 19,49 13,36 17,39 7,22 0,28 0,780 Cu 5,80 5,09 6,33 3,01 0,28 0,780 Mn 30,46 19,87 24,72 7,81 0,96 0,335 Pb 2,64 1,71 1,01 0,29 3,13 0,002 Cd 0,16 0,24 0,14 0,09 -0,84 0,401

SOD 110,04 100,51 171,65 146,89 -1,93 0,053 CAT 0,39 0,32 0,15 0,14 1,88 0,061 APOX 34,44 34,77 21,68 17,38 0,55 0,583 MDA 2,48 0,69 4,04 0,98 -3,64 0,000 Na 44235,06 32261,31 49327,20 22546,83 -0,78 0,436 Ca 22766,03 20946,63 25326,90 12844,86 -0,49 0,624 Fe 378,85 482,21 239,69 159,86 -0,38 0,707 Zn 19,98 10,44 13,38 6,51 1,82 0,069 Cu 8,25 18,29 4,07 1,62 -0,89 0,371 Mn 36,92 36,70 127,42 84,06 -3,20 0,001 Pb 1,32 0,55 0,27 0,08 4,13 0,000 Cd 0,20 0,29 0,20 0,18 -0,49 0,624 **Table 3.** Elements concentrations, activity of antioxidant enzymes, and degree of lipoperoxidation in the organs of populations of Common glasswort *Salicornia europaea* in sodium manufacture (SM), and anthropogenic environments (AE); mean ± SD. Significant differences among environments at p<0.05.

Our results indicated significant differences only in the activity of SOD (glycophytes) in both organs. SOD activity was higher in roots from agricultural environments comparatively with sodium manufactures and wetlands. Simultaneously, SOD activity was lower in leaves from wetlands in comparison with remaining environments. We also found lower MDA content in roots from agricultural environments than from remaining examined areas. Instead the highest degree of lipid peroxidation was in the green parts of plants from anthropogenic environments (Table 2). We did not found differences in the biochemical parameters in organs of Common glasswort *Salicornia europaea* except of MDA content in the green parts of plants (higher level in the AE than in the SM) from different environments (Tables 4, 5).

**4.3. Antioxidant enzymes activity and lipoperoxidation** 

**roots** 

**green parts** 

**Table 2.** Elements concentrations, activity of antioxidant enzymes, and degree of lipoperoxidation in organs of populations of glycophytes in sodium manufacture (SM), anthropogenic environments (AE), wetlands (WE), agricultural environments (AGRE) and control (C); mean ± SD. Significant differences among environments (p<0.05).


**Table 3.** Elements concentrations, activity of antioxidant enzymes, and degree of lipoperoxidation in the organs of populations of Common glasswort *Salicornia europaea* in sodium manufacture (SM), and anthropogenic environments (AE); mean ± SD. Significant differences among environments at p<0.05.

#### **4.3. Antioxidant enzymes activity and lipoperoxidation**

44 Plant Science

**Table 2.** Elements concentrations, activity of antioxidant enzymes, and degree of lipoperoxidation in organs of populations of glycophytes in sodium manufacture (SM), anthropogenic environments (AE), wetlands (WE), agricultural environments (AGRE) and control (C); mean ± SD. Significant differences

among environments (p<0.05).

Our results indicated significant differences only in the activity of SOD (glycophytes) in both organs. SOD activity was higher in roots from agricultural environments comparatively with sodium manufactures and wetlands. Simultaneously, SOD activity was lower in leaves from wetlands in comparison with remaining environments. We also found lower MDA content in roots from agricultural environments than from remaining examined areas. Instead the highest degree of lipid peroxidation was in the green parts of plants from anthropogenic environments (Table 2). We did not found differences in the biochemical parameters in organs of Common glasswort *Salicornia europaea* except of MDA content in the green parts of plants (higher level in the AE than in the SM) from different environments (Tables 4, 5).

## **4.4. The impact of metals on the antioxidant enzymes activity in roots and leaves of plants**

Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 47

**SM (N=34) AE (N=49) WE (N=72) AGRE (N=34) C (N=31)** 

**roots** 

**green parts** 

**Roots** 

**green parts** 

 MDA & Zn -0,43 0,002 MDA & Cd -0,32 0,023 MDA & Na 0,49 0,000 MDA & Ca -0,41 0,003 APOX & MDA -0,51 0,000 CAT & MDA 0,55 0,000

**relations R p relations R p relations R p relations R p relations R p** 

SOD & Cu 0,43 0,011 SOD & Fe 0,46 0,001 SOD & Fe 0,29 0,015 APOX & Cu -0,37 0,031 SOD & Cu 0,364 0,044 SOD & Na 0,61 0,000 SOD & Cu 0,59 0,000 SOD & Cu 0,42 0,000 APOX & Cd -0,51 0,002 APOX & Cu -0,565 0,001 APX & Na 0,36 0,036 SOD & Pb 0,34 0,016 MDA & Cu 0,37 0,001 APOX & Na 0,61 0,000 APOX & Cd -0,360 0,040 MDA & Mn -0,35 0,040 APX & Mn 0,32 0,024 MDA & Cd 0,39 0,001 CAT & MDA 0,63 0,000 CAT & Mn 0,421 0,040 APX & Zn 0,32 0,025 MDA & Pb 0,33 0,005 CAT & Zn 0,528 0,040 CAT & Cu 0,43 0,002 MDA & Na 0,32 0,006 MDA & Cu 0,498 0,040 CAT & Na 0,34 0,018 MDA & Ca -0,27 0,021 MDA & Cd 0,720 0,040 MDA & Cu 0,35 0,014 MDA & Na -0,510 0,040

SOD & Cu 0,34 0,047 SOD & Na 0,35 0,013 SOD & Cd 0,42 0,000 SOD & Mn -0,41 0,017 SOD & Mn -0,521 0,003 SOD & Zn 0,38 0,025 APOX & Cd -0,45 0,001 APOX & Fe 0,34 0,003 SOD & Zn 0,60 0,000 SOD & Na 0,411 0,022 SOD & Cd 0,35 0,044 APOX & Ca 0,59 0,000 APOX & Cu 0,29 0,014 SOD & Cd 0,57 0,000 APOX & Zn -0,473 0,007 APOX & Cd -0,36 0,036 CAT & Cu -0,35 0,012 APOX & Ca 0,31 0,008 SOD & Pb -0,48 0,004 APOX & Cd -0,649 0,000 APOX & Ca 0,41 0,017 MDA & Zn -0,43 0,002 CAT & Fe -0,33 0,004 APOX & Fe 0,39 0,024 APOX & Na -0,367 0,043 CAT & Mn -0,50 0,002 MDA & Cd -0,43 0,002 CAT & Pb -0,31 0,007 APOX & Ca 0,38 0,029 APOX & Ca 0,545 0,002 CAT & Fe -0,40 0,020 MDA & Ca 0,50 0,000 CAT & Ca -0,23 0,049 cat & Fe -0,47 0,005 MDA & Cu -0,628 0,000 CAT & Cu 0,38 0,028 APOX & MDA 0,54 0,000 MDA & Cu 0,24 0,040 CAT & Zn 0,50 0,002 MDA & Zn -0,378 0,036

**Table 4.** Concentration of element–activity of enzyme, content of MDA interactions and content of MDA–activity of enzyme interaction related changes of elements level and antioxidant enzymes activity, the level of lipoperoxidation in roots and green parts of glycophytes in different environments.

**SM (N=70) AE (N=80) Relations R p relations R p** 

SOD & Zn 0,65 0,023 ns

SOD & Cd 0,82 0,001 APOX & Fe 0,59 0,044 APOX & Cu 0,65 0,023 APOX & Cd 0,79 0,002 APOX & Ca 0,60 0,041 CAT & Pb 0,60 0,039

SOD & Fe 0,66 0,036 CAT & Fe -0,77 0,002 SOD & Cu 0,64 0,046 CAT & Cu -0,76 0,003 SOD & Zn 0,74 0,015 CAT & Zn -0,62 0,023 APOX & Cd -0,82 0,004 CAT & Cd -0,67 0,012 APOX & MDA -0,64 0,048 CAT & Na -0,71 0,006

**Table 5.** Concentration of element–activity of enzyme, content of MDA interactions and content of MDA– activity of enzyme interactions related changes of elements level and antioxidant enzymes activity, level of lipoperoxidation in roots and green parts of Common glasswort *Salicornia europaea* in different environments.

MDA & Fe -0,46 0,006 MDA & Ca 0,40 0,000 MDA & Mn 0,34 0,048 MDA & Cu -0,60 0,000 APOX & MDA 0,42 0,000 MDA & Fe 0,59 0,000 MDA & Zn -0,52 0,002 MDA & Cd -0,61 0,000 MDA & Cd -0,61 0,000 SOD & MDA -0,49 0,003 APOX & MDA 0,51 0,002 CAT & MDA -0,63 0,000

Spearman coefficient analyses were performed with concentrations of each of the metals studied in roots and green parts of glycophytes and Common glasswort *Salicornia europaea* versus the activity of antioxidant enzymes and degree of lipoperoxidation. The activity of enzymes was also correlated with MDA content. However, these relationships were depending on the type of environment (Tables 4-6).

#### *4.4.1. Glycophytes*

We found more relationships between activity of antioxidant enzymes and metals in the green parts than in roots, depending on the type of environment. However, concentrations of Na (SM), Fe (AE, WE), Cu (SM, AE, WE, C) and Pb (AE) were positively correlated with SOD activity. Instead, CAT activity had also positive relationships with Na, Cu (AE) and Zn, Mn (C). Simultaneously, the activity of another enzyme, APOX, was positively related with level of Na (SM, AGRE), Zn, and Mn (AE) and negatively – with Cu (C), and Cd (AGRE, C).

Our results indicated positive correlations of SOD activity with concentrations of Zn (SM, AGRE), Cu (SM) and Cd (SM, WE, AGRE) but also negative with Mn (AGRE, C) and Pb (AGRE). Concentrations of Na were also related with SOD activity, but were positive (AE) or negative (C). We found negative relationships between CAT activity and concentrations of Ca (WE), Fe (SM, WE, AGRE), Mn (SM), and Pb (WE, AGRE) but also positive with Zn (AGRE). The level of Cu was positive related with activity of CAT in the leaves of plants from sodium manufactures but also negative in the leaves from anthropogenic areas. APOX activity was positively correlated with Ca concentrations in the leaves of plants from all environments and with Fe in the leaves collected at wetland and agriculture environments. However, the activity of this enzyme was negatively related with Na, Zn (C) and Cd (SM, AE, C). Concentrations of Cu had both positive (WE) and negative (AE) relationships with APOX in the leaves.

We found positive relations between concentrations of Na (AE), Cu, (AE, C), Pb (WE) and Cd (WE, C) and degree of lipoperoxidation processes in roots. However, we also found correlations between Na, Zn, Mn, and Cd but in the control environment (Na), AE, WE (Ca), AE (Zn, Cd), and SM (Mn). It should be emphasized that we did not state relation between concentrations of Na, and also Mn, and Pb and MDA content in the leaves. Concentrations of Ca were positively correlated with level of MDA (AE, WE) in the green parts in opposite to metals – Zn and Cd (AE, AGRE). Iron and copper stimulated lipoperoxidation (Fe in AGRE, Cu in WE) and was mutually negatively correlated (SM, SM, C) with content of MDA but it depend on the type of environment (Tables 4, 5, Figs. 2, 3). MDA content was positively correlated with CAT activity in roots (AE, AGRE) and APOX in leaves (AE, WE, AGRE). On the other hand, we found negative relations between MDA and the activity of APOX in roots (AE) and SOD, and CAT in leaves (AGRE); Table 4.

Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 47


46 Plant Science

**of plants** 

*4.4.1. Glycophytes* 

(AGRE, C).

APOX in the leaves.

**4.4. The impact of metals on the antioxidant enzymes activity in roots and leaves** 

Spearman coefficient analyses were performed with concentrations of each of the metals studied in roots and green parts of glycophytes and Common glasswort *Salicornia europaea* versus the activity of antioxidant enzymes and degree of lipoperoxidation. The activity of enzymes was also correlated with MDA content. However, these relationships were

We found more relationships between activity of antioxidant enzymes and metals in the green parts than in roots, depending on the type of environment. However, concentrations of Na (SM), Fe (AE, WE), Cu (SM, AE, WE, C) and Pb (AE) were positively correlated with SOD activity. Instead, CAT activity had also positive relationships with Na, Cu (AE) and Zn, Mn (C). Simultaneously, the activity of another enzyme, APOX, was positively related with level of Na (SM, AGRE), Zn, and Mn (AE) and negatively – with Cu (C), and Cd

Our results indicated positive correlations of SOD activity with concentrations of Zn (SM, AGRE), Cu (SM) and Cd (SM, WE, AGRE) but also negative with Mn (AGRE, C) and Pb (AGRE). Concentrations of Na were also related with SOD activity, but were positive (AE) or negative (C). We found negative relationships between CAT activity and concentrations of Ca (WE), Fe (SM, WE, AGRE), Mn (SM), and Pb (WE, AGRE) but also positive with Zn (AGRE). The level of Cu was positive related with activity of CAT in the leaves of plants from sodium manufactures but also negative in the leaves from anthropogenic areas. APOX activity was positively correlated with Ca concentrations in the leaves of plants from all environments and with Fe in the leaves collected at wetland and agriculture environments. However, the activity of this enzyme was negatively related with Na, Zn (C) and Cd (SM, AE, C). Concentrations of Cu had both positive (WE) and negative (AE) relationships with

We found positive relations between concentrations of Na (AE), Cu, (AE, C), Pb (WE) and Cd (WE, C) and degree of lipoperoxidation processes in roots. However, we also found correlations between Na, Zn, Mn, and Cd but in the control environment (Na), AE, WE (Ca), AE (Zn, Cd), and SM (Mn). It should be emphasized that we did not state relation between concentrations of Na, and also Mn, and Pb and MDA content in the leaves. Concentrations of Ca were positively correlated with level of MDA (AE, WE) in the green parts in opposite to metals – Zn and Cd (AE, AGRE). Iron and copper stimulated lipoperoxidation (Fe in AGRE, Cu in WE) and was mutually negatively correlated (SM, SM, C) with content of MDA but it depend on the type of environment (Tables 4, 5, Figs. 2, 3). MDA content was positively correlated with CAT activity in roots (AE, AGRE) and APOX in leaves (AE, WE, AGRE). On the other hand, we found negative relations between MDA and the activity of

APOX in roots (AE) and SOD, and CAT in leaves (AGRE); Table 4.

depending on the type of environment (Tables 4-6).

**Table 4.** Concentration of element–activity of enzyme, content of MDA interactions and content of MDA–activity of enzyme interaction related changes of elements level and antioxidant enzymes activity, the level of lipoperoxidation in roots and green parts of glycophytes in different environments.


**Table 5.** Concentration of element–activity of enzyme, content of MDA interactions and content of MDA– activity of enzyme interactions related changes of elements level and antioxidant enzymes activity, level of lipoperoxidation in roots and green parts of Common glasswort *Salicornia europaea* in different environments.

Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 49

Our results indicated relationships between activity of antioxidant enzymes and concentrations of metals in roots of plants in both environments and only in shoots from sodium manufactures. Concentrations of Fe, Cu, and Cd were positively related with SOD activity in the roots of plants from sodium manufactures in opposite to activity of CAT in roots of plants from anthropogenic areas. However, Na and Cd concentrations were also negatively correlated with CAT activity in shoots of plants from anthropogenic environments. We also found negative relations between concentrations of Cd and APOX activity in roots of plants from sodium manufactures. On the other hand, the activity of antioxidant enzymes was positively correlated with concentrations of Zn, Cd (SOD), Pb (CAT), Ca, Fe, Cu, and Cd (APOX) in shoots of plants. We did not find relations between MDA content and chemical elements concentration in both studied organs of Common glasswort *Salicornia europaea* (Tables 4, 5, Figs. 4, 5). However, we found negative relations between MDA content and APOX activity in the roots of plants from sodium

**Figure 4.** Interrelationships between CAT activity and the concentration of Cu, and Zn in roots (mg\*kg-1)

of Common glasswort *Salicornia europaea* from anthropogenic environment.

*4.4.2. Common glasswort Salicornia europaea* 

manufactures.

**Figure 2.** Interrelationships between SOD activity and the concentration of Cu, and Na (mg\*kg-1) in roots of glycophytes from sodium manufacture.

**Figure 3.** Interrelationships between APOX activity and the concentration of Ca, and Cd in (mg\*kg-1) the green parts of glycophytes from anthropogenic environment.

#### *4.4.2. Common glasswort Salicornia europaea*

48 Plant Science

**Figure 2.** Interrelationships between SOD activity and the concentration of Cu, and Na (mg\*kg-1) in

**Figure 3.** Interrelationships between APOX activity and the concentration of Ca, and Cd in (mg\*kg-1)

the green parts of glycophytes from anthropogenic environment.

roots of glycophytes from sodium manufacture.

Our results indicated relationships between activity of antioxidant enzymes and concentrations of metals in roots of plants in both environments and only in shoots from sodium manufactures. Concentrations of Fe, Cu, and Cd were positively related with SOD activity in the roots of plants from sodium manufactures in opposite to activity of CAT in roots of plants from anthropogenic areas. However, Na and Cd concentrations were also negatively correlated with CAT activity in shoots of plants from anthropogenic environments. We also found negative relations between concentrations of Cd and APOX activity in roots of plants from sodium manufactures. On the other hand, the activity of antioxidant enzymes was positively correlated with concentrations of Zn, Cd (SOD), Pb (CAT), Ca, Fe, Cu, and Cd (APOX) in shoots of plants. We did not find relations between MDA content and chemical elements concentration in both studied organs of Common glasswort *Salicornia europaea* (Tables 4, 5, Figs. 4, 5). However, we found negative relations between MDA content and APOX activity in the roots of plants from sodium manufactures.

**Figure 4.** Interrelationships between CAT activity and the concentration of Cu, and Zn in roots (mg\*kg-1) of Common glasswort *Salicornia europaea* from anthropogenic environment.

Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 51

**SM AE SM AE** 

SOD ns ns ns ns

Ca -2,53 \* -3,86 \*\*\* ns ns Fe ns ns ns ns

glasswort *Salicornia europaea* from different environments (p<0.005).

glasswort *Salicornia europea* from both environments (Table 7).

**of elements in plants** 

Z p Z p Z p Z p

CAT ns ns ns -2,02 \* APX 3,23 \*\* 3,54 \*\*\* -2,14 **\*** -2,06 \* MDA -3,25 \*\* -4,00 \*\*\* -5,20 \*\*\* -5,82 \*\*\* Na 4,19 \*\*\* 5,24 \*\*\* 5,09 \*\*\* 5,32 \*\*\*

Zn ns ns ns -3,66 \*\*\* Cu -2,53 \* ns -2,51 \* -3,53 \*\*\* Mn ns ns ns 3,86 \*\*\* Pb ns -3,10 \*\*\* 3,01 \*\* -5,00 \*\*\* Cd ns 0,14 ns 2,22 \*\*\* **Table 6.** Differences among biochemical parameters: activity of enzymes (SOD, CAT, APOX), content of MDA, concentrations of elements between root and green part of glycophytes and Common

**4.6. Differences between enzymes activity, lipoperoxidation and concentrations** 

We noted higher activity of SOD and APOX in the green parts of glycophytes from sodium manufactures. However MDA level was significantly higher only in green parts of glycophytes than in roots from AE and AGRE. On the other hand, APOX activity in shoots of Common glasswort *Salicornia europaea* was lower, similarly to MDA level of these plants from SM, but the intensity of lipoperoxidation was significantly higher in roots also at AE. Concentrations of Na were higher in roots of glycophytes in opposite to halophyte. Simultaneously, Ca concentrations were higher in leaves of both glycophytes and Common glasswort in opposite to level of Fe, which was rather cumulated in roots (not significant at SM in Common glasswort *Salicornia europea*). Simultaneously, Zn was found in higher concentrations only in glycophytes but in non salted environments (WE, AGRE, C). Cu and Mn were rather mobile metals but we found significant higher Cu concentrations in roots of glycophytes (SM) and – for both elements – in roots of Common glasswort (AE). Lead, as toxic metal was accumulated in roots of plants. Glycophytes also accumulated Cd in roots but it is interesting that we did not find the differences among organs in plants from control environments. Instead, concentrations of Cd were not differs amongst organs of Common

 **roots green parts** 

**Figure 5.** Interrelationships between Cd concentration (mg\*kg-1) and SOD, and APOX (U/mg) activity in the green parts of Common glasswort *Salicornia europaea* from sodium manufacture.

#### **4.5. Differences between glycophytes and Common glasswort** *Salicornia europaea* **in their natural environments**

We found significant higher activity of APOX in roots of glycophytes than in Common glasswort *Salicornia europaea* from both examined environments (SM, AE) in opposite to green parts. CAT activity was similarly higher in the green parts of glycophytes than in the halophyte (Common glasswort *Salicornia europaea*) but only in the anthropogenic environments. Instead, the level of MDA was higher in the glycophytes except green parts in the anthropogenic areas. Common glasswort *Salicornia europea* cumulated more sodium in both organs in all environments studied and also had higher level of Zn, Cu, Mn, and Cd in the green parts from AE than glycophytes. However, Cu and Pb concentrations were also higher in the green parts of this halophyte in sodium manufactures. On the other hand, glycophytes accumulated more calcium in roots from both environments, and more copper in roots from sodium manufactures and also Pb in both organs from anthropogenic environments than Common glasswort *Salicornia europaea*. The differences between Fe concentration and SOD activity in the organs of examined plants from two various environments did not differ (Table 6).


**Figure 5.** Interrelationships between Cd concentration (mg\*kg-1) and SOD, and APOX (U/mg) activity

We found significant higher activity of APOX in roots of glycophytes than in Common glasswort *Salicornia europaea* from both examined environments (SM, AE) in opposite to green parts. CAT activity was similarly higher in the green parts of glycophytes than in the halophyte (Common glasswort *Salicornia europaea*) but only in the anthropogenic environments. Instead, the level of MDA was higher in the glycophytes except green parts in the anthropogenic areas. Common glasswort *Salicornia europea* cumulated more sodium in both organs in all environments studied and also had higher level of Zn, Cu, Mn, and Cd in the green parts from AE than glycophytes. However, Cu and Pb concentrations were also higher in the green parts of this halophyte in sodium manufactures. On the other hand, glycophytes accumulated more calcium in roots from both environments, and more copper in roots from sodium manufactures and also Pb in both organs from anthropogenic environments than Common glasswort *Salicornia europaea*. The differences between Fe concentration and SOD activity in the organs of examined plants from two various

in the green parts of Common glasswort *Salicornia europaea* from sodium manufacture.

*europaea* **in their natural environments** 

environments did not differ (Table 6).

**4.5. Differences between glycophytes and Common glasswort** *Salicornia* 

**Table 6.** Differences among biochemical parameters: activity of enzymes (SOD, CAT, APOX), content of MDA, concentrations of elements between root and green part of glycophytes and Common glasswort *Salicornia europaea* from different environments (p<0.005).

#### **4.6. Differences between enzymes activity, lipoperoxidation and concentrations of elements in plants**

We noted higher activity of SOD and APOX in the green parts of glycophytes from sodium manufactures. However MDA level was significantly higher only in green parts of glycophytes than in roots from AE and AGRE. On the other hand, APOX activity in shoots of Common glasswort *Salicornia europaea* was lower, similarly to MDA level of these plants from SM, but the intensity of lipoperoxidation was significantly higher in roots also at AE. Concentrations of Na were higher in roots of glycophytes in opposite to halophyte. Simultaneously, Ca concentrations were higher in leaves of both glycophytes and Common glasswort in opposite to level of Fe, which was rather cumulated in roots (not significant at SM in Common glasswort *Salicornia europea*). Simultaneously, Zn was found in higher concentrations only in glycophytes but in non salted environments (WE, AGRE, C). Cu and Mn were rather mobile metals but we found significant higher Cu concentrations in roots of glycophytes (SM) and – for both elements – in roots of Common glasswort (AE). Lead, as toxic metal was accumulated in roots of plants. Glycophytes also accumulated Cd in roots but it is interesting that we did not find the differences among organs in plants from control environments. Instead, concentrations of Cd were not differs amongst organs of Common glasswort *Salicornia europea* from both environments (Table 7).


Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 53

respectively). Similar level of Pb, and Cd in plant's organs in our study was below ranges of 30-300 mg\*kg-1, and 5-30 mg\*kg-1, respectively, considered by [68] as phyototoxic. However, iron and manganese concentration in the range of 40-500 mg\*kg-1 and 50-500 mg\*kg-1, respectively, according to [70] are found as toxic for plants, so our result indicate that Fe level is high in both organs of plants, but high concentrations of Mn in roots and green parts

Stressful factors (acidity, salinity, toxic heavy metals) adversely affects the associated ecological balance and increases the level of free radicals and reactive oxygen species (ROS), which is related with changes in activities of antioxidant enzymes [71], [72], [73]. Most studies show that the higher activities of these enzymes were positively correlated with plant stress-resistance [74], [6], [73]. However, the changes of enzyme activities also depend

Based on the results obtained by us, we can conclude that physiological activity of important antioxidant enzymes can maintain oxidative homeostasis and also reduce the membrane damages in the plant organism (mainly by lipoperoxidation). Moreover, SOD, CAT, and APOX is related with concentration of examined elements (Na, Ca, Fe, Zn. Cu, Mn, Pb, Cd), depending on the status of the environment. These relations were positive and negative, and they depend on the ecological group of plants, the type of organ and on stress intensity (various degree of anthropogenic impact). Glycophytes studied by us were subjected on many stressors at disturbed environments in Kujawy region. These plant species strive to maintain their oxidative homeostasis and develop efficient antioxidative responses. Thus we found positive relationships between sodium concentrations and the content of MDA as indicator of oxidant injury, but also between CAT activity in roots of plants from anthropogenic environments, and also similar between calcium concentrations and the level of lipoperoxidation and APOX in green parts of plants from anthropogenic and wetland environments. Copper concentration was also related with APOX activity and MDA level in the wetlands. Instead, Fe was correlated with MDA and APOX in the agricultural areas (Table 4). We can thus concluded that Na, Ca, Cu and also Fe stimulated lipoperoxidation processes but also mobilization antioxidant mechanisms of glycophytes in Kujawy region. Furthermore, negative relation of Cd with APOX in both organs from controls and also positive correlations with degree of lipoperoxidation in roots (Table 4) indicate that discrimination of one of important enzymes involved in antioxidant mechanism and impact on ROS generation. Similar to Cd, also Cu stimulated higher level of MDA and negatively affect the activity of APOX in opposite to activity of SOD in roots of glycophytes in control sites (Table 4). In the organs of Common glasswort *Salicornia europea* we did not find these relations (Table 5). Reports of [8], [76], and [6] showed correlations between stress level and MDA content. Moreover, lipid peroxidation and H2O2 levels, and also SOD, CAT, APOX and GR activities increased in pea roots and leaves under Cd stress [77], whilst APOX and CAT decreased at high Cd concentrations [78]. Also [47] reported

that Cd-induced oxidative stress in *Arabidopsis* is due to H2O2 accumulation.

It should be emphasized that we found more correlations between concentrations of chemical elements and studied biochemical parameters (Tables 4, 5), which indicate the

of glycophytes was connected with environmental factors.

on plant genotypes and stress intensity [74], [75].

**Table 7.** Differences among the content of biochemical parameters: MDA, proline and concentration of elements in organs of plant groups from the same environment (p<0.005).

## **5. Discussion and conclusions**

The presence of a sodium factory and industrialization of Kujawy region affected the salinity, alkalinity and higher content of Na, Ca but also Fe, Cu, and Zn in the environments of this region, especially neighbored with sodium manufactures and waste dumping sites. On the other hand, higher concentration of microelements in the agricultural environments than in controls was probably reflected the use of fertilizers in the agricultural practices. It should be emphasized that we did not find differences in the concentration of toxic elements (Pb, Cd) in plants and soils between control and disturbed environments (SM, AE); Table 1. However, concentration of chemical elements studied in plants differed among environments but relations were not directly similar to those occurred in soils (Tables 2, 3). It probably depends on the environmental conditions. We should consider that bioavailability of chemical elements by plant is affected by numerous basal environmental characteristics of soil: pH, red-ox potential, salinity, the content of organic matter, etc. [62], [63]. Simultaneously, different factors than edaphic conditions of soils, i.e. seasonal physiology, the condition of plants, species-species capacities for uptake, translocation and compartmentalization, may contribute to the differential bioaccumulation of elements [64]. The roots of glycophytes accumulated about 3 fold higher concentrations of Na than collected at control environments, whilst green parts of plants – even 17 fold higher (Table 2). We can thus conclude that at sodium manufactures, especially anthropogenic excess of sodium affects ionic balance and primarily impacts upon metabolic processes [65]. Instead, calcium concentration in the organs of plants do not exceed average concentrations recommended by [66]; 0.2-5% d.w. However, the higher Ca concentrations, especially in salted environments, could be related with higher level of this element in these environments. On the other hand, increased intake of calcium could be connected with excess of sodium and mobilization of defence mechanisms [67]. Simultaneously, our results indicated that concentration of Zn and Cu in roots and leaves of examined plants do not exceed the phytotoxic range recommended by [68] and [69], i.e., 100-400 mg\*kg-1; 30 mg\*kg-1, respectively). Similar level of Pb, and Cd in plant's organs in our study was below ranges of 30-300 mg\*kg-1, and 5-30 mg\*kg-1, respectively, considered by [68] as phyototoxic. However, iron and manganese concentration in the range of 40-500 mg\*kg-1 and 50-500 mg\*kg-1, respectively, according to [70] are found as toxic for plants, so our result indicate that Fe level is high in both organs of plants, but high concentrations of Mn in roots and green parts of glycophytes was connected with environmental factors.

52 Plant Science

**glycophytes Common glasswort SM AE WE AGRE C SM AE**  SOD -1,98 \* ns ns ns ns ns ns CAT ns ns ns ns ns ns ns

Cd 5,15 \*\*\* 4,860 \*\*\* 4,37 \*\*\* 3,72 \*\*\* ns ns ns

elements in organs of plant groups from the same environment (p<0.005).

**5. Discussion and conclusions** 

**Table 7.** Differences among the content of biochemical parameters: MDA, proline and concentration of

The presence of a sodium factory and industrialization of Kujawy region affected the salinity, alkalinity and higher content of Na, Ca but also Fe, Cu, and Zn in the environments of this region, especially neighbored with sodium manufactures and waste dumping sites. On the other hand, higher concentration of microelements in the agricultural environments than in controls was probably reflected the use of fertilizers in the agricultural practices. It should be emphasized that we did not find differences in the concentration of toxic elements (Pb, Cd) in plants and soils between control and disturbed environments (SM, AE); Table 1. However, concentration of chemical elements studied in plants differed among environments but relations were not directly similar to those occurred in soils (Tables 2, 3). It probably depends on the environmental conditions. We should consider that bioavailability of chemical elements by plant is affected by numerous basal environmental characteristics of soil: pH, red-ox potential, salinity, the content of organic matter, etc. [62], [63]. Simultaneously, different factors than edaphic conditions of soils, i.e. seasonal physiology, the condition of plants, species-species capacities for uptake, translocation and compartmentalization, may contribute to the differential bioaccumulation of elements [64]. The roots of glycophytes accumulated about 3 fold higher concentrations of Na than collected at control environments, whilst green parts of plants – even 17 fold higher (Table 2). We can thus conclude that at sodium manufactures, especially anthropogenic excess of sodium affects ionic balance and primarily impacts upon metabolic processes [65]. Instead, calcium concentration in the organs of plants do not exceed average concentrations recommended by [66]; 0.2-5% d.w. However, the higher Ca concentrations, especially in salted environments, could be related with higher level of this element in these environments. On the other hand, increased intake of calcium could be connected with excess of sodium and mobilization of defence mechanisms [67]. Simultaneously, our results indicated that concentration of Zn and Cu in roots and leaves of examined plants do not exceed the phytotoxic range recommended by [68] and [69], i.e., 100-400 mg\*kg-1; 30 mg\*kg-1,

APX -2,46 \* ns ns ns ns 2,67 \* 3,78 \*\*\* MDA ns -3,26 \*\* ns -4,02 \*\*\* ns 3,73 \*\*\* 2,38 \* Na 5,61 \*\*\* 3,091 \*\* 6,41 \*\*\* 6,38 \*\*\* 4,55 \*\*\* -3,13 \*\* -2,75 \*\* Ca -2,59 \*\* -5,492 \*\*\* -7,79 \*\*\* -5,01 \*\*\* -5,14 \*\*\* -2,14 \* -4,22 \*\*\* Fe 5,17 \*\*\* 5,372 \*\*\* 5,86 \*\*\* 4,63 \*\*\* 3,01 \*\* ns 2,09 \* Zn ns ns -4,25 \*\*\* -4,94 \*\*\* -2,25 \* ns ns Cu 3,05 \*\* ns ns ns ns ns 2,26 \* Mn ns ns ns ns ns ns -4,16 \*\*\* Pb 5,83 \*\*\* 5,642 \*\*\* 4,26 \*\*\* 2,39 \* 3,63 \*\*\* 2,27 \* 4,22 \*\*\*

Stressful factors (acidity, salinity, toxic heavy metals) adversely affects the associated ecological balance and increases the level of free radicals and reactive oxygen species (ROS), which is related with changes in activities of antioxidant enzymes [71], [72], [73]. Most studies show that the higher activities of these enzymes were positively correlated with plant stress-resistance [74], [6], [73]. However, the changes of enzyme activities also depend on plant genotypes and stress intensity [74], [75].

Based on the results obtained by us, we can conclude that physiological activity of important antioxidant enzymes can maintain oxidative homeostasis and also reduce the membrane damages in the plant organism (mainly by lipoperoxidation). Moreover, SOD, CAT, and APOX is related with concentration of examined elements (Na, Ca, Fe, Zn. Cu, Mn, Pb, Cd), depending on the status of the environment. These relations were positive and negative, and they depend on the ecological group of plants, the type of organ and on stress intensity (various degree of anthropogenic impact). Glycophytes studied by us were subjected on many stressors at disturbed environments in Kujawy region. These plant species strive to maintain their oxidative homeostasis and develop efficient antioxidative responses. Thus we found positive relationships between sodium concentrations and the content of MDA as indicator of oxidant injury, but also between CAT activity in roots of plants from anthropogenic environments, and also similar between calcium concentrations and the level of lipoperoxidation and APOX in green parts of plants from anthropogenic and wetland environments. Copper concentration was also related with APOX activity and MDA level in the wetlands. Instead, Fe was correlated with MDA and APOX in the agricultural areas (Table 4). We can thus concluded that Na, Ca, Cu and also Fe stimulated lipoperoxidation processes but also mobilization antioxidant mechanisms of glycophytes in Kujawy region. Furthermore, negative relation of Cd with APOX in both organs from controls and also positive correlations with degree of lipoperoxidation in roots (Table 4) indicate that discrimination of one of important enzymes involved in antioxidant mechanism and impact on ROS generation. Similar to Cd, also Cu stimulated higher level of MDA and negatively affect the activity of APOX in opposite to activity of SOD in roots of glycophytes in control sites (Table 4). In the organs of Common glasswort *Salicornia europea* we did not find these relations (Table 5). Reports of [8], [76], and [6] showed correlations between stress level and MDA content. Moreover, lipid peroxidation and H2O2 levels, and also SOD, CAT, APOX and GR activities increased in pea roots and leaves under Cd stress [77], whilst APOX and CAT decreased at high Cd concentrations [78]. Also [47] reported that Cd-induced oxidative stress in *Arabidopsis* is due to H2O2 accumulation.

It should be emphasized that we found more correlations between concentrations of chemical elements and studied biochemical parameters (Tables 4, 5), which indicate the complexity of antioxidative processes and simultaneously the participation of chemical elements in stimulation and modification of lipoperoxidation processes and SOD, CAT or APOX activities. Simultaneously, the same elements could impact upon the activation of many oxidative pathways in organs of glycophytes (Fig. 5) and it depends on the environmental conditions. In disturbed sodium manufactures the level of Na stimulated activity of SOD and APOX, but in the other environments it stimulated the activity of CAT. Similarly, copper concentration is positively correlated with CAT in roots but negatively in the green parts of plants from anthropogenic environments. Furthermore, Na and Ca concentrations stimulated activity of antioxidant mechanisms, especially in disturbed environments. On the other hand, calcium is also related with APOX activity in all studied environments. Simultaneously, concentrations of transition metals could impact the increase or decrease of antioxidant enzymes activity (Figs. 2-5). We suggest it depends on the interactions amongst elements, the intensity of stressors or impact of other not examined factors. However, more analysis will require for estimation these problems. We also should consider that Cu, Zn, Mn, and also Fe are cofactors of metalloprotein, i.e. SOD. Simultaneously, cadmium and lead as toxic metals especially defected the activity of APOX and CAT, respectively, in the organs of glycophytes, which is in opposite to SOD activity (Tab. 4). Similarly, Fe, Zn, and Cu stimulate activity of SOD in sodium manufactures in roots and shoots of Common glasswort *Salicornia europea*. Moreover, the same metals decrease CAT activity in roots from anthropogenic environments. Cadmium and lead concentration are also defected the activity of APOX and CAT and impacted the increase of SOD. We also found correlations between APOX and Ca concentrations in shoots of Common glasswort *Salicornia europaea* in sodium manufactures, alike as in the green parts of glycophytes. Moreover, sodium concentrations influence the decrease of CAT in roots of this halophyte (Table 5). Similarly, CAT activity is also higher in green parts of glycophytes than in halophytes. This indicate that glycophytes are better adaptation to stressful conditions, however there are many indistinct questions required to estimation.

Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 55

We can consider that heavy metals toxicity is occurred in the ability to bind strongly the oxygen, nitrogen and sulphur ions. This process is related with the free enthalpy of the formation of the product of metals and ligands. By this features heavy metals can inactivate the enzymes by binding to cysteine residues. Heavy metals can also displace one metal with another, which can lead to inhibition or loss the enzyme activities [4], [13]. On the other hand, [85] examined the role of antioxidative enzyme system. They investigated the relation to Cd stress in hyperaccumulator plants of the genus *Alyssum*. In both species superoxide dismutase activity was elevated at high cadmium concentrations, whilst ascorbate peroxidase activity remained unchanged [7], whilst [86] indicated that activity of antioxidant enzymes was estimated as a function of time and concentrations of Pb in roots of lupin. The results of [25] suggested that lead induces oxidative stress in growing rice plants and that SOD and APOX could serve as important components of antioxidative defence mechanism against Pb induced oxidative injury in rice. They observed a Pb dependent increase in the activities of SOD from root tip extract, whilst CAT and APOX activities decreased at higher lead concentrations. Cadmium treatment induced lipooxygenase with simultaneous inhibition of antioxidative enzymes, SOD and CAT [41]. In particular, CAT activity often decreased following exposure to elevated cadmium concentrations [78], [46]. On the other hand, [87] indicated that CAT activities and specific isoenzymes of SOD increased in the leaves and roots of a resistant variety of radish, following exposure to increasing (between 0.25 and 1 mM) concentrations of cadmium. A severe suppression of SOD and CAT, and almost complete loss of APOX activities after 48 h of exposure to 50 μM Cd was observed in pine roots [88]. Cd-induced inhibition of APOX and CAT was also associated with H2O2 accumulation and growth retardation in the poplar roots [4]. Also [89] studied the involvement of H2O2 and O2.- in the signaling events that resulted in variation of the

transcript levels of CAT, GR and Cu-Zn-SOD in pea plants under Cd stress.

the membrane to its normal function [31].

stress in plants.

Considering the influence of chemical elements on the defence mechanisms of plants [90] we can conclude the unique importance of Ca2+ for stabilization of membranes. High salinity results in increased cytosolic Ca2+, which is transported from the apoplast and the intracellular compartments [91]. This transient increase in the cytosolic Ca2+ initiates stresssignal transduction leading to salt adaptation. Adequate levels of calcium are necessary for

Most of the interest in calcium participation in plants has centered on its role in the cytoplasm in controlling the developmental processes [92]. [32] reported the effects of calcium chloride on the sodium chloride-stressed plants of Mung bean *Vigna radiatae* (L.). We could confirm that when CaCl2 was combined with NaCl, CaCl2 altered the overall plant metabolism to ameliorate the deleterious effects of NaCl stress and increased the vegetative growth of plants. [93] indicated that Ca2+ prevented Cd-induced increasing of the activity of SOD and restored CAT activity. These results suggested that exogenous application of Ca2+ could be advantageous against Cd2+ toxicity, and could confer tolerance to heavy metal's

Our results confirm also the disruption of oxidative homeostasis related with environments of glycophytes through higher degree of lipid peroxidation in leaves in the anthropogenic

There is much evidence that salinity and heavy metals enhanced or decreased activity of antioxidant enzymes but usually under control conditions. Both increase as well as decrease in the activity of SOD has been reported in plants in response to salinity stress [8], [79]. It appears that the activity of SOD under salinity varies depending upon plant species, organ analyzed as well as upon the level of salinity [80], [81]. [9] showed that in Cotton *Gossypium hirsutum* L. under salted NaCl stress the increases of SOD activities occurs, as well as decreases of the activities of catalase and ascorbate peroxidase. In the leaves of rice plant, salt stress preferentially enhances the content of H2O2 and the activities of SOD, APOX, whereas it decreases catalase activity [82]. These authors reported that NaCl treatment increases the activities of catalase but does not affected the activity of SOD in cucumber plants. The tomato under high salt concentration showed higher among others antioxidant enzyme activities such as SOD, catalase, and ascorbate peroxidase [83]. Similarly, [84] showed that activity of cytosolic CuZn-SOD II, chloroplastic CuZn- SOD II, and mitochondrial and/or peroxisomal Mn-SOD were correlated with increasing concentration of NaCl in pea at the higher NaCl concentrations.

We can consider that heavy metals toxicity is occurred in the ability to bind strongly the oxygen, nitrogen and sulphur ions. This process is related with the free enthalpy of the formation of the product of metals and ligands. By this features heavy metals can inactivate the enzymes by binding to cysteine residues. Heavy metals can also displace one metal with another, which can lead to inhibition or loss the enzyme activities [4], [13]. On the other hand, [85] examined the role of antioxidative enzyme system. They investigated the relation to Cd stress in hyperaccumulator plants of the genus *Alyssum*. In both species superoxide dismutase activity was elevated at high cadmium concentrations, whilst ascorbate peroxidase activity remained unchanged [7], whilst [86] indicated that activity of antioxidant enzymes was estimated as a function of time and concentrations of Pb in roots of lupin. The results of [25] suggested that lead induces oxidative stress in growing rice plants and that SOD and APOX could serve as important components of antioxidative defence mechanism against Pb induced oxidative injury in rice. They observed a Pb dependent increase in the activities of SOD from root tip extract, whilst CAT and APOX activities decreased at higher lead concentrations. Cadmium treatment induced lipooxygenase with simultaneous inhibition of antioxidative enzymes, SOD and CAT [41]. In particular, CAT activity often decreased following exposure to elevated cadmium concentrations [78], [46]. On the other hand, [87] indicated that CAT activities and specific isoenzymes of SOD increased in the leaves and roots of a resistant variety of radish, following exposure to increasing (between 0.25 and 1 mM) concentrations of cadmium. A severe suppression of SOD and CAT, and almost complete loss of APOX activities after 48 h of exposure to 50 μM Cd was observed in pine roots [88]. Cd-induced inhibition of APOX and CAT was also associated with H2O2 accumulation and growth retardation in the poplar roots [4]. Also [89] studied the involvement of H2O2 and O2.- in the signaling events that resulted in variation of the transcript levels of CAT, GR and Cu-Zn-SOD in pea plants under Cd stress.

54 Plant Science

complexity of antioxidative processes and simultaneously the participation of chemical elements in stimulation and modification of lipoperoxidation processes and SOD, CAT or APOX activities. Simultaneously, the same elements could impact upon the activation of many oxidative pathways in organs of glycophytes (Fig. 5) and it depends on the environmental conditions. In disturbed sodium manufactures the level of Na stimulated activity of SOD and APOX, but in the other environments it stimulated the activity of CAT. Similarly, copper concentration is positively correlated with CAT in roots but negatively in the green parts of plants from anthropogenic environments. Furthermore, Na and Ca concentrations stimulated activity of antioxidant mechanisms, especially in disturbed environments. On the other hand, calcium is also related with APOX activity in all studied environments. Simultaneously, concentrations of transition metals could impact the increase or decrease of antioxidant enzymes activity (Figs. 2-5). We suggest it depends on the interactions amongst elements, the intensity of stressors or impact of other not examined factors. However, more analysis will require for estimation these problems. We also should consider that Cu, Zn, Mn, and also Fe are cofactors of metalloprotein, i.e. SOD. Simultaneously, cadmium and lead as toxic metals especially defected the activity of APOX and CAT, respectively, in the organs of glycophytes, which is in opposite to SOD activity (Tab. 4). Similarly, Fe, Zn, and Cu stimulate activity of SOD in sodium manufactures in roots and shoots of Common glasswort *Salicornia europea*. Moreover, the same metals decrease CAT activity in roots from anthropogenic environments. Cadmium and lead concentration are also defected the activity of APOX and CAT and impacted the increase of SOD. We also found correlations between APOX and Ca concentrations in shoots of Common glasswort *Salicornia europaea* in sodium manufactures, alike as in the green parts of glycophytes. Moreover, sodium concentrations influence the decrease of CAT in roots of this halophyte (Table 5). Similarly, CAT activity is also higher in green parts of glycophytes than in halophytes. This indicate that glycophytes are better adaptation to stressful conditions,

however there are many indistinct questions required to estimation.

of NaCl in pea at the higher NaCl concentrations.

There is much evidence that salinity and heavy metals enhanced or decreased activity of antioxidant enzymes but usually under control conditions. Both increase as well as decrease in the activity of SOD has been reported in plants in response to salinity stress [8], [79]. It appears that the activity of SOD under salinity varies depending upon plant species, organ analyzed as well as upon the level of salinity [80], [81]. [9] showed that in Cotton *Gossypium hirsutum* L. under salted NaCl stress the increases of SOD activities occurs, as well as decreases of the activities of catalase and ascorbate peroxidase. In the leaves of rice plant, salt stress preferentially enhances the content of H2O2 and the activities of SOD, APOX, whereas it decreases catalase activity [82]. These authors reported that NaCl treatment increases the activities of catalase but does not affected the activity of SOD in cucumber plants. The tomato under high salt concentration showed higher among others antioxidant enzyme activities such as SOD, catalase, and ascorbate peroxidase [83]. Similarly, [84] showed that activity of cytosolic CuZn-SOD II, chloroplastic CuZn- SOD II, and mitochondrial and/or peroxisomal Mn-SOD were correlated with increasing concentration Considering the influence of chemical elements on the defence mechanisms of plants [90] we can conclude the unique importance of Ca2+ for stabilization of membranes. High salinity results in increased cytosolic Ca2+, which is transported from the apoplast and the intracellular compartments [91]. This transient increase in the cytosolic Ca2+ initiates stresssignal transduction leading to salt adaptation. Adequate levels of calcium are necessary for the membrane to its normal function [31].

Most of the interest in calcium participation in plants has centered on its role in the cytoplasm in controlling the developmental processes [92]. [32] reported the effects of calcium chloride on the sodium chloride-stressed plants of Mung bean *Vigna radiatae* (L.). We could confirm that when CaCl2 was combined with NaCl, CaCl2 altered the overall plant metabolism to ameliorate the deleterious effects of NaCl stress and increased the vegetative growth of plants. [93] indicated that Ca2+ prevented Cd-induced increasing of the activity of SOD and restored CAT activity. These results suggested that exogenous application of Ca2+ could be advantageous against Cd2+ toxicity, and could confer tolerance to heavy metal's stress in plants.

Our results confirm also the disruption of oxidative homeostasis related with environments of glycophytes through higher degree of lipid peroxidation in leaves in the anthropogenic environments and lower level of MDA in roots of plants from wetlands than from remaining environments. However, we found a higher SOD activity in the roots of glycophytes from agriculture areas in comparison with sodium manufactures and wetlands. Simultaneously, the activity of the same enzymes in green parts of plants was lower in wetland areas than in remaining environments (Table 2). All of these results indicated that plants probably can mitigate the oxidative damage initiated by ROS (especially at agriculture) by the complex of defensive antioxidative system [71]. Moreover, [15] indicated that both roots and shoots of plants had malonyldialdehyde (MDA) contents cultivated at the optimal salt concentration (50 mM NaCl) were lower than in the control areas. This was related to enhanced activities of antioxidant enzymes, like superoxide dismutase, catalase and peroxidase, especially in shoots. SOD and CAT activities have been reported to be negatively correlated with the degree of damage to plasmalemma, chloroplasts and mitochondrial membrane systems and positively correlated with stress resistance indices [7], [32]. Simultaneously, we can not exclude other factors as draught or climate conditions, which could generate free radicals or reactive oxygen species (i.e., draught, climate conditions) and thus impact on high activity of antioxidant enzymes in organs in the control environments (C). We should also note that the level of cadmium was higher in leaves of plants from Tuchola Forestry than from remaining environments. Similarly, in roots of plants from these areas, cadmium concentrations did not differ from other environments, but was higher than in the agricultures. Cadmium is probably a factor, which can indirectly upset cell membrane. Simultaneously, we found relationships between biochemical parameters and concentrations of Na, Ca, Cu, Zn and Mn, which stimulated the activity of CAT, and Na-SOD and Ca-SOD and APOX. Instead, we found differences between the content of MDA (higher in shoots of plants from anthropogenic than from sodium manufactures) but the activity of SOD, CAT and APOX did not differ in the organs of Common glasswort *Salicornia europaea* between two examined environments. It could indicate that Common glasswort, as obligatory halophyte, is adapted to high salted environments (sodium manufactures). Thus mutual and differentiated relationships between chemical elements and antioxidant enzymatic mechanisms as plant responses to the environmental disturbs, are probably another factor influenced the higher level of lipoperoxidation in anthropogenic environments.

Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 57

osmoregulation); [13]. It is interesting we found the higher level of MDA and higher lead concentrations in roots, similar to activity of APOX. However, we could not confirm relations among these parameters because we did not find significant correlation. Furthermore, the roots are more markedly affected by saline conditions than leaves, because of being the first part at the plant to encounter soil salinity [94]. [81] confirm that roots of halophyte Sea fennel *Crithmum maritimum* were distinguished from leaves by malondialdehyde concentration, however activity of APOX was higher in leaves, despite the fact that stress factor was only a high salinity. Simultaneously, [95] and [96] stated that root tissues markedly exhibited by higher APOX activity in comparison of shoots of plants under

The mechanisms of salt tolerance are of two main types: those minimizing the entry of salt into the plant, and those minimizing the concentration of salt in the cytoplasm. Halophytes, as naturally salt tolerant plants, have both types of mechanisms. They exclude salt well, but effectively compartmentalize in vacuoles the salt that inevitably gets in. This allows them to growth of long period of time in saline soil. Instead, glycophytes avoid sodium to maintain ionic homeostasis [13], [15]. We can thus conclude basing also on our results showed in Table 5 that higher concentrations of sodium in roots and leaves of Common glasswort *Salicornia europea* than in glycophytes are resulting from the saline determinations of the environment and predominantly from the interactions with sodium. We also conclude that glycophytes are subjected on higher salinity stress, which cause probably higher level of ROS, and lipoperoxidation in the organs (except green parts at AE) than in halophytes. On the other hand, activity of APOX is higher in the roots of halophyte than in glycophytes in both environments in opposite to green parts. We could suspect that this were connected with higher concentrations of Ca, elevated salinity effect [32] and indicated the activity of APOX in the green parts of plants more sensitive on salt damages than roots [97]. Similar activity of CAT is also higher in green parts of our glycophytes than halophyte. It is indicated that glycophytes are well adapted to

stressful conditions however there are still many indistinct topics, required to estimation.

The area of mutual relationships: plants-environmental stressors and conducted research in this field has been gaining ground in the recent years. However, in the view of considerable variations in the protective mechanisms against activated oxygen species in different plant species, the further work, especially in natural conditions under many stressors is required to establish the general validity of various regularities and processes in salinity tolerance

The positive and negative correlations between the level of chemical elements and antioxidative responses of plants indicate the complexity of enzymatic antioxidative processes and simultaneously the participation of chemical elements, which does not influence lipid peroxidation but stimulate or injure SOD, CAT or APOX activity. However, the concentrations of Na, Ca, Cu and Fe stimulate lipoperoxidation but can also mobilize antioxidant responses of glycophytes in natural salted Pomeranian region, whilst Cd in the control environment influence MDA content and increasing APOX activity in roots of glycophytes. Simultaneously this toxic metal may impact the activation of many oxidant ways in organs of glycophytes and it depend on the environmental conditions (pH, Ec). Na

salinity stress.

and also disturbed ionic homeostasis.

We also found that Na, Fe, and Pb are accumulated in higher level in roots of glycophytes in opposite to Ca. Zn, Cu and Mn at all examined environments and they showed more mobility, which depends on the type of environment. Interestingly, the concentrations of Cd and Pb, as toxic elements were also higher in roots of plants at all environments of Kujawy region but not at the control sites, whilst the activity of antioxidant enzymes did not differ significantly between organs of plants in the examined environments, except of SOD and APOX at SM. We found higher level of these enzymes in the leaves than in roots of plants. It was probably related with the concentrations of Ca and also Cu, Zn, and Cd. We could also conclude that Zn and Cu are co-factors of SOD so further analyses are needed. However, the location of chemical elements rather not deflected on the activity of enzymes in glycophytes. On the other hand, the concentrations of Na were higher in shoots than roots of Common glasswort *Salicornia europaea*, which is connected with adaptation to life in high salinity (e.g., osmoregulation); [13]. It is interesting we found the higher level of MDA and higher lead concentrations in roots, similar to activity of APOX. However, we could not confirm relations among these parameters because we did not find significant correlation. Furthermore, the roots are more markedly affected by saline conditions than leaves, because of being the first part at the plant to encounter soil salinity [94]. [81] confirm that roots of halophyte Sea fennel *Crithmum maritimum* were distinguished from leaves by malondialdehyde concentration, however activity of APOX was higher in leaves, despite the fact that stress factor was only a high salinity. Simultaneously, [95] and [96] stated that root tissues markedly exhibited by higher APOX activity in comparison of shoots of plants under salinity stress.

56 Plant Science

environments and lower level of MDA in roots of plants from wetlands than from remaining environments. However, we found a higher SOD activity in the roots of glycophytes from agriculture areas in comparison with sodium manufactures and wetlands. Simultaneously, the activity of the same enzymes in green parts of plants was lower in wetland areas than in remaining environments (Table 2). All of these results indicated that plants probably can mitigate the oxidative damage initiated by ROS (especially at agriculture) by the complex of defensive antioxidative system [71]. Moreover, [15] indicated that both roots and shoots of plants had malonyldialdehyde (MDA) contents cultivated at the optimal salt concentration (50 mM NaCl) were lower than in the control areas. This was related to enhanced activities of antioxidant enzymes, like superoxide dismutase, catalase and peroxidase, especially in shoots. SOD and CAT activities have been reported to be negatively correlated with the degree of damage to plasmalemma, chloroplasts and mitochondrial membrane systems and positively correlated with stress resistance indices [7], [32]. Simultaneously, we can not exclude other factors as draught or climate conditions, which could generate free radicals or reactive oxygen species (i.e., draught, climate conditions) and thus impact on high activity of antioxidant enzymes in organs in the control environments (C). We should also note that the level of cadmium was higher in leaves of plants from Tuchola Forestry than from remaining environments. Similarly, in roots of plants from these areas, cadmium concentrations did not differ from other environments, but was higher than in the agricultures. Cadmium is probably a factor, which can indirectly upset cell membrane. Simultaneously, we found relationships between biochemical parameters and concentrations of Na, Ca, Cu, Zn and Mn, which stimulated the activity of CAT, and Na-SOD and Ca-SOD and APOX. Instead, we found differences between the content of MDA (higher in shoots of plants from anthropogenic than from sodium manufactures) but the activity of SOD, CAT and APOX did not differ in the organs of Common glasswort *Salicornia europaea* between two examined environments. It could indicate that Common glasswort, as obligatory halophyte, is adapted to high salted environments (sodium manufactures). Thus mutual and differentiated relationships between chemical elements and antioxidant enzymatic mechanisms as plant responses to the environmental disturbs, are probably another factor influenced the higher level of

We also found that Na, Fe, and Pb are accumulated in higher level in roots of glycophytes in opposite to Ca. Zn, Cu and Mn at all examined environments and they showed more mobility, which depends on the type of environment. Interestingly, the concentrations of Cd and Pb, as toxic elements were also higher in roots of plants at all environments of Kujawy region but not at the control sites, whilst the activity of antioxidant enzymes did not differ significantly between organs of plants in the examined environments, except of SOD and APOX at SM. We found higher level of these enzymes in the leaves than in roots of plants. It was probably related with the concentrations of Ca and also Cu, Zn, and Cd. We could also conclude that Zn and Cu are co-factors of SOD so further analyses are needed. However, the location of chemical elements rather not deflected on the activity of enzymes in glycophytes. On the other hand, the concentrations of Na were higher in shoots than roots of Common glasswort *Salicornia europaea*, which is connected with adaptation to life in high salinity (e.g.,

lipoperoxidation in anthropogenic environments.

The mechanisms of salt tolerance are of two main types: those minimizing the entry of salt into the plant, and those minimizing the concentration of salt in the cytoplasm. Halophytes, as naturally salt tolerant plants, have both types of mechanisms. They exclude salt well, but effectively compartmentalize in vacuoles the salt that inevitably gets in. This allows them to growth of long period of time in saline soil. Instead, glycophytes avoid sodium to maintain ionic homeostasis [13], [15]. We can thus conclude basing also on our results showed in Table 5 that higher concentrations of sodium in roots and leaves of Common glasswort *Salicornia europea* than in glycophytes are resulting from the saline determinations of the environment and predominantly from the interactions with sodium. We also conclude that glycophytes are subjected on higher salinity stress, which cause probably higher level of ROS, and lipoperoxidation in the organs (except green parts at AE) than in halophytes. On the other hand, activity of APOX is higher in the roots of halophyte than in glycophytes in both environments in opposite to green parts. We could suspect that this were connected with higher concentrations of Ca, elevated salinity effect [32] and indicated the activity of APOX in the green parts of plants more sensitive on salt damages than roots [97]. Similar activity of CAT is also higher in green parts of our glycophytes than halophyte. It is indicated that glycophytes are well adapted to stressful conditions however there are still many indistinct topics, required to estimation.

The area of mutual relationships: plants-environmental stressors and conducted research in this field has been gaining ground in the recent years. However, in the view of considerable variations in the protective mechanisms against activated oxygen species in different plant species, the further work, especially in natural conditions under many stressors is required to establish the general validity of various regularities and processes in salinity tolerance and also disturbed ionic homeostasis.

The positive and negative correlations between the level of chemical elements and antioxidative responses of plants indicate the complexity of enzymatic antioxidative processes and simultaneously the participation of chemical elements, which does not influence lipid peroxidation but stimulate or injure SOD, CAT or APOX activity. However, the concentrations of Na, Ca, Cu and Fe stimulate lipoperoxidation but can also mobilize antioxidant responses of glycophytes in natural salted Pomeranian region, whilst Cd in the control environment influence MDA content and increasing APOX activity in roots of glycophytes. Simultaneously this toxic metal may impact the activation of many oxidant ways in organs of glycophytes and it depend on the environmental conditions (pH, Ec). Na

and Ca concentrations rather stimulate enzymatic antioxidant mechanisms activity, especially in disturbed environments, and they can also stimulate the transition metals which causing increase or decrease of antioxidant enzymatic activity. However, Cd and Pb can defect the activity of APOX and CAT in organs of glycophytes, which is opposite to SOD activity. Similarly, in the organs of Common glasswort *Salicornia europaea*, Fe, Zn, and Cu level can stimulate SOD activity but they can also inhibit CAT activity. These processes depend on the environmental factors (pH, Ec) and types of plant organs and toxic heavy metals Cd and Pb concentration. They defect APOX and CAT activity but influence the increase of SOD. We also found correlation between APOX and calcium concentration (alcalinity) in shoots of Common glasswort and in green parts of glycophytes.

Enzymatic Antioxidant Responses of Plants in Saline Anthropogenic Environments 59

*Government Forestry in Toruń; Zamrzenica Forestry District, Bysław, Poland* 

*University of Zielona Góra, Faculty of Biological Sciences, Institute of Biotechnology and* 

University of Bahrain) for his help with improving English language of the paper.

controlling cross-tolerance. Trends Plant Sci. 5: 241-246.

*Environment Protection, Department of Environmental Protection and Biodiversity, Zielona Góra,* 

We thank Professor Brendan P. Kavanagh (Royal College of Surgeons in Ireland, Medical

[1] Mittler R. 2002. Oxidative stress, antioxidant and stress tolerance. Trends Plant Sci. 7:

[2] Bowler C., Fluhr R. 2000. The role of calcium and activated oxygens as signals for

[3] Panda S.K., Khan M.H. 2004. Changes in growth and superoxide dismutase activity in *Hydrilla verticillata* L. under abiotic stress. Braz. J. Plant Physiol., 16(2):115-118. [4] Schutzendubel A., Polle A. 2002. Plant responses to abiotic stresses: heavy-metal induced

[7] Elkahoui S., Hernandez J.A., Abdelly Ch., Ghrir R., Limam F. 2005. Effects of salt on lipid peroxidation and antioxidant enzyme activities of *Catharanthus roseus* suspension cells.

[8] Dionisio-Sese M.L., Tobita S. 1998. Antioxidant responses of rice seedlings to salinity

[9] Gossett D.R., Millhollon E.P., Lucas M.C. 1994. Antioxidant responses toNaCl stress in

[10] Sreenivasulu N., Ramanjulu S., Ramachandra-Kini K., Prakash H.S., Shekar-Shetty H., Savitri H.S., Sudhakar C. 1999. Total peroxidase activity and peroxidase isoforms as modified by salt stress in two cultivars of fox-tail millet with differential salt tolerance.

[11] Loureiro S., Santos C., Pinto G., Costa A., Monteiro M., Nogueira A.J.A., Soares A.M.V.M. 2006. Toxicity assessment of two soils from Jales Mine (Portugal) using plants: growth and biochemical parameters. Arch. Environ. Contam. Toxicol. 50: 182-

[12] Bidar G., Pruvot Ch., Garçon G., Verdin A., Shirali P., Douay F. 2009. Seasonal and annual variations of metal uptake, bioaccumulation, and toxicity in *Trifolium repens* and

salt tolerant and salt-sensitive cultivars of cotton, Crop Sci. 34: 706-714.

oxidative stress and protection by mycorrhization. J. Exp. Bot. 53: 1351-1365. [5] Scandalios J.G. 1993. Oxygen stress and superoxide dismutase. Plant Physiol. 101: 7-12. [6] Sairam R.K., Srivastava G.C. 2002. Changes in antioxidant activity in sub-cellular fractions of tolerant and susceptible wheat genotypes in response to long term salt

Piotr Puchowski

Leszek Jerzak

**Acknowledgement** 

**6. References** 

405-410.

stress. Plant Sci. 162: 897-904.

Plant Sci. 168: 607-613.

stress. Plant Sci. 135: 1-9.

Plant Sci. 141: 1–9.

190.

*Poland* 

The differences in MDA content and SOD activity either in roots or in green parts of glycophytes indicate that plants can probably mitigate the oxidative damage initiated by reactive oxygen species (ROS) by complexes of defensive enzymatic antioxidative system. Instead we found only differences between MDA content (higher in shoots from anthropogenic than in more salted environments) but the activity of SOD, CAT and APOX didn't differ in organs of Common glasswort *Salicornia europaea*, which indicates that the impact of other factors than examined elements influenced higher level of lipoperoxidation in anthropogenic environments. Antioxidant enzymatic activity didn't differ significantly between plants organs in examined environments except of SOD and APOX in strong salted environment (SM). We found higher level of these enzymes in leaves than in roots of plants studied. It was probably related with Ca and also Cu, Zn, and Cd concentrations. However, the level of lipoperoxidation was significant higher in green parts of glycophytes (anthropogenic, agricultural environments), which is opposite to common glasswort (both environments). Instead in glycophytes avoiding sodium to maintain ionic homeostasis we found higher Na concentrations in roots and leaves of halophytes (Common glasswort *Salicornia europaea*) than in glycophytes. We can conclude that glycophytes subjected to higher salinity stress develop probably higher level of ROS, thus lipoperoxidation in their organs (except of green parts in AE) is higher than in halophytes. On the other hand, APOX activity is higher in roots of halophytes than in glycophytes in both environments in opposite to their green parts. We thus can suspect that this is connected with higher concentrations of Ca (alcalinity) and elevated salinity effect indicated APOX activity in green parts of plants more sensitive on salt damages than roots. Similarly, CAT activity is also higher in green parts of glycophytes than in halophytes. This indicate that glycophytes are better adaptation to stressful conditions, however there are many indistinct questions which require further study.

#### **Author details**

Piotr Kamiński, Beata Koim-Puchowska, Monika Wieloch and Karolina Bombolewska *Nicolaus Copernicus University, Collegium Medium in Bydgoszcz, Department of Ecology and Environmental Protection, Bydgoszcz, Poland* 

Piotr Kamiński *University of Zielona Góra, Faculty of Biological Sciences, Institute of Biotechnology and Environment Protection, Department of Biotechnology, Zielona Góra, Poland* 

Piotr Puchowski *Government Forestry in Toruń; Zamrzenica Forestry District, Bysław, Poland* 

Leszek Jerzak

58 Plant Science

**Author details** 

Piotr Kamiński

*Environmental Protection, Bydgoszcz, Poland* 

and Ca concentrations rather stimulate enzymatic antioxidant mechanisms activity, especially in disturbed environments, and they can also stimulate the transition metals which causing increase or decrease of antioxidant enzymatic activity. However, Cd and Pb can defect the activity of APOX and CAT in organs of glycophytes, which is opposite to SOD activity. Similarly, in the organs of Common glasswort *Salicornia europaea*, Fe, Zn, and Cu level can stimulate SOD activity but they can also inhibit CAT activity. These processes depend on the environmental factors (pH, Ec) and types of plant organs and toxic heavy metals Cd and Pb concentration. They defect APOX and CAT activity but influence the increase of SOD. We also found correlation between APOX and calcium concentration

The differences in MDA content and SOD activity either in roots or in green parts of glycophytes indicate that plants can probably mitigate the oxidative damage initiated by reactive oxygen species (ROS) by complexes of defensive enzymatic antioxidative system. Instead we found only differences between MDA content (higher in shoots from anthropogenic than in more salted environments) but the activity of SOD, CAT and APOX didn't differ in organs of Common glasswort *Salicornia europaea*, which indicates that the impact of other factors than examined elements influenced higher level of lipoperoxidation in anthropogenic environments. Antioxidant enzymatic activity didn't differ significantly between plants organs in examined environments except of SOD and APOX in strong salted environment (SM). We found higher level of these enzymes in leaves than in roots of plants studied. It was probably related with Ca and also Cu, Zn, and Cd concentrations. However, the level of lipoperoxidation was significant higher in green parts of glycophytes (anthropogenic, agricultural environments), which is opposite to common glasswort (both environments). Instead in glycophytes avoiding sodium to maintain ionic homeostasis we found higher Na concentrations in roots and leaves of halophytes (Common glasswort *Salicornia europaea*) than in glycophytes. We can conclude that glycophytes subjected to higher salinity stress develop probably higher level of ROS, thus lipoperoxidation in their organs (except of green parts in AE) is higher than in halophytes. On the other hand, APOX activity is higher in roots of halophytes than in glycophytes in both environments in opposite to their green parts. We thus can suspect that this is connected with higher concentrations of Ca (alcalinity) and elevated salinity effect indicated APOX activity in green parts of plants more sensitive on salt damages than roots. Similarly, CAT activity is also higher in green parts of glycophytes than in halophytes. This indicate that glycophytes are better adaptation to stressful conditions, however there are many indistinct questions which require further study.

Piotr Kamiński, Beata Koim-Puchowska, Monika Wieloch and Karolina Bombolewska *Nicolaus Copernicus University, Collegium Medium in Bydgoszcz, Department of Ecology and* 

*University of Zielona Góra, Faculty of Biological Sciences, Institute of Biotechnology and* 

*Environment Protection, Department of Biotechnology, Zielona Góra, Poland* 

(alcalinity) in shoots of Common glasswort and in green parts of glycophytes.

*University of Zielona Góra, Faculty of Biological Sciences, Institute of Biotechnology and Environment Protection, Department of Environmental Protection and Biodiversity, Zielona Góra, Poland* 

#### **Acknowledgement**

We thank Professor Brendan P. Kavanagh (Royal College of Surgeons in Ireland, Medical University of Bahrain) for his help with improving English language of the paper.

#### **6. References**


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**1. Introduction**

http://dx.doi.org/10.5772/53604

and this is hundreds and thousands of tons.

cited.

respiration and metabolism.

It is extremely important to reduce the action of man-made factors on the environment and vegetables and crops grown in greenhouses. On the one hand, the whole year round, greenhouse technologies provide the production of vegetables and other crops, which contain micro- and macroelements necessary for the vital activity of human being. On the other hand, the use of the technologies implies the use of mineral fertilizers for extra root and foliar nutrition in order to intensify the production. However, despite the use of drop irrigation and small doses of mineral feeding, the concentrations of various heavy metal compounds, radioactive and poisonous substances in the soil of greenhouses reach the limit values after 2–3 years of its service. Then the exhausted soil should be disposed and replaced by new soil,

**The Plant Nutrition from the Gas Medium in** 

**Chapter 3**

**Greenhouses: Multilevel Simulation and** 

**Experimental Investigation** 

V.B. Golubchikov and E.V. Golubchikov

Additional information is available at the end of the chapter

A.V. Vakhrushev, A.Yu. Fedotov, A.A. Vakhrushev,

At the same time, the problem of the neutralization of the used soil arises, since it cannot simply be stored in the open air due to the formation of the dust fractions of the above compounds. In the rain and during snow melting, water-soluble substances penetrate into soil and water sources together with run-off water. From the aforesaid it follows that searching new ways for the nutrition of plants, which allow reducing soil contamination, is urgent. In this regard, the technologies for plant nutrition from the gas phase are quite promising, since the plants receive significant portion of nutrients (up to 80%) through their foliage and stems. It should be noted that controlled gas media have already been used for quite a long time for storing vegetables and fruit. The method is based on the formation of special concentrations of nitrogen, oxygen, carbon dioxide and water vapours in order to reduce the intensity of

Join Stocks Company Nord, (Perm, Russia) has developed a new nanobiotechnology involving nanoparticles for the production of ecologically pure vegetables in greenhouses.

> ©2012 Vakhrushev et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly

©2012 Vakhrushev et al., licensee InTech. This is a paper distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

