Corrected data following equation (*x* + 0.5)0.5.

ΨMeans followed by the same lowercase letters in the column and same uppercase letters in the line do not differ by Tukey at 0.05 probability level.

**Table 10.** Inoculation with *A. brasilense* and N sources interaction in the AE in wheat crop.

Regarding AE, according to Dobbelaere et al. [23], positive responses to inoculation with *A. brasilense* are obtained even when the crops are cultivated in soils with high levels of available N, which indicates that the plant responses do not only occur due to the fixed N2 but mainly as a function of the production of growth‐promoting phytohormones such as cytokinin, gibberellin, and indole acetic acid. This fact could possibly have affected the root development of wheat, which according to Novakowiski et al. [54] would improve the efficiency of utilization of residual N, water, and other nutrients uptake, directly reflecting a greater agronomic efficiency of the wheat crop with inoculation with *A. brasilense*, as verified in the present work.

The increment in N rates in association with *A. brasilense* inoculation increases the N grain concentration up to 165 kg ha−1 N, whereas without this inoculation occurred a linear increase with lower maximum N grain concentration. That is, the inoculation afforded higher N grain concentration applying less nitrogen fertilizers in topdressing. Therefore, it can increase more

The Effect of N Fertilization on Wheat under Inoculation with *Azospirillum brasilense*

http://dx.doi.org/10.5772/intechopen.68904

181

With *A. brasilense* inoculation, the increment in N rates increases the wheat yield up to 142 kg ha−1 N, whereas without this inoculation increases occurred up to 134 kg ha−1. However, even

Inoculation with *A. brasilense* increased the agronomic efficiency, apparent N recovery, and N utilization efficiency. This research demonstrated that inoculation with *A. brasilense* associated with nitrogen fertilization in topdressing is beneficial to N nutrition and wheat yield,

For further increasing the efficiency of nitrogen fertilization, new researches of complementary inoculation with *A. brasilense* during the vegetative phase of the plant would be interesting.

Marcelo Carvalho Minhoto Teixeira Filho\*, Fernando Shintate Galindo, Salatiér Buzetti and

Universidade Estadual Paulista (UNESP), Campus de Ilha Solteira, Ilha Solteira, São Paulo

[1] Marini N, Tunes LM, Silva JI, Moraes DM, Cantos FAA. Carboxim Tiram fungicide effect in wheat seeds physiological quality (*Triticum aestivum* L.). Revista Brasileira de Ciência Agrárias. 2011;**6**:17‐22. DOI: 10.5039/agraria.v6i1a737 (in Portuguese with abstract in

[2] USDA. United States Department of Agriculture. Databases: Production, Supply and Distribution Online. 2016. Available from: http://apps.fas.usda.gov/psdonline/

[3] Zagonel J, Venancio WS, Kunz RP, Tanamati H. Nitrogen doses and plant densities with and without a growth regulator affecting wheat, cultivar OR‐1. Ciência Rural. 2002;**32**:25‐29. DOI: dx.doi.org/10.1590/S0103‐84782002000100005 (in Portuguese with

sustainably the protein content in the wheat grain.

increasing nitrogen fertilization efficiency.

Eduardo Henrique Marcandalli Boleta

[Accessed: December 23, 2016]

abstract in English)

**Author details**

State, Brazil

**References**

English)

at the highest doses, the inoculation afforded higher grain yield.

\*Address all correspondence to: mcmteixeirafilho@agr.feis.unesp.br

N sources did not differ in AE (**Table 6**), which is due in part to the similar concentrations of foliar nutrients obtained with urea and Super N and can be explained by the non‐efficacy of NBPT action due to high activity of the urease enzyme as a function of the straw of the predecessor cultures and the high temperatures that are recorded (**Figure 1**). Another possible explanation would be the uptake of a small part of the urea applied before the action of urease and NH3 formation [37]. Comparisons between several nitrogen fertilizers were made by several authors, and, in general, with satisfactory conditions of soil moisture, no differences have been found in the efficiency of these sources such as grain yield of wheat in the Cerrado for sources of N ammonium sulfonitrate, uran, and urea [36] between urea and ammonium sulfonitrate in the no‐tillage system [55] and between urea, urea + NBPT, and coated urea [56].

The efficiency of the use of N sources by annual crops, such as wheat, is low, around 50%, and the causes for this low value are related to the inadequate dose and timing of application associated with volatilization, leaching, as well as degradation, immobilization, and soil erosion [57] and differs with cultivars [58]. Thus, N fertilization strategy should aim to improve the synchronization between the season of application and the season of greater demand for the plant, in order to maximize N uptake and grain yield [59]. The improvement of N use and recovery efficiencies is desirable to increase productivity, reduce production costs, and maintain environmental quality [44].

It is worth noting that the tendency of agriculture is to seek to enrich food from the nutritional point of view, that is, to increase the availability of nutrients in the parts that will be used as food for humans and animals such as wheat grains. This research demonstrated that inoculation with *A. brasilense* associated with nitrogen fertilization in topdressing is beneficial not only to N nutrition and wheat yield but also to increase the nutritional quality of the grains more sustainably, like the protein content of this important cereal. Therefore, as the inoculation is a low‐cost technique, easy to apply and use, non‐polluting, and which falls within the desired sustainable context in actuality, the trend is that this technology can be increasingly used in wheat crops.

## **4. Conclusions**

Urea provides higher N utilization efficiency, while the Super N obtains greater LCI and recovery of the applied nitrogen, being the last one only when inoculated. However, the N sources provide similar N accumulations in straw and grains yield of wheat; thus, it is recommended to use urea at the best cost‐benefit ratio.

N leaf concentration, LCI, and N straw accumulation increase with the nitrogen fertilization increment, regardless of the N source or *A. brasilense* inoculation.

The increment in N rates in association with *A. brasilense* inoculation increases the N grain concentration up to 165 kg ha−1 N, whereas without this inoculation occurred a linear increase with lower maximum N grain concentration. That is, the inoculation afforded higher N grain concentration applying less nitrogen fertilizers in topdressing. Therefore, it can increase more sustainably the protein content in the wheat grain.

With *A. brasilense* inoculation, the increment in N rates increases the wheat yield up to 142 kg ha−1 N, whereas without this inoculation increases occurred up to 134 kg ha−1. However, even at the highest doses, the inoculation afforded higher grain yield.

Inoculation with *A. brasilense* increased the agronomic efficiency, apparent N recovery, and N utilization efficiency. This research demonstrated that inoculation with *A. brasilense* associated with nitrogen fertilization in topdressing is beneficial to N nutrition and wheat yield, increasing nitrogen fertilization efficiency.

For further increasing the efficiency of nitrogen fertilization, new researches of complementary inoculation with *A. brasilense* during the vegetative phase of the plant would be interesting.

## **Author details**

gibberellin, and indole acetic acid. This fact could possibly have affected the root development of wheat, which according to Novakowiski et al. [54] would improve the efficiency of utilization of residual N, water, and other nutrients uptake, directly reflecting a greater agronomic efficiency of the wheat crop with inoculation with *A. brasilense*, as verified in the present work. N sources did not differ in AE (**Table 6**), which is due in part to the similar concentrations of foliar nutrients obtained with urea and Super N and can be explained by the non‐efficacy of NBPT action due to high activity of the urease enzyme as a function of the straw of the predecessor cultures and the high temperatures that are recorded (**Figure 1**). Another possible explanation would be the uptake of a small part of the urea applied before the action of ure-

several authors, and, in general, with satisfactory conditions of soil moisture, no differences have been found in the efficiency of these sources such as grain yield of wheat in the Cerrado for sources of N ammonium sulfonitrate, uran, and urea [36] between urea and ammonium sulfonitrate in the no‐tillage system [55] and between urea, urea + NBPT, and coated urea [56]. The efficiency of the use of N sources by annual crops, such as wheat, is low, around 50%, and the causes for this low value are related to the inadequate dose and timing of application associated with volatilization, leaching, as well as degradation, immobilization, and soil erosion [57] and differs with cultivars [58]. Thus, N fertilization strategy should aim to improve the synchronization between the season of application and the season of greater demand for the plant, in order to maximize N uptake and grain yield [59]. The improvement of N use and recovery efficiencies is desirable to increase productivity, reduce production costs, and

It is worth noting that the tendency of agriculture is to seek to enrich food from the nutritional point of view, that is, to increase the availability of nutrients in the parts that will be used as food for humans and animals such as wheat grains. This research demonstrated that inoculation with *A. brasilense* associated with nitrogen fertilization in topdressing is beneficial not only to N nutrition and wheat yield but also to increase the nutritional quality of the grains more sustainably, like the protein content of this important cereal. Therefore, as the inoculation is a low‐cost technique, easy to apply and use, non‐polluting, and which falls within the desired sustainable context in actuality, the trend is that this technology can be increasingly

Urea provides higher N utilization efficiency, while the Super N obtains greater LCI and recovery of the applied nitrogen, being the last one only when inoculated. However, the N sources provide similar N accumulations in straw and grains yield of wheat; thus, it is recom-

N leaf concentration, LCI, and N straw accumulation increase with the nitrogen fertilization

formation [37]. Comparisons between several nitrogen fertilizers were made by

ase and NH3

180 Nitrogen in Agriculture - Updates

maintain environmental quality [44].

mended to use urea at the best cost‐benefit ratio.

increment, regardless of the N source or *A. brasilense* inoculation.

used in wheat crops.

**4. Conclusions**

Marcelo Carvalho Minhoto Teixeira Filho\*, Fernando Shintate Galindo, Salatiér Buzetti and Eduardo Henrique Marcandalli Boleta

\*Address all correspondence to: mcmteixeirafilho@agr.feis.unesp.br

Universidade Estadual Paulista (UNESP), Campus de Ilha Solteira, Ilha Solteira, São Paulo State, Brazil

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[51] Silva DRG, Costa KAP, Faquin V, de Oliveira IP, de Souza MRF, Souza MAS. Nutritional efficiency and nitrogen uptake by capim‐marandu at pasture in moderate stage of degradation under doses and sources of nitrogen. Ciência e Agrotecnologia. 2011;**35**:242‐249. DOI: http://dx.doi.org/10.1590/S1413‐70542011000200003 (in Portuguese with abstract in English)

**Chapter 10**

**Provisional chapter**

**Nitrogen Use Efficiency in Rice**

Shuangjie Huang, Chunfang Zhao,

Yali Zhang and Cailin Wang

**Nitrogen Use Efficiency in Rice**

Additional information is available at the end of the chapter

improve NUE, is a major objective in the future.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69052

Cailin Wang

**Abstract**

N remobilization

**1. Introduction**

Shuangjie Huang, Chunfang Zhao, Yali Zhang and

DOI: 10.5772/intechopen.69052

Food security is a major global issue because of the growing population and decreasing land area. Rice (*Oryza sativa* L.) is the most important staple cereal crop in the world. Application of nitrogen (N) fertilizer has improved crop yield in the world during the past five decades but with considerable negative impacts on the environment. New solutions are therefore urgently needed to simultaneously increase yields while maintaining or preferably decreasing applied N to maximize the nitrogen use efficiency (NUE) of crops. Plant NUE is inherently complex with each step (including N uptake, translocation, assimilation, and remobilization) governed by multiple interacting genetic and environmental factors. Based on the current knowledge, we propose some possible approaches enhancing NUE, by molecular manipulation selecting candidate genes and agricultural integrated management practices for NUE improvement. Developing an integrated research program combining approaches, mainly based on whole-plant physiology, quantitative genetics, forward and reverse genetics, and agronomy approaches to

**Keywords:** rice, nitrogen use efficiency, nitrate, ammonium, N uptake, N assimilation,

The global population is predicted to reach 9 billion, and food supplies are projected to increase by 70–100% by 2050 [1, 2]. Given the limited capacity for arable land expansion, it requires sustaining yield improvement in existing land to meet the increasing food demand [3]. Rice is one of the staple food crops for approximately half of the global population. Therefore, rice production must be increased significantly to satisfy the requirements of the growing world population. However, we are facing challenges in increasing rice production under

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is 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.

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


## **Chapter 10**

**Provisional chapter**

## **Nitrogen Use Efficiency in Rice**

**Nitrogen Use Efficiency in Rice**

Shuangjie Huang, Chunfang Zhao, Yali Zhang and Cailin Wang Yali Zhang and Cailin Wang Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69052

Shuangjie Huang, Chunfang Zhao,

#### **Abstract**

[51] Silva DRG, Costa KAP, Faquin V, de Oliveira IP, de Souza MRF, Souza MAS. Nutritional efficiency and nitrogen uptake by capim‐marandu at pasture in moderate stage of degradation under doses and sources of nitrogen. Ciência e Agrotecnologia. 2011;**35**:242‐249. DOI: http://dx.doi.org/10.1590/S1413‐70542011000200003 (in Portuguese with abstract in

[52] Sant'Ana EVP, dos Santos AB, da Silveira PM. The efficiency of use of nitrogen applied in top dressing in irrigated bean. Revista Brasileira de Engenharia Agrícola e Ambiental. 2011;**15**;458‐462. DOI: http://www.scielo.br/pdf/rbeaa/v15n5/v15n5a04.pdf

[53] Dupas E, Buzetti S, Rabêlo FHS, Sarto AL, Cheng NC, Teixeira Filho MCM, Galindo FS, Dinalli RP, Gazola RN. Nitrogen recovery, use efficiency, dry matter yield, and chemical composition of palisade grass fertilized with nitrogen sources in the Cerrado biome. Australian Journal of Crop Science. 2016;**10**:1330‐1338. DOI: 10.21475/ajcs.2016.10.09.

[54] Novakowiski JH, Sandini IE, Falbo MK, Moraes A, Novakowiski JH, Cheng NC. Residual effect of nitrogen fertilization and *Azospirillum brasilense* inoculation in the maize culture. Semina: Ciências Agrárias. 2011;**32**:1687‐1698. DOI: dx.doi.org/10.5433/1679‐

[55] Silva SA, Arf O, Buzetti S, Silva MG. Nitrogen sources and application times in no‐till wheat on Cerrado soil. Revista Brasileira de Ciência do Solo. 2008;**32**:2717‐2722. DOI: http://dx.doi.org/10.1590/S0100‐06832008000700015 (in Portuguese with abstract in

[56] Prando AM, Zucarelli C, Fronza V, Oliveira FA, Oliveira Júnior A. Productive characteristics of wheat according to nitrogen sources and levels. Pesquisa Agropecuária Tropical. 2013;**43**:34‐41. DOI: http://dx.doi.org/10.1590/S1983‐40632013000100009 (in Portuguese

[57] Fageria NK, Baligar VC. Enhancing nitrogen use efficiency in crop plants. Advances in Agronomy. 2005;**88**:97‐185. DOI: http://dx.doi.org/10.1016/S0065‐2113(05)88004‐6 [58] Furtini IV, Ramalho MAP, Abreu AFB, Furtini Neto AEF. Differential response of breeding lines of common bean to nitrogen. Ciência Rural. 2006;**36**:1696‐1700. DOI: http:// dx.doi.org/10.1590/S0103‐84782006000600005 (in Portuguese with abstract in English)

[59] dos Santos AB, Fageria NK. Physiological characteristics of common bean in tropical varzea soils as affected by rate and nitrogen management. Ciência e Agrotecnologia. 2008;**32**:23‐31. DOI: http://www.scielo.br/pdf/cagro/v32n1/a03v32n1.pdf (in Portuguese

0359.2011v32n4Sup1p1687 (in Portuguese with abstract in English)

English)

186 Nitrogen in Agriculture - Updates

p7854

English)

with abstract in English)

with abstract in English)

(in Portuguese with abstract in English)

Food security is a major global issue because of the growing population and decreasing land area. Rice (*Oryza sativa* L.) is the most important staple cereal crop in the world. Application of nitrogen (N) fertilizer has improved crop yield in the world during the past five decades but with considerable negative impacts on the environment. New solutions are therefore urgently needed to simultaneously increase yields while maintaining or preferably decreasing applied N to maximize the nitrogen use efficiency (NUE) of crops. Plant NUE is inherently complex with each step (including N uptake, translocation, assimilation, and remobilization) governed by multiple interacting genetic and environmental factors. Based on the current knowledge, we propose some possible approaches enhancing NUE, by molecular manipulation selecting candidate genes and agricultural integrated management practices for NUE improvement. Developing an integrated research program combining approaches, mainly based on whole-plant physiology, quantitative genetics, forward and reverse genetics, and agronomy approaches to improve NUE, is a major objective in the future.

DOI: 10.5772/intechopen.69052

**Keywords:** rice, nitrogen use efficiency, nitrate, ammonium, N uptake, N assimilation, N remobilization

## **1. Introduction**

The global population is predicted to reach 9 billion, and food supplies are projected to increase by 70–100% by 2050 [1, 2]. Given the limited capacity for arable land expansion, it requires sustaining yield improvement in existing land to meet the increasing food demand [3]. Rice is one of the staple food crops for approximately half of the global population. Therefore, rice production must be increased significantly to satisfy the requirements of the growing world population. However, we are facing challenges in increasing rice production under

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. © 2018 The Author(s). Licensee InTech. This chapter is 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.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

the pressures of decreased arable land area, global climate change, intensified natural disasters, and frequent occurrence of diseases and pests [4]. Nitrogen (N) is one of the essential macroelements required for plant growth and development. Soil N availability usually limits plant yields in most agricultural cropping systems [5]. Thus, application of N fertilizer has become an important, cost-effective strategy to increase crop yields in intensive agricultural systems worldwide [6]. However, traditionally adding N fertilizer to improve crop yields may have reached a plateau. Excessive application of nitrogen fertilizer may not result in yield improvements but will lead to serious environmental problems [7, 8]. From 1960 to 2012, the global N fertilizer consumption increased by 800%, and the annual N consumption in China increased from 8 to 35% of the world's N consumption [4]. Although the rate of cereal grain yield increased by 65% between 1980 and 2010, the consumption of chemical fertilizers increased by 512% [9]. High N fertilizer input leads to low nitrogen use efficiency (NUE) due to the rapid N losses from ammonia volatilization, denitrification, surface runoff, and leaching in the soil-flood water system. As a consequence, significant environmental problems (i.e., soil acidification, air pollution, water eutrophication) occurred [10–12]. To achieve further high crop productivity and high NUE under well-fertilized conditions, new solutions are urgently needed to increase yields while maintaining or preferably decreasing applied N [13].

In this chapter, we outlined the definition of NUE, the genes related to NUE, as well as the effect of the factors on the expression of those genes, with an emphasis on rice research. Based on the current knowledge, we proposed some possible strategies enhancing NUE, by breeding, molecular manipulation selecting candidate genes, and developing a range of optimized crop management practices for NUE improvement.

**3. Genes responsible for nitrogen use efficiency**

**Table 1.** Some definitions of NUE mostly used with respect to nitrogen.

**Abbreviation Term Definitions**

NUE N use efficiency NUpE × NUtE = yield/N available

NUEg N use efficiency of grain Grain production/available N

UI Utilization index Total plant biomass/total plant N

FUE Fertilizer use efficiency (NUp/N applied) × 100

NUpE N uptake efficiency NUp/Nav (soil + fertilizer) = acquired N/N available NUtE N utilization efficiency Yield/NUp (assimilation of plant N to produce grain)

AE Agronomy N efficiency Net increased yield of the plant with and without N

NpUE N physiological use efficiency Net increased yield/net increased N uptake with and

HI Harvest index Grain weight(vegetative organ weight + grain weight)

NHI Nitrogen harvest index Grain N accumulation/total N accumulation in aboveground

NTE N transport efficiency Total N transported into the aboveground parts/total N in

the whole plant

ANR Apparent N recovery rate Net increased total N uptake by the plant with and without

N fertilization/total amount of fertilizer N

fertilization/total amount of fertilizer N

without application of fertilizer N

biomass (e.g., grain + straw)

sink leaves or developing grains (seeds)

N remobilization from source or senescent leaves/that of

rapid nitrification on their surface, and thus absorb N as NO<sup>3</sup>

uptake [17, 18]. Direct molecular evidence for NO<sup>3</sup>

or directly from NH4

N is available for plants as nitrate (NO3

NRE Nitrogen remobilization efficiency

−

NH4 +

NH4 + or NO3 −

derived from NO3

Generally, NUE can be divided into two parts: assimilation efficiency involved in N uptake and assimilation, and utilization efficiency involved in N remobilization. Understanding the mechanisms regulating these processes is crucial for improving crop NUE. In soil, inorganic

wetland or acidic soils. Rice roots in paddy soils release oxygen via their aerenchyma, generate

nitrate transporters (NAR2/NRTs) is assimilated in the roots, the other larger part transported to the shoots, where it is reduced to ammonium by a range of enzymes (**Figure 1**). The NH4

ilated into amino acids via the glutamine synthetase (GS)/glutamine-2-oxoglutarate aminotransferase (GOGAT) cycle and then is exported to sink organs [14]. Therefore, regulating gene function in N metabolism processes including N uptake, assimilation, compartmenta-

) in aerobic uplands and ammonium (NH4

−

uptake by ammonium transporters (AMTs) is assim-

−

uptake by roots commonly results in acidification or alkalization of the rhizo-

+

Nitrogen Use Efficiency in Rice

189

http://dx.doi.org/10.5772/intechopen.69052

−

at a rate comparable with that of

uptake in rice has been presented [19].

) in flooded

taken up by

+

−

sphere, which in turn changes the soil N availability [14]. For many plants, some NO<sup>3</sup>

+

tion, translocation, and remobilization may be essential for improving NUE.

## **2. Defining nitrogen use efficiency**

NUE is inherently complex determined by the interaction of multiple genes with the environment factors. A number of different definitions and calculations of NUE include N utilization, N content, and N availability as NUE equation components (**Table 1**) [13, 14]. In general, plant NUE comprises two key components: N uptake efficiency (NUpE), which is the efficiency of absorption/uptake of supplied N, and N utilization efficiency (NUtE), which is the efficiency of assimilation and remobilization of plant N to ultimately produce grain [13, 14]. The simplest definition of plant NUE is the grain yield per unit of supplied N, also an integration of NUpE and NUtE. Another method to describe NUE is the utilization index (UI), which means the absolute amount of biomass produced per unit of N. NUE can also be described as NUEg, which is grain production per unit of N available, and HI, which is grain production of the total plant biomass. However, a crop plant could produce large amounts of biomass per unit N (high UI) without converting the acquired N to seed production and therefore have a low NUEg and HI. There are other NUE calculations taking various agronomic and physiological variations into account described elsewhere [14–16]. In summary, improving NUE could be achieved by improving either NUpE, NUtE, or both. However, owing to the fluctuations in the rhizosphere that influenced by microorganism, root exudates, and the volatile loss of gaseous N from the soil/plant canopy, it is difficult to quantify the "real" amount of N fertilizer available or actually acquired by plants.


**Table 1.** Some definitions of NUE mostly used with respect to nitrogen.

the pressures of decreased arable land area, global climate change, intensified natural disasters, and frequent occurrence of diseases and pests [4]. Nitrogen (N) is one of the essential macroelements required for plant growth and development. Soil N availability usually limits plant yields in most agricultural cropping systems [5]. Thus, application of N fertilizer has become an important, cost-effective strategy to increase crop yields in intensive agricultural systems worldwide [6]. However, traditionally adding N fertilizer to improve crop yields may have reached a plateau. Excessive application of nitrogen fertilizer may not result in yield improvements but will lead to serious environmental problems [7, 8]. From 1960 to 2012, the global N fertilizer consumption increased by 800%, and the annual N consumption in China increased from 8 to 35% of the world's N consumption [4]. Although the rate of cereal grain yield increased by 65% between 1980 and 2010, the consumption of chemical fertilizers increased by 512% [9]. High N fertilizer input leads to low nitrogen use efficiency (NUE) due to the rapid N losses from ammonia volatilization, denitrification, surface runoff, and leaching in the soil-flood water system. As a consequence, significant environmental problems (i.e., soil acidification, air pollution, water eutrophication) occurred [10–12]. To achieve further high crop productivity and high NUE under well-fertilized conditions, new solutions are urgently needed to increase yields while maintaining or preferably decreasing applied N [13]. In this chapter, we outlined the definition of NUE, the genes related to NUE, as well as the effect of the factors on the expression of those genes, with an emphasis on rice research. Based on the current knowledge, we proposed some possible strategies enhancing NUE, by breeding, molecular manipulation selecting candidate genes, and developing a range of optimized

NUE is inherently complex determined by the interaction of multiple genes with the environment factors. A number of different definitions and calculations of NUE include N utilization, N content, and N availability as NUE equation components (**Table 1**) [13, 14]. In general, plant NUE comprises two key components: N uptake efficiency (NUpE), which is the efficiency of absorption/uptake of supplied N, and N utilization efficiency (NUtE), which is the efficiency of assimilation and remobilization of plant N to ultimately produce grain [13, 14]. The simplest definition of plant NUE is the grain yield per unit of supplied N, also an integration of NUpE and NUtE. Another method to describe NUE is the utilization index (UI), which means the absolute amount of biomass produced per unit of N. NUE can also be described as NUEg, which is grain production per unit of N available, and HI, which is grain production of the total plant biomass. However, a crop plant could produce large amounts of biomass per unit N (high UI) without converting the acquired N to seed production and therefore have a low NUEg and HI. There are other NUE calculations taking various agronomic and physiological variations into account described elsewhere [14–16]. In summary, improving NUE could be achieved by improving either NUpE, NUtE, or both. However, owing to the fluctuations in the rhizosphere that influenced by microorganism, root exudates, and the volatile loss of gaseous N from the soil/plant canopy, it is difficult to quantify the "real" amount of N fertilizer

crop management practices for NUE improvement.

**2. Defining nitrogen use efficiency**

188 Nitrogen in Agriculture - Updates

available or actually acquired by plants.

## **3. Genes responsible for nitrogen use efficiency**

Generally, NUE can be divided into two parts: assimilation efficiency involved in N uptake and assimilation, and utilization efficiency involved in N remobilization. Understanding the mechanisms regulating these processes is crucial for improving crop NUE. In soil, inorganic N is available for plants as nitrate (NO3 − ) in aerobic uplands and ammonium (NH4 + ) in flooded wetland or acidic soils. Rice roots in paddy soils release oxygen via their aerenchyma, generate rapid nitrification on their surface, and thus absorb N as NO<sup>3</sup> − at a rate comparable with that of NH4 + uptake [17, 18]. Direct molecular evidence for NO<sup>3</sup> − uptake in rice has been presented [19]. NH4 + or NO3 − uptake by roots commonly results in acidification or alkalization of the rhizosphere, which in turn changes the soil N availability [14]. For many plants, some NO<sup>3</sup> − taken up by nitrate transporters (NAR2/NRTs) is assimilated in the roots, the other larger part transported to the shoots, where it is reduced to ammonium by a range of enzymes (**Figure 1**). The NH4 + derived from NO3 − or directly from NH4 + uptake by ammonium transporters (AMTs) is assimilated into amino acids via the glutamine synthetase (GS)/glutamine-2-oxoglutarate aminotransferase (GOGAT) cycle and then is exported to sink organs [14]. Therefore, regulating gene function in N metabolism processes including N uptake, assimilation, compartmentation, translocation, and remobilization may be essential for improving NUE.

developed transporters for both nitrate and ammonium. These transporters are divided into high-affinity transporter system (HATS) and low-affinity transport system (LATS) [20]. Under low nitrogen concentrations (<1 mM), HATS mediates most of the N uptake, while under high concentrations of N (>1 mM), LATS plays roles in N uptake [21, 22]. Each high- and low-affinity nitrate transport system is composed of constitutive and nitrate-inducible components (cHATS and iHATS), respectively [20, 23]. So far, four families of nitrate transporters/channels have been identified: nitrate transporter 1/peptide transporter family (NPF, also known as the NRT1/PTR family), nitrate transporter 2 family (NRT2), the chloride channel family (CLC),

Nitrogen Use Efficiency in Rice

191

http://dx.doi.org/10.5772/intechopen.69052

In rice, two transporter families NPF and NRT2 (or NAR2/NRT2) for uptake and translocation of nitrate have been identified (**Table 2** and **Figure 1**) [14, 25, 26]. At least 80 genes belong to NPF family in rice genome [27]. Most NPF family members characterized so far are low-affinity nitrate transporters, except that OsNPF6.5 (NRT1.1b) showed dual-affinity nitrate transport activity, associated with enhancing nitrate uptake and root-to-shoot transport [28]. OsNPF6.5, considered as a putative mRNA splicing product of OsNPF8.9 (NRT1/ NRT1.1/NRT1.1a), has a significant impact on both NUE and yield [26–29]. OsNPF8.9, mainly expressed in root epidermis and hairs, has been cloned contribution to N uptake [30]. The role of OsNPF4.1 (SP1) has been demonstrated to function in rice panicle elongation [31] and OsNPF8.20 (OsPTR9) function in ammonium uptake, promotion of lateral root formation, and increased grain yield [32]. However, their substrates are still unknown. Eight peptide transporters, OsPTR1 (OsNPF8.2), OsPTR2 (OsNPF2.2), OsPTR3 (OsNPF5.5), OsPTR4 (OsNPF7.1), OsPTR5 (OsNPF7.4), OsPTR6 (OsNPF7.3), OsPTR7 (OsNPF8.1), and OsPTR8 (OsNPF8.5), were investigated in a yeast ptr2 mutant strain, and their expression patterns were evaluated in plants. Only OsNPF7.3 transports Gly-His and Gly-His-Gly, showing substrate selectivity for di-/tripeptides. However, the other seven proteins did not transport the five tested di-/tripeptides [33]. Elevated expression of *OsNPF7.3* promoted rice growth through increasing ammonium transporter expression and glutamine synthetase activity [34]. Recently, OsNPF2.4 [35] and OsNPF2.2 [36] involved in long-distance root-to-shoot nitrate transport have been identified. Knockout of *OsNPF2.4* impaired potassium (K)-coupled nitrate upward transport and nitrate redistribution from old leaves to other organs [35]. OsNPF2.2 can unload nitrate from the xylem affecting the root-to-shoot nitrate transport and plant development [36]. In addition, a tonoplast-localized low-affinity nitrate transporter OsNPF7.2 has been characterized playing a pivotal role in intracellular allocation of nitrate in roots [37]. To date, five *NRT2s* (*OsNRT2.1/2.2/2.3a/2.3b/2.4*) and two *NAR2s* (*OsNAR2.1/2.2*) genes encoding HATS components have been identified in rice, each showing different expression and regulation patterns (**Table 2**) [19, 38]. Among the five *OsNRT2s* genes, *OsNRT2.1* and *OsNRT2.2* share an identical coding region sequence with different 5′- and 3′-untranscribed regions [38–40]. *OsNRT2.3a* and *OsNRT2.3b* are derived from the alternative splicing of *OsNRT2.3* [38]. *OsNRT2.3a* is mainly expressed in the xylem parenchyma of root participating in long-distance nitrate transport from root to shoot at low nitrate concentrations [41]. *OsNRT2.3b* is mainly expressed in the phloem of shoot, sensitive to pH. Elevated expression of *OsNRT2.3b* increased N, Fe, and P uptake and improved grain yield and NUE [42]. *OsNAR2.1*, *OsNRT2.1*, and *OsNRT2.2* were expressed abundantly throughout the primary and lateral roots. Overexpression of *OsNRT2.1*

and slow anion channel-associated homologues (SLAC/SLAH) [24].

**Figure 1.** Schematic representation of characterized and predicted functions of the rice nitrate transporters of NRT and NPF families, ammonium transporters of AMT families, and nitrogen assimilation proteins of GS and GOGAT.

#### **3.1. Nitrogen acquisition**

Owing to the heterogeneity and dynamic variations of nitrate and ammonium concentrations, which range from lower than 100 μM to higher than 10 mM in soil solutions, plants have developed transporters for both nitrate and ammonium. These transporters are divided into high-affinity transporter system (HATS) and low-affinity transport system (LATS) [20]. Under low nitrogen concentrations (<1 mM), HATS mediates most of the N uptake, while under high concentrations of N (>1 mM), LATS plays roles in N uptake [21, 22]. Each high- and low-affinity nitrate transport system is composed of constitutive and nitrate-inducible components (cHATS and iHATS), respectively [20, 23]. So far, four families of nitrate transporters/channels have been identified: nitrate transporter 1/peptide transporter family (NPF, also known as the NRT1/PTR family), nitrate transporter 2 family (NRT2), the chloride channel family (CLC), and slow anion channel-associated homologues (SLAC/SLAH) [24].

In rice, two transporter families NPF and NRT2 (or NAR2/NRT2) for uptake and translocation of nitrate have been identified (**Table 2** and **Figure 1**) [14, 25, 26]. At least 80 genes belong to NPF family in rice genome [27]. Most NPF family members characterized so far are low-affinity nitrate transporters, except that OsNPF6.5 (NRT1.1b) showed dual-affinity nitrate transport activity, associated with enhancing nitrate uptake and root-to-shoot transport [28]. OsNPF6.5, considered as a putative mRNA splicing product of OsNPF8.9 (NRT1/ NRT1.1/NRT1.1a), has a significant impact on both NUE and yield [26–29]. OsNPF8.9, mainly expressed in root epidermis and hairs, has been cloned contribution to N uptake [30]. The role of OsNPF4.1 (SP1) has been demonstrated to function in rice panicle elongation [31] and OsNPF8.20 (OsPTR9) function in ammonium uptake, promotion of lateral root formation, and increased grain yield [32]. However, their substrates are still unknown. Eight peptide transporters, OsPTR1 (OsNPF8.2), OsPTR2 (OsNPF2.2), OsPTR3 (OsNPF5.5), OsPTR4 (OsNPF7.1), OsPTR5 (OsNPF7.4), OsPTR6 (OsNPF7.3), OsPTR7 (OsNPF8.1), and OsPTR8 (OsNPF8.5), were investigated in a yeast ptr2 mutant strain, and their expression patterns were evaluated in plants. Only OsNPF7.3 transports Gly-His and Gly-His-Gly, showing substrate selectivity for di-/tripeptides. However, the other seven proteins did not transport the five tested di-/tripeptides [33]. Elevated expression of *OsNPF7.3* promoted rice growth through increasing ammonium transporter expression and glutamine synthetase activity [34]. Recently, OsNPF2.4 [35] and OsNPF2.2 [36] involved in long-distance root-to-shoot nitrate transport have been identified. Knockout of *OsNPF2.4* impaired potassium (K)-coupled nitrate upward transport and nitrate redistribution from old leaves to other organs [35]. OsNPF2.2 can unload nitrate from the xylem affecting the root-to-shoot nitrate transport and plant development [36]. In addition, a tonoplast-localized low-affinity nitrate transporter OsNPF7.2 has been characterized playing a pivotal role in intracellular allocation of nitrate in roots [37]. To date, five *NRT2s* (*OsNRT2.1/2.2/2.3a/2.3b/2.4*) and two *NAR2s* (*OsNAR2.1/2.2*) genes encoding HATS components have been identified in rice, each showing different expression and regulation patterns (**Table 2**) [19, 38]. Among the five *OsNRT2s* genes, *OsNRT2.1* and *OsNRT2.2* share an identical coding region sequence with different 5′- and 3′-untranscribed regions [38–40]. *OsNRT2.3a* and *OsNRT2.3b* are derived from the alternative splicing of *OsNRT2.3* [38]. *OsNRT2.3a* is mainly expressed in the xylem parenchyma of root participating in long-distance nitrate transport from root to shoot at low nitrate concentrations [41]. *OsNRT2.3b* is mainly expressed in the phloem of shoot, sensitive to pH. Elevated expression of *OsNRT2.3b* increased N, Fe, and P uptake and improved grain yield and NUE [42]. *OsNAR2.1*, *OsNRT2.1*, and *OsNRT2.2* were expressed abundantly throughout the primary and lateral roots. Overexpression of *OsNRT2.1*

**3.1. Nitrogen acquisition**

190 Nitrogen in Agriculture - Updates

Owing to the heterogeneity and dynamic variations of nitrate and ammonium concentrations, which range from lower than 100 μM to higher than 10 mM in soil solutions, plants have

**Figure 1.** Schematic representation of characterized and predicted functions of the rice nitrate transporters of NRT and NPF families, ammonium transporters of AMT families, and nitrogen assimilation proteins of GS and GOGAT.


gene alone did not increase nitrate uptake in rice [43], owing to that the nitrate uptake activity of *OsNRT2.1*, *OsNRT2.2*, and *OsNRT2.3a* requires a partner protein, OsNAR2.1 [19, 38, 44]. The transcripts of *OsNAR2.2* and *OsNRT2.4* were detected in roots and shoots, accumulation

**Accession no. Gene Regulation Expression pattern Substrates References**

<sup>+</sup> Root central cylinder and cell surface of root tips

of primary root

Root exodermis, sclerenchyma, endodermis, and pericycle cells

<sup>+</sup> Leaves NH4

<sup>+</sup> Roots NH4

premature leaf blade, spikelet at the early stage of ripening

<sup>+</sup> Unknown Unknown [46, 55]

NH4

NH4

<sup>+</sup> [46, 50]

http://dx.doi.org/10.5772/intechopen.69052

<sup>+</sup> [46]

<sup>+</sup> [46]

, Glu [58, 59]

, Glu [58, 59]

, Glu [58, 60]

Gln, 2-OG [60, 61]

Gln, 2-OG [60, 61, 68]

, Glu [58, 59, 64]

+

+

+

+

<sup>+</sup> [46, 47, 50, 53]

Nitrogen Use Efficiency in Rice

193

AF289478 *OsAMT1;2* NH4

NM 190445 *OsAMT2;2* NO3

AB037664 *OsGS1;1* NH4

AB180688 *OsGS1;2* NH4

*GOGAT1*

*GOGAT2*

AB008845 *OsNADH-*

AB274818 *OsNADH-*

AF289479 *OsAMT1;3* Repressed,

circadian rhythm

> − , NH<sup>4</sup>

AB051864 *OsAMT2;1* Unknown Constitutive expression NH4

AB083582 *OsAMT3;1* Unknown Roots, shoots NH4

AB180689 *OsGS1;3* Unknown Spikelets NH4

X14246 *OsGS2* Unknown Leaves NH4

NH4 +

NH4

NM\_001051237 *OsAMT2;3* Unknown Unknown Unknown [46]

AC104487 *OsAMT3;2* Unknown Unknown Unknown [46] AP004775 *OsAMT3;3* Unknown Unknown Unknown [46] AC091811 *OsAMT4* Unknown Unknown Unknown [46]

AB024716 *OsFd-GOGAT* Light Shoots Gln, 2-OG [60, 61]

, Gln Developing tissues: root tip,

<sup>+</sup> Mature leaf blade and sheath: phloem companion and parenchyma cells

Ammonium uptake is mainly mediated by proteins of the ammonia transport protein (AMT)/ transports methylammonium (MEP)/rhesus (RH) superfamily [45]. There are uncertainties regarding the exact chemical species transported by AMT, which can be in the form of either hydrophobic NH3 or charged ammonium [14, 45]. The activity of AMT members may play a more important role in NUpE in ammonium-preferring rice than in nitrate-utilizing crops. In rice, there are at least ten putative *OsAMT*-like genes grouped into four subfamilies (i.e*.,* three each for *OsAMT1*, *OsAMT2*, and *OsAMT3*, respectively, and one for *OsAMT4*) (**Table 2**) [46]. So far, studies on expression regulation of *AMT* genes in rice are mainly focused on *OsAMT1* gene family, which displayed different spatiotemporal expression patterns in response to changes in N levels or daily irradiance (**Table 2**) [47, 48]. *OsAMT1;1* is constitutively expressed

induced by nitrate [38–40]. However, their functions remain unknown.

**Table 2.** Literature summary of the tissue expression and regulation of genes responsible for NUE.


**Accession no. Gene Regulation Expression pattern Substrates References**

NO3

NO3

NO3

NO3

NO3

Gly-His-Gly

Unknown [32]

Unknown [31]

<sup>−</sup> [38–40]

<sup>−</sup> [38–40]

<sup>−</sup> [38, 41]

<sup>−</sup> [38, 42]

<sup>+</sup> [46, 48, 50, 52]

<sup>−</sup> [29, 30]

<sup>−</sup> [28, 29]

<sup>−</sup> [35]

<sup>−</sup> [36]

<sup>−</sup> [37]

[33, 34]

roots

cells

xylem

<sup>−</sup> Root hairs, epidermis, and vascular tissues

> parenchyma, and phloem companion cells, leaf phloem

> Parenchyma cells around the

<sup>−</sup> Root, seeds Gly-His

tips, cortical fiber cells of lateral

Root tip, meristem NO3

Root tip, meristem NO3

Root stele NO3

Shoot phloem NO3

Constitutive expression NH4

Root, shoot Unknown [38–40]

Root epidermal cells Unknown [19, 38–40]

Seeds, leaf, panicle Unknown [33]

<sup>−</sup> Root sclerenchyma, cortex, and stele cells

roots, stems

AK101055 *OsNPF5.5* Unknown Seeds, leaf Unknown [33] AK101099 *OsNPF7.1* Unknown Constitutive expression Unknown [33] AK070216 *OsNPF7.4* Drought, salt Root, panicle, node Unknown [33] AK070036 *OsNPF8.1* Drought, salt Shoot, leaf, panicle, seeds Unknown [33] AK072691 *OsNPF8.5* Drought, salt Constitutive expression Unknown [33]

young panicles

<sup>−</sup> Root epidermis, xylem

AF140606 *OsNPF8.9* Unknown Constitutively expressed in

− , drought,

AK064899 *OsNPF8.20* N, light Leaves, panicles, young root

AK100802 *OsNPF4.1* Unknown Phloem of the branches of

cold

− , light, sucrose

− , light, sucrose

− , light, sucrose

− , light, sucrose, pH, NAA

− , light, sucrose

+ , circadian rhythm

AK109571 *OsNAR2.2* Light, sucrose Root, shoot None [19, 38, 39]

pH

salt

AK066920 *OsNPF6.5* NO3

192 Nitrogen in Agriculture - Updates

AK099321.1 *OsNPF2.4* NO3

AK068351 *OsNPF2.2* NO3

XM\_015767550 *OsNPF7.2* NO3

AK101480 *OsNPF7.3* NO3

AK100112 *OsNPF8.2* Drought, salt,

AB008519 *OsNRT2.1* NO3

AK109733 *OsNRT2.2* NO3

AK109776 *OsNRT2.3a* NO3

NM\_193361 *OsNRT2.4* NO3

NM\_001053852.2 *OsNAR2.1* NO3

AF289477 *OsAMT1;1* NH4

AK072215 *OsNRT2.3b* Light, sucrose,

**Table 2.** Literature summary of the tissue expression and regulation of genes responsible for NUE.

gene alone did not increase nitrate uptake in rice [43], owing to that the nitrate uptake activity of *OsNRT2.1*, *OsNRT2.2*, and *OsNRT2.3a* requires a partner protein, OsNAR2.1 [19, 38, 44]. The transcripts of *OsNAR2.2* and *OsNRT2.4* were detected in roots and shoots, accumulation induced by nitrate [38–40]. However, their functions remain unknown.

Ammonium uptake is mainly mediated by proteins of the ammonia transport protein (AMT)/ transports methylammonium (MEP)/rhesus (RH) superfamily [45]. There are uncertainties regarding the exact chemical species transported by AMT, which can be in the form of either hydrophobic NH3 or charged ammonium [14, 45]. The activity of AMT members may play a more important role in NUpE in ammonium-preferring rice than in nitrate-utilizing crops. In rice, there are at least ten putative *OsAMT*-like genes grouped into four subfamilies (i.e*.,* three each for *OsAMT1*, *OsAMT2*, and *OsAMT3*, respectively, and one for *OsAMT4*) (**Table 2**) [46]. So far, studies on expression regulation of *AMT* genes in rice are mainly focused on *OsAMT1* gene family, which displayed different spatiotemporal expression patterns in response to changes in N levels or daily irradiance (**Table 2**) [47, 48]. *OsAMT1;1* is constitutively expressed in rice roots and shoots showing a positive feedback regulation by endogenous glutamine [49]. It has been reported that *OsAMT1;1*, showing a higher expression level in roots under ammonium supply, contributes to NH<sup>4</sup> + uptake and plays an important role in NK homeostasis [48, 50–52]. *OsAMT1;2* showed root-specific expression, is induced by ammonium, and may function as a nitrogen assimilator [49, 53]. Root-specific and nitrogen-derepressible expression for *OsAMT1;3* may function as a nitrogen sensor [49, 53]. Overexpression *OsAMT1;3* displayed significant decreases in growth but with poor nitrogen uptake ability, accompanied with a higher leaf C/N ratio [54]. *OsAMT2;1* showed constitutive expression in both roots and shoots, and *OsAMT3;1* showed very weak expression in roots and shoots [46]. *OsAMT2;2* is evenly expressed in roots and shoots and is induced by nitrogen [55].

of glutamine generation in senescing leaves for the remobilization of leaf nitrogen through phloem to the panicle during natural senescence. *OsNADH-GOGAT2* mutants had marked

Although these observed phenotypes and those observed for GS enzymes have been identified, the interaction between isozymes of GOGAT and the GS isozymes, how they affect NUE, as well as posttranscriptional regulation of these enzymes needs to be further investigated.

During the vegetative stage, the leaves are a sink for N; later, during senescence, this N is remobilized for reuse in the developing seeds, mainly as amino acids (**Figure 1**) [69]. Up to 95% of seed protein is derived from amino acids that are exported to the seed after the degradation of existing proteins in leaves [14], and the rest is supplemented from the soil and late top-dressed fertilizers [70]. Gln and asparagine (Asn) are major forms of total amino acids in phloem and xylem sap of rice plants [14, 71]. Increases of both Asn and Gln concentrations during senescence in the phloem sap suggest their key role in rendering N available for remobilization from the senescing leaves. Some isoforms of GS1, NADH-glutamate dehydrogenase (GDH), and asparagine synthetase (AS) are strongly activated during N remobilization [72]. The nature of the amino acid transporters, belonging to complex multigene families, is

poorly understood in phloem loading for N redistribution during senescence [69].

important in the development of active tillers through the assimilation of NH4

expressed in root surface (epidermis, exodermis, and sclerenchyma) in an NH<sup>4</sup>

is apparently coupled with the primary assimilation of NH4

against aromatic amino acids [77].

The importance of GS/GOGAT activity in N remobilization, reassimilation, growth rate, yield, and grain filling has been emphasized previously. OsGS1;1 and OsNADH-GOGAT2 are important in remobilization of nitrogen during natural senescence [62]. GS1;2 is also

during lignin synthesis [64]. Together with GS, AS is believed to play a crucial role in primary N metabolism, catalyzing the formation of Asn and Glu from Gln and aspartate [14, 64]. There are two genes (i.e., *OsAS1* and *OsAS2*) identified encoding AS in rice. *OsAS1* is mainly

manner, which are very similar with *OsGS1;2* and *NADH-GOGAT1* in rice roots. Thus, AS1

phloem companion and parenchyma cells [71, 73] is abundant in leaf blades and sheathes, along with the GS1;1 protein [61]. These suggest that AS2 in rice leaves is probably important in the long-distance transport of asparagine from rice leaves during natural senescence. In addition, the mitochondrial GDH plays a major role in reassimilation of photorespiratory ammonia and can alternatively incorporate ammonium into Glu in response to high levels of ammonium under stress [72]. Although there are a large number of amino acid permeases (AAPs) presented in rice [74, 75], no transporters have been functionally characterized with an exception for *OsAAP6,* which is mainly expressed in seeds for grain protein content [76]. Recently, the transport function of four rice AAP genes (*OsAAP1*, *OsAAP3*, *OsAAP7*, and *OsAAP16*) has been analyzed by expression in *Xenopus laevis oocytes*, electrophysiology, and cellular localization. *OsAAP1*, *OsAAP7*, and *OsAAP16* functioned as general AAPs and could transport all amino acids well except aspartate and β-alanine. While *OsAAP3* had a distinct substrate specificity transporting the basic amino acids lysine and arginine well but selected

+

+

Nitrogen Use Efficiency in Rice

195

http://dx.doi.org/10.5772/intechopen.69052

+

in rice roots. *OsAS2* detected in

generated


reduction in spikelet number per panicle [62, 68].

**3.3. Nitrogen remobilization and reassimilation**

#### **3.2. Nitrogen assimilation**

After taken up by the roots, nitrate is assimilated in the roots, the other larger part transported to the shoots, where it is first reduced to nitrite catalyzed by nitrate reductase (NR) in the cytoplasm and then further to ammonium by nitrite reductase (NiR) in the plastids. The ammonium derived from nitrate or directly from ammonium uptake by AMTs is finally assimilated into amino acids via the GS/GOGAT cycle (**Figure 1**) [14, 22]. GOGAT catalyzes the transfer of the amide group of glutamine (Gln) formed by GS to 2-oxoglutarate (2-OG) to yield two molecules of glutamate (Glu). One of the Gln molecules can be cycled back as a substrate for the GS reaction, and the other can be used for many synthetic reactions [56, 57].

Rice possesses three homologous but distinct genes for cytosolic glutamine synthetase (i.e., *OsGS1;1*, *OsGS1;2*, and *OsGS1;3*) and one chloroplastic gene (*OsGS2*). *OsGS1;1* and *OsGS1;2* both showed a high substrate affinity for ammonium and were induced by ammonium within the central cylinder of rice-elongating zone [58]. *OsGS1;1* was constitutively expressed , with higher expression profile in leaf blade and participated in rice normal growth and grain filling [59, 60]. *OsGS1;1* also functions in coordinating the global metabolic network in rice plants grown using ammonium as the nitrogen source [60] and is important for remobilization of nitrogen during natural senescence [61, 62]. *OsGS1;2* is constitutively expressed in surface cells of roots responsible for the primary assimilation of ammonium, and knockout of *OsGS1;2* showed severe reduction in active tiller number [63]. However, Ohashi et al. thought that the reduction in tiller number is an NH4 + -specific event and the outgrowth of the axillary buds was severely suppressed caused by metabolic disorder in *OsGS1;2* mutants [64]. *OsGS1;3* is exclusively expressed in spikelet [59], indicating that it is probably important in grain ripening and/ or germination. The OsGS2 subunit protein was present in leaves but was hardly detectable in roots [58]. There is also a small gene family for GOGAT: one ferredoxin (Fd)-dependent type and two NADH-dependent types [65]. *OsFd-GOGAT* is highly abundant in mesophyll cells and other chloroplast-containing cells regulated by light [56] and is important in reassimilation of ammonium generated by photorespiration in chloroplasts [65]. Recently, participating in nitrogen assimilation, C/N balance, [66], leaf senescence, and the nitrogen remobilization has been reported [67]. *OsNADH-GOGAT1* is mainly expressed in surface cells of rice roots in an NH4 + dependent manner and is important for primary ammonium assimilation in roots at the seedling stage and development of active tiller number until the harvest [62, 65]. *OsNADH-GOGAT2* is mainly expressed in vascular tissues of mature leaf blades and is important in the process of glutamine generation in senescing leaves for the remobilization of leaf nitrogen through phloem to the panicle during natural senescence. *OsNADH-GOGAT2* mutants had marked reduction in spikelet number per panicle [62, 68].

Although these observed phenotypes and those observed for GS enzymes have been identified, the interaction between isozymes of GOGAT and the GS isozymes, how they affect NUE, as well as posttranscriptional regulation of these enzymes needs to be further investigated.

#### **3.3. Nitrogen remobilization and reassimilation**

in rice roots and shoots showing a positive feedback regulation by endogenous glutamine [49]. It has been reported that *OsAMT1;1*, showing a higher expression level in roots under ammo-

[48, 50–52]. *OsAMT1;2* showed root-specific expression, is induced by ammonium, and may function as a nitrogen assimilator [49, 53]. Root-specific and nitrogen-derepressible expression for *OsAMT1;3* may function as a nitrogen sensor [49, 53]. Overexpression *OsAMT1;3* displayed significant decreases in growth but with poor nitrogen uptake ability, accompanied with a higher leaf C/N ratio [54]. *OsAMT2;1* showed constitutive expression in both roots and shoots, and *OsAMT3;1* showed very weak expression in roots and shoots [46]. *OsAMT2;2* is

After taken up by the roots, nitrate is assimilated in the roots, the other larger part transported to the shoots, where it is first reduced to nitrite catalyzed by nitrate reductase (NR) in the cytoplasm and then further to ammonium by nitrite reductase (NiR) in the plastids. The ammonium derived from nitrate or directly from ammonium uptake by AMTs is finally assimilated into amino acids via the GS/GOGAT cycle (**Figure 1**) [14, 22]. GOGAT catalyzes the transfer of the amide group of glutamine (Gln) formed by GS to 2-oxoglutarate (2-OG) to yield two molecules of glutamate (Glu). One of the Gln molecules can be cycled back as a substrate for

Rice possesses three homologous but distinct genes for cytosolic glutamine synthetase (i.e., *OsGS1;1*, *OsGS1;2*, and *OsGS1;3*) and one chloroplastic gene (*OsGS2*). *OsGS1;1* and *OsGS1;2* both showed a high substrate affinity for ammonium and were induced by ammonium within the central cylinder of rice-elongating zone [58]. *OsGS1;1* was constitutively expressed , with higher expression profile in leaf blade and participated in rice normal growth and grain filling [59, 60]. *OsGS1;1* also functions in coordinating the global metabolic network in rice plants grown using ammonium as the nitrogen source [60] and is important for remobilization of nitrogen during natural senescence [61, 62]. *OsGS1;2* is constitutively expressed in surface cells of roots responsible for the primary assimilation of ammonium, and knockout of *OsGS1;2* showed severe reduction in active tiller number [63]. However, Ohashi et al. thought that the

severely suppressed caused by metabolic disorder in *OsGS1;2* mutants [64]. *OsGS1;3* is exclusively expressed in spikelet [59], indicating that it is probably important in grain ripening and/ or germination. The OsGS2 subunit protein was present in leaves but was hardly detectable in roots [58]. There is also a small gene family for GOGAT: one ferredoxin (Fd)-dependent type and two NADH-dependent types [65]. *OsFd-GOGAT* is highly abundant in mesophyll cells and other chloroplast-containing cells regulated by light [56] and is important in reassimilation of ammonium generated by photorespiration in chloroplasts [65]. Recently, participating in nitrogen assimilation, C/N balance, [66], leaf senescence, and the nitrogen remobilization has been reported [67]. *OsNADH-GOGAT1* is mainly expressed in surface cells of rice roots in an NH4

dependent manner and is important for primary ammonium assimilation in roots at the seedling stage and development of active tiller number until the harvest [62, 65]. *OsNADH-GOGAT2* is mainly expressed in vascular tissues of mature leaf blades and is important in the process

the GS reaction, and the other can be used for many synthetic reactions [56, 57].

+

uptake and plays an important role in NK homeostasis


+ -

+

evenly expressed in roots and shoots and is induced by nitrogen [55].

nium supply, contributes to NH<sup>4</sup>

194 Nitrogen in Agriculture - Updates

**3.2. Nitrogen assimilation**

reduction in tiller number is an NH4

During the vegetative stage, the leaves are a sink for N; later, during senescence, this N is remobilized for reuse in the developing seeds, mainly as amino acids (**Figure 1**) [69]. Up to 95% of seed protein is derived from amino acids that are exported to the seed after the degradation of existing proteins in leaves [14], and the rest is supplemented from the soil and late top-dressed fertilizers [70]. Gln and asparagine (Asn) are major forms of total amino acids in phloem and xylem sap of rice plants [14, 71]. Increases of both Asn and Gln concentrations during senescence in the phloem sap suggest their key role in rendering N available for remobilization from the senescing leaves. Some isoforms of GS1, NADH-glutamate dehydrogenase (GDH), and asparagine synthetase (AS) are strongly activated during N remobilization [72]. The nature of the amino acid transporters, belonging to complex multigene families, is poorly understood in phloem loading for N redistribution during senescence [69].

The importance of GS/GOGAT activity in N remobilization, reassimilation, growth rate, yield, and grain filling has been emphasized previously. OsGS1;1 and OsNADH-GOGAT2 are important in remobilization of nitrogen during natural senescence [62]. GS1;2 is also important in the development of active tillers through the assimilation of NH4 + generated during lignin synthesis [64]. Together with GS, AS is believed to play a crucial role in primary N metabolism, catalyzing the formation of Asn and Glu from Gln and aspartate [14, 64]. There are two genes (i.e., *OsAS1* and *OsAS2*) identified encoding AS in rice. *OsAS1* is mainly expressed in root surface (epidermis, exodermis, and sclerenchyma) in an NH<sup>4</sup> + -dependent manner, which are very similar with *OsGS1;2* and *NADH-GOGAT1* in rice roots. Thus, AS1 is apparently coupled with the primary assimilation of NH4 + in rice roots. *OsAS2* detected in phloem companion and parenchyma cells [71, 73] is abundant in leaf blades and sheathes, along with the GS1;1 protein [61]. These suggest that AS2 in rice leaves is probably important in the long-distance transport of asparagine from rice leaves during natural senescence. In addition, the mitochondrial GDH plays a major role in reassimilation of photorespiratory ammonia and can alternatively incorporate ammonium into Glu in response to high levels of ammonium under stress [72]. Although there are a large number of amino acid permeases (AAPs) presented in rice [74, 75], no transporters have been functionally characterized with an exception for *OsAAP6,* which is mainly expressed in seeds for grain protein content [76]. Recently, the transport function of four rice AAP genes (*OsAAP1*, *OsAAP3*, *OsAAP7*, and *OsAAP16*) has been analyzed by expression in *Xenopus laevis oocytes*, electrophysiology, and cellular localization. *OsAAP1*, *OsAAP7*, and *OsAAP16* functioned as general AAPs and could transport all amino acids well except aspartate and β-alanine. While *OsAAP3* had a distinct substrate specificity transporting the basic amino acids lysine and arginine well but selected against aromatic amino acids [77].

## **4. Enhancing nitrogen use efficiency**

As mentioned above, molecular studies have provided a general validation of the physiological conceptual framework of NUE in rice. However, besides genetics, there are other factors needed to consider such as the interactions between N uptake and water availability, the interaction between N utilization and carbon metabolism, and the interaction between different macronutrients and micronutrients [13]. Understanding the mechanisms regulating nitrogen movement in rice is crucial for improvement of NUE. Improvements in NUE result from NUpE, NUtE, or both. We describe approaches for increasing NUE with special consideration to genetics and agricultural management.

Water is another key factor determining crop yield and NUE. Without sufficient water, plants cannot extract nutrients from the soil. Yield is constrained by moisture availability, not N availability, especially in maize [89]. In contrast to upland crops, alternate wetting and drying (AWD, flooding the soil and then allowing to dry down before being reflooded) to reduce total water for irrigation in rice has been developed for a number of decades. A number of studies have shown that AWD increases grain yield when compared to continuous flooding

Nitrogen Use Efficiency in Rice

197

http://dx.doi.org/10.5772/intechopen.69052

On the base of current knowledge, scientists have developed a range of optimized crop management practices, such as site-specific nutrient management (SSNM) [92], real-time N management (RTNM) [93], and preliminary integrated precision rice management (PRM) system combining SSNM with alternate drying and wetting irrigation and optimized transplanting density [94]. Only integrated N management strategies are allowed for the achievement of production goals while minimizing the risk of environmental pollution. Sources of N and timing of application determine the most suitable method for application. The interest in implementing new knowledge about the methods of application is to develop sensors to diagnose the N status of crops in real time throughout large areas and decision support systems to help

A number of physiological traits can affect the NUtE in crops, including the effect of N on carbohydrate partitioning, the storage of N, and the remobilization of N from senescent tissues, and these have been subdivided into a number of components by researchers [95, 96].

Increasing nitrogen utilization capacity can be achieved through overexpression of candidate genes in the pathways relating to N assimilation, translocation, remobilization, and reassimilation. As mentioned above, changes in the expression and activity of GS and GOGAT would have an effect on N assimilation, recycle, reassimilation, C/N balance, and senescence in rice, potentially affecting grain filling, yield, and NUE [62, 64, 66]. Identifying candidate genes cosegregate with NUE in genetic crosses is another efficient method. One of the first QTL studies conducted analyzing NUE in rice was carried out [97]. They looked at QTLs associated with NUE and determined whether they cosegregated with GS1 and NADH-GOGAT. The analysis identified seven loci that cosegregated with GS1 activity and six loci that cosegregated with NADH-GOGAT activity. A number of QTLs for agronomic traits related to N use and yield have been mapped to the chromosomal regions containing GS2 in rice [97, 98], suggesting that the genomic region surrounding GS2 may be valuable for breeding rice with improved agronomic performance and NUE. However, to date, no one has been able to introduce a GS gene into a NUE-inefficient background and show either

C and N metabolisms are tightly linked with each other in plants. N assimilation requires carbon metabolism to provide adenosine triphosphate (ATP), reductants, and C skeletons through photosynthesis, photorespiration, and respiration. Large amounts of N are used in photosynthesis, particularly during ribulose 1,5-bisphosphate carboxylase-oxygenase

(CF) [90, 91].

determine N fertilizer recommendations [88].

**4.2. Increasing utilization efficiency**

enhanced NUE or yield.

#### **4.1. Increasing uptake capacity**

Increased nitrogen uptake capacity may be achieved through better nitrogen transporters, more effective regulation of the transport systems, or better storage and assimilation. A simple example to improve NUpE would be to increase uptake by overexpressing more efficient transporters or all the transporters using transgenic methods [28, 42, 48, 78]. However, only increasing the uptake capacity of roots is not simple because of the tight regulation of N uptake, N taken up surplus to requirements increasing plant N status, which, in turn, leads to feedback regulation and reduction in uptake capacity [20].

Physiological traits that may also affect NUpE including root architecture and any other characteristic play a pivotal role in extracting available N from the soil [13, 79]. The capacity of the root for uptake depends on the degree to which the root extends and its absorption area, which is determined by complex root morphology. A common example is to target genes related to root morphology through a mapping approach, whereby traits are identified through genetic crosses using distinct populations, and then quantitative trait loci (QTLs) can be cloned by positional cloning [79–81]. To date, studies have been carried out to identify root morphological features such as root mass and depth, root axis length, and lateral branching related to NUE [82–84].

However, ammonium or nitrate uptake by rice roots commonly results in acidification or alkalization of the rhizosphere, which in turn changes the soil N availability for plants. In the rhizosphere, rice roots can also release oxygen and exudates that greatly influence local redox potential and the density and activity of microbial populations, which in turn can interconvert soil N forms, including those derived from fertilizer [14]. Thus, soil N availability fluctuating greatly in both space and time affects root morphology, which could make plants uptake N efficiently [14]. Studies in rice have been confirmed that compared to sole NH<sup>4</sup> + nutrition, a mixture of NH<sup>4</sup> + and NO3 − promoted root growth as well as N absorption and assimilation [85, 86]. In the course of agricultural management, fertilizer type (i.e., controlled N release fertilizers, new potential N sources), methods of applying N fertilizers (e.g., the 4R nutrient stewardship framework: right source, right rate, right time, and right placement), soil types, tillage, transplanting density, cropping system, and microorganisms are governed to avoid nitrogen loss increasing fertilizer nitrogen use efficiency [87, 88].

Water is another key factor determining crop yield and NUE. Without sufficient water, plants cannot extract nutrients from the soil. Yield is constrained by moisture availability, not N availability, especially in maize [89]. In contrast to upland crops, alternate wetting and drying (AWD, flooding the soil and then allowing to dry down before being reflooded) to reduce total water for irrigation in rice has been developed for a number of decades. A number of studies have shown that AWD increases grain yield when compared to continuous flooding (CF) [90, 91].

On the base of current knowledge, scientists have developed a range of optimized crop management practices, such as site-specific nutrient management (SSNM) [92], real-time N management (RTNM) [93], and preliminary integrated precision rice management (PRM) system combining SSNM with alternate drying and wetting irrigation and optimized transplanting density [94]. Only integrated N management strategies are allowed for the achievement of production goals while minimizing the risk of environmental pollution. Sources of N and timing of application determine the most suitable method for application. The interest in implementing new knowledge about the methods of application is to develop sensors to diagnose the N status of crops in real time throughout large areas and decision support systems to help determine N fertilizer recommendations [88].

#### **4.2. Increasing utilization efficiency**

**4. Enhancing nitrogen use efficiency**

196 Nitrogen in Agriculture - Updates

to genetics and agricultural management.

feedback regulation and reduction in uptake capacity [20].

+

and NO3

avoid nitrogen loss increasing fertilizer nitrogen use efficiency [87, 88].

−

**4.1. Increasing uptake capacity**

related to NUE [82–84].

nutrition, a mixture of NH<sup>4</sup>

As mentioned above, molecular studies have provided a general validation of the physiological conceptual framework of NUE in rice. However, besides genetics, there are other factors needed to consider such as the interactions between N uptake and water availability, the interaction between N utilization and carbon metabolism, and the interaction between different macronutrients and micronutrients [13]. Understanding the mechanisms regulating nitrogen movement in rice is crucial for improvement of NUE. Improvements in NUE result from NUpE, NUtE, or both. We describe approaches for increasing NUE with special consideration

Increased nitrogen uptake capacity may be achieved through better nitrogen transporters, more effective regulation of the transport systems, or better storage and assimilation. A simple example to improve NUpE would be to increase uptake by overexpressing more efficient transporters or all the transporters using transgenic methods [28, 42, 48, 78]. However, only increasing the uptake capacity of roots is not simple because of the tight regulation of N uptake, N taken up surplus to requirements increasing plant N status, which, in turn, leads to

Physiological traits that may also affect NUpE including root architecture and any other characteristic play a pivotal role in extracting available N from the soil [13, 79]. The capacity of the root for uptake depends on the degree to which the root extends and its absorption area, which is determined by complex root morphology. A common example is to target genes related to root morphology through a mapping approach, whereby traits are identified through genetic crosses using distinct populations, and then quantitative trait loci (QTLs) can be cloned by positional cloning [79–81]. To date, studies have been carried out to identify root morphological features such as root mass and depth, root axis length, and lateral branching

However, ammonium or nitrate uptake by rice roots commonly results in acidification or alkalization of the rhizosphere, which in turn changes the soil N availability for plants. In the rhizosphere, rice roots can also release oxygen and exudates that greatly influence local redox potential and the density and activity of microbial populations, which in turn can interconvert soil N forms, including those derived from fertilizer [14]. Thus, soil N availability fluctuating greatly in both space and time affects root morphology, which could make plants uptake N efficiently [14]. Studies in rice have been confirmed that compared to sole NH<sup>4</sup>

assimilation [85, 86]. In the course of agricultural management, fertilizer type (i.e., controlled N release fertilizers, new potential N sources), methods of applying N fertilizers (e.g., the 4R nutrient stewardship framework: right source, right rate, right time, and right placement), soil types, tillage, transplanting density, cropping system, and microorganisms are governed to

promoted root growth as well as N absorption and

+

A number of physiological traits can affect the NUtE in crops, including the effect of N on carbohydrate partitioning, the storage of N, and the remobilization of N from senescent tissues, and these have been subdivided into a number of components by researchers [95, 96].

Increasing nitrogen utilization capacity can be achieved through overexpression of candidate genes in the pathways relating to N assimilation, translocation, remobilization, and reassimilation. As mentioned above, changes in the expression and activity of GS and GOGAT would have an effect on N assimilation, recycle, reassimilation, C/N balance, and senescence in rice, potentially affecting grain filling, yield, and NUE [62, 64, 66]. Identifying candidate genes cosegregate with NUE in genetic crosses is another efficient method. One of the first QTL studies conducted analyzing NUE in rice was carried out [97]. They looked at QTLs associated with NUE and determined whether they cosegregated with GS1 and NADH-GOGAT. The analysis identified seven loci that cosegregated with GS1 activity and six loci that cosegregated with NADH-GOGAT activity. A number of QTLs for agronomic traits related to N use and yield have been mapped to the chromosomal regions containing GS2 in rice [97, 98], suggesting that the genomic region surrounding GS2 may be valuable for breeding rice with improved agronomic performance and NUE. However, to date, no one has been able to introduce a GS gene into a NUE-inefficient background and show either enhanced NUE or yield.

C and N metabolisms are tightly linked with each other in plants. N assimilation requires carbon metabolism to provide adenosine triphosphate (ATP), reductants, and C skeletons through photosynthesis, photorespiration, and respiration. Large amounts of N are used in photosynthesis, particularly during ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) and light-harvesting complexes to support the light-dependent use of CO2 , inorganic N, and water to produce sugars, amino acids, and organic acids [99]. Photorespiration, a side reaction of photosynthesis, has crucial implications in N reassimilation, which is catalyzed by the Rubisco. During photorespiration, NH<sup>4</sup> + is produced during methylenetetrahydrofolate synthesis from glycine [100]. Respiration is a third fundamental process of energy metabolism in the dark and in nonphotosynthetic tissues, as well as in the light. In the respiratory pathways, the C skeletons for N assimilation are generated in different sectors, such as the oxidative pentose phosphate pathway (OPPP), glycolysis, and TCA cycle [101]. The operation of the TCA cycle in illuminated leaves is critical for the provision of 2-OG, which is necessary for glutamate and glutamine production [101–103]. Evidence has shown that the synthesis of 2-OG is induced by the activity of phosphoenolpyruvate carboxylase (PEPC), citrate synthase, isocitrate dehydrogenase, and aconitase, while the subsequent conversion of 2-OG to fumarate may be repressed in the light [101].

**5. Conclusions**

of N and C metabolism.

**Acknowledgements**

**Author details**

Shuangjie Huang<sup>1</sup>

**References**

uptake, assimilation, and remobilization.

approaches to improve NUE, is a major objective in the future.

\*, Chunfang Zhao<sup>1</sup>

\*Address all correspondence to: huangdeifan@163.com

Agriculture, Nanjing Agricultural University, China

Plant NUE is a complex trait determined by quantitative trait loci and influenced by environmental changes and is the integration of NUpE and NUtE. There is a complex regulation of N

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Enhanced NUE can be achieved by genetically modifying plants and integrated agricultural management practices. The former is the most effective biotechnological method for increasing NUE. This can be achieved by overexpression of nitrate and ammonium transporters responsible for N uptake by roots and by manipulation of key genes controlling the balance

Developing an integrated research program combining approaches, mainly based on whole-plant physiology, quantitative genetics, forward and reverse genetics, and agronomy

We thank Dr. Chang Li for his comments on **Figure 1** and for his helpful discussions on this timely topic. We apologize to all colleagues whose work could not be cited owing for space limitations. This work is supported by grants from China's Agriculture Research System

(#CARS-01-47) and the National Key Technology Support Program (2015BAD01B02).

, Yali Zhang<sup>2</sup>

1 Institute of Food Crops of Jiangsu Academy of Agricultural Sciences, Nanjing, China

2 State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of

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and Cailin Wang<sup>1</sup>

Thus, exploiting candidate genes involved in C/N metabolism is another approach to improve NUE. To date, there are two key genes identified to contribute to NUE in rice. Chloroplastic proteins are known to make up approximately 80% of the stored N in leaf tissues, with Rubisco accounting for up to 50% and 20% of the stored N in C3 and C4 plants, respectively [104]. Thus, Rubisco is an excellent N storage molecule, and its autophagic degradation in rice leaves may contribute to an efficient and rapid N remobilization by facilitating protein degradation for N mobilization in senescent leaves [70]. Rubisco is also involved in photorespiratory losses which can be as high as 20% of the total carbon fixation in C<sup>3</sup> plants and also liberates ammonia, which is required for reassimilation [105]. However, when rice plants overexpressing the Rubisco (rbcS) gene were analyzed, Rubisco-N to leaf-N increased, but there was no change in the rate of photosynthesis [106]. PEPC is a component of primary metabolism in plants and has a nonphotosynthetic role as one of its products is OAA, a component of the TCA cycle [107]. RNAi knockdown experiments of the chloroplastic isoform in rice have indicated that PEPC plays an important role in N assimilation, specifically when the main N source is NH4 + [108].

Growth and yield of rice plants are markedly affected by increased CO<sup>2</sup> concentration and temperature [109, 110]. Numerous studies have indicated that an increase in CO2 generally stimulates photosynthesis, reduces stomatal conductance, and changes the rhizosphere conditions of plants, leading to increases in biomass and yield of crops [111–113], whereas an increase in temperature accelerates crop phenological development and shortens grainfilling period of crops, leading to decrease grain yield and reduce crop production in many regions of the world [114, 115]. Furthermore, high temperature, if occurring at critical stages of crop development (such as meiosis and flowering stages), reduces spikelet fertility [115]. Owing to elevated CO2 under future climate change is associated with an increase in air temperature, many studies about plant response to the interaction of CO<sup>2</sup> and temperature have been reported [109, 110, 116]. Increases in CO2 were unable to compensate for the negative impact of increases in temperature on biomass and yield in rice [109, 110]. Thus, selecting high-temperature-tolerant germplasm will be required to realize yield benefits in the future.

## **5. Conclusions**

, inor-

is produced during methylenetetrahy-

and C4

under future climate change is associated with an increase

plants, respectively

concentration and

generally

and tem-

were unable to compensate

plants and

(Rubisco) and light-harvesting complexes to support the light-dependent use of CO2

lyzed by the Rubisco. During photorespiration, NH<sup>4</sup>

2-OG to fumarate may be repressed in the light [101].

main N source is NH4

198 Nitrogen in Agriculture - Updates

ity [115]. Owing to elevated CO2

efits in the future.

+ [108].

Rubisco accounting for up to 50% and 20% of the stored N in C3

ganic N, and water to produce sugars, amino acids, and organic acids [99]. Photorespiration, a side reaction of photosynthesis, has crucial implications in N reassimilation, which is cata-

drofolate synthesis from glycine [100]. Respiration is a third fundamental process of energy metabolism in the dark and in nonphotosynthetic tissues, as well as in the light. In the respiratory pathways, the C skeletons for N assimilation are generated in different sectors, such as the oxidative pentose phosphate pathway (OPPP), glycolysis, and TCA cycle [101]. The operation of the TCA cycle in illuminated leaves is critical for the provision of 2-OG, which is necessary for glutamate and glutamine production [101–103]. Evidence has shown that the synthesis of 2-OG is induced by the activity of phosphoenolpyruvate carboxylase (PEPC), citrate synthase, isocitrate dehydrogenase, and aconitase, while the subsequent conversion of

Thus, exploiting candidate genes involved in C/N metabolism is another approach to improve NUE. To date, there are two key genes identified to contribute to NUE in rice. Chloroplastic proteins are known to make up approximately 80% of the stored N in leaf tissues, with

[104]. Thus, Rubisco is an excellent N storage molecule, and its autophagic degradation in rice leaves may contribute to an efficient and rapid N remobilization by facilitating protein degradation for N mobilization in senescent leaves [70]. Rubisco is also involved in photo-

also liberates ammonia, which is required for reassimilation [105]. However, when rice plants overexpressing the Rubisco (rbcS) gene were analyzed, Rubisco-N to leaf-N increased, but there was no change in the rate of photosynthesis [106]. PEPC is a component of primary metabolism in plants and has a nonphotosynthetic role as one of its products is OAA, a component of the TCA cycle [107]. RNAi knockdown experiments of the chloroplastic isoform in rice have indicated that PEPC plays an important role in N assimilation, specifically when the

respiratory losses which can be as high as 20% of the total carbon fixation in C<sup>3</sup>

Growth and yield of rice plants are markedly affected by increased CO<sup>2</sup>

temperature [109, 110]. Numerous studies have indicated that an increase in CO2

in air temperature, many studies about plant response to the interaction of CO<sup>2</sup>

perature have been reported [109, 110, 116]. Increases in CO2

stimulates photosynthesis, reduces stomatal conductance, and changes the rhizosphere conditions of plants, leading to increases in biomass and yield of crops [111–113], whereas an increase in temperature accelerates crop phenological development and shortens grainfilling period of crops, leading to decrease grain yield and reduce crop production in many regions of the world [114, 115]. Furthermore, high temperature, if occurring at critical stages of crop development (such as meiosis and flowering stages), reduces spikelet fertil-

for the negative impact of increases in temperature on biomass and yield in rice [109, 110]. Thus, selecting high-temperature-tolerant germplasm will be required to realize yield ben-

+

Plant NUE is a complex trait determined by quantitative trait loci and influenced by environmental changes and is the integration of NUpE and NUtE. There is a complex regulation of N uptake, assimilation, and remobilization.

Enhanced NUE can be achieved by genetically modifying plants and integrated agricultural management practices. The former is the most effective biotechnological method for increasing NUE. This can be achieved by overexpression of nitrate and ammonium transporters responsible for N uptake by roots and by manipulation of key genes controlling the balance of N and C metabolism.

Developing an integrated research program combining approaches, mainly based on whole-plant physiology, quantitative genetics, forward and reverse genetics, and agronomy approaches to improve NUE, is a major objective in the future.

## **Acknowledgements**

We thank Dr. Chang Li for his comments on **Figure 1** and for his helpful discussions on this timely topic. We apologize to all colleagues whose work could not be cited owing for space limitations. This work is supported by grants from China's Agriculture Research System (#CARS-01-47) and the National Key Technology Support Program (2015BAD01B02).

## **Author details**

Shuangjie Huang<sup>1</sup> \*, Chunfang Zhao<sup>1</sup> , Yali Zhang<sup>2</sup> and Cailin Wang<sup>1</sup>

\*Address all correspondence to: huangdeifan@163.com

1 Institute of Food Crops of Jiangsu Academy of Agricultural Sciences, Nanjing, China

2 State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Nanjing Agricultural University, China

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[99] Zhu XG, Long SP, Ort DR. What is the maximum efficiency with which photosynthesis can convert solar energy into biomass?. Current Opinion in Biotechnology. 2008;**19**:153-

[100] Guo SW, Yi Z, Gao YX. New insights into the nitrogen form effect on photosynthesis and photorespiration. Pedosphere. 2007;**17**:601-610. DOI: 10.1016/S1002-0160(07)60071-X [101] Nunesnesi A, Fernie AR, Stitt M. Metabolic and signaling aspects underpinning the regulation of plant carbon nitrogen interactions. Molecular Plant. 2010;**3**:973-996. DOI:

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[106] Suzuki YJ, Ohkubo M, Hatakeyama H. Increased Rubisco content in transgenic rice transformed with the 'sense' rbcS gene. Plant & Cell Physiology. 2007;**48**:626-637. DOI:

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interaction with rising temperature or nitrogen supply: A meta-analysis. Climatic

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**Chapter 11**

**Provisional chapter**

**Prospects of N Fertilization in Medicinal Plants**

**Prospects of N Fertilization in Medicinal Plants** 

DOI: 10.5772/intechopen.68165

High global demand for plant-derived medicines is threatening the existence of many wild indigenous plant species. However, the high demand of medicinal plants has also created huge business opportunities in commercial farming of medicinal plants. Largescale production of secondary metabolites by plants and medicinal materials will be crucial in the medicinal plant industry. As commercial cultivation of medicinal plants gains traction among farmers, N fertilizers will be increasingly used to enhance plant growth and yield. Therefore, the implementation of better nitrogen use efficiency is critically important. Excessive use of N can lead to many problems; it is costly, it can cause environmental pollution and its high levels in plant tissues can be toxic to plants, herbivores and humans. This chapter discusses the potential risks, opportunities and setbacks asso-

**Keywords:** nitrogen fertilizer, medicinal plants, toxicity, yield, secondary metabolite

Exploitation of plant resources for the treatment of human and animal diseases has placed significant pressure on plant biodiversity. It has been reported that more than 3.5 billion people in the developing world rely on plants as components of their primary health care [1]. However, the use of medicinal plants is not only limited to the developing world, in fact demand for herbal medicine is also rising in many developed countries, for example, in Germany, it is estimated that 600–700 plant-based medicines are available and prescribed by 70% of physicians [2]. The demand for plant-derived medicines has created a large business in indigenous plants in South Africa, which is estimated to be worth R270 million annually [3, 4].

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is 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.

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

**Cultivation**

**Cultivation**

Charles P. Laubscher

**Abstract**

**1. Introduction**

Charles P. Laubscher

Felix Nchu, Yonela Matanzima and

Felix Nchu, Yonela Matanzima and

http://dx.doi.org/10.5772/intechopen.68165

Additional information is available at the end of the chapter

ciated with the use of N in cultivation of medicinal plants.

Additional information is available at the end of the chapter


**Provisional chapter**

## **Prospects of N Fertilization in Medicinal Plants Cultivation Cultivation**

**Prospects of N Fertilization in Medicinal Plants** 

DOI: 10.5772/intechopen.68165

Felix Nchu, Yonela Matanzima and Charles P. Laubscher Charles P. Laubscher Additional information is available at the end of the chapter

Felix Nchu, Yonela Matanzima and

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68165

#### **Abstract**

[110] Cai C, Yin XY, He SQ. Responses of wheat and rice to factorial combinations of ambi-

[111] Ainsworth EA, Rogers A. The response of photosynthesis and stomatal conductance to

[112] Rajkumar M, Prasad MNV, Swaminathan S. Climate change driven plant-metal-microbe interactions. Environment International. 2013;**53**:74-86. DOI: 10.1016/j.envint.2012.12.009

ronmental factors. Frontiers in Plant Science 2016;**7**:657. DOI: 10.3389/fpls.2016.00657

[114] Wheeler TR, Craufurd PQ, Ellis RH. Temperature variability and the yield of annual crops. Agriculture Ecosystems & Environment. 2000;**82**:159-167. DOI: 10.1016/S0167-8809(00)

[115] Jagadish SVK, Craufurd PQ, Wheeler TR. High temperature stress and spikelet fertility in rice (*Oryza sativa* L.). Journal of Experimental Botany. 2007;**58**:1627-1635. DOI:

ature. Current Opinion in Plant Biology. 2016;**31**:36-43. DOI: 10.1016/j.pbi.2016.03.006

and temperature in face experiments. Global Change Biology.

response of stomata and its dependence on envi-

O, N, and temper-

and interactions with H2

]: Mechanisms and environmental interactions. Plant Cell & Environment.

ent and elevated CO2

rising [CO2

208 Nitrogen in Agriculture - Updates

00224-3

10.1093/jxb/erm003

2016;**22**:856-874. DOI: 10.1111/gcb.13065

[113] Xu ZZ, Jiang YL, Jia BR. Elevated-CO<sup>2</sup>

[116] Kimball BA. Crop responses to elevated CO<sup>2</sup>

2007;**30**:258-270. DOI: 10.1111/j.1365-3040.2007.01641.x

High global demand for plant-derived medicines is threatening the existence of many wild indigenous plant species. However, the high demand of medicinal plants has also created huge business opportunities in commercial farming of medicinal plants. Largescale production of secondary metabolites by plants and medicinal materials will be crucial in the medicinal plant industry. As commercial cultivation of medicinal plants gains traction among farmers, N fertilizers will be increasingly used to enhance plant growth and yield. Therefore, the implementation of better nitrogen use efficiency is critically important. Excessive use of N can lead to many problems; it is costly, it can cause environmental pollution and its high levels in plant tissues can be toxic to plants, herbivores and humans. This chapter discusses the potential risks, opportunities and setbacks associated with the use of N in cultivation of medicinal plants.

**Keywords:** nitrogen fertilizer, medicinal plants, toxicity, yield, secondary metabolite

## **1. Introduction**

Exploitation of plant resources for the treatment of human and animal diseases has placed significant pressure on plant biodiversity. It has been reported that more than 3.5 billion people in the developing world rely on plants as components of their primary health care [1]. However, the use of medicinal plants is not only limited to the developing world, in fact demand for herbal medicine is also rising in many developed countries, for example, in Germany, it is estimated that 600–700 plant-based medicines are available and prescribed by 70% of physicians [2]. The demand for plant-derived medicines has created a large business in indigenous plants in South Africa, which is estimated to be worth R270 million annually [3, 4].

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. © 2018 The Author(s). Licensee InTech. This chapter is 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.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

In South Africa alone, there are some 27 million indigenous medicinal consumers [5]. The wellknown examples of plant species that are currently traded in South Africa include *Artemisia afra* (Asteraceae), *Melianthus comosus* (Melianthaceae), *Aloe ferox* (Asphodelaceae), *Aloe arborescens* (Asphodelaceae), *Salvia Africana-caerulea* (Lamiaceae) and *Helichrysum cymosum* (Asteraceae) [6].

used in traditional practices worldwide and their use has been increasing steadily. Medicinal plants constitute an important component of health care systems, globally. The trade of medicinal plants is estimated to be worth R270 million annually [3]. According to Sobiecke [11], globally, products that are derived from traditional medicine are estimated to be worth R2.9 billion per year. On the demand for medicinal plants is increasing worldwide and it is estimated up to 700,000 tonnes of plant material are consumed annually to the value of about 150 million US dollars [4]. The World Health Organization estimates that 21,000 species are used for medicinal purposes around the world and in India 150 species are used commercially [12]. In Zimbabwe, herbal medicine is the most affordable and easily accessible form of treatment in primary health care and up to 93 medicinal plant species are used in the southcentral region of Zimbabwe [13]. In Pakistan, more than 500 species of plants are used in herbal medicine [14]. Street and Prinsloo [15] presented 10 highly used South African medicinal plants, such as *Agathosma betulina* (Rutaceae), *A. ferox* (Asphodelaceae), *Aspalathus linearis* (Fabaceae), *Harpagophytum procumbens* (Pedaliaceae), *Hypoxis hemerocallidea* (Hypoxidaceae), *Merwilla natalensis* (Hyacinthaceae), *Pelargonium sidoides* (Geraniaceae), *Siphonochilus aethiopicus* (Zingiberaceae) and *Sutherlandia frutescens* (Fabaceae) in a review paper. Although some critics have argued that traditionally it is not acceptable to use cultivated medicinal plants, a recent report on the perception of cultivation of medicinal plant species indicated that very high proportions (over 69%) of respondents are willing to buy and make use of cultivated medicinal plants [16]. This trend suggests that developing efficient and sustainable agro-tech-

Prospects of N Fertilization in Medicinal Plants Cultivation

http://dx.doi.org/10.5772/intechopen.68165

211

Cultivation of medicinal plants is gaining momentum among subsistence and commercial farmers [17]. Farming of medicinal plants has many advantages, for examples it can contribute to job creation and improvement of household earnings, and it can reduce overexploitation and harvesting of some wild and endangered species. Similar to the cultivation of food crops, medicinal plant cultivation programmes should have specific goals, which include to increase medicinal plant yield and plant growth rate, increase and standardized quality and quality of secondary metabolites produced and reduce toxicity to humans. It is worth noting that commercial cultivation may inadvertently lead to environmental degradation and loss of genetic diversity as well as loss of incentives to conserve wild populations [18]. However, Wiersum et al. [4] argued that the impact of the cultivation of medicinal plant can be beneficial if it is done within the context of protecting and strengthening the cultural values of biodiversity and creating a positive attitude towards biodiversity conser-

Nitrogen is one of the most important nutrients needed by plants; it is an important element for the formation amino acids, it is essential for plant cell division, it is directly involved in photosynthesis, it is an important component of vitamins and it aids in the production of carbohydrates. Physiologically, N is mostly available to plants in the forms of ammonium and nitrate and preference for one of the two forms to be taken up by plants tend to be influenced by the plant species and soil conditions, including pH and soil temperatures [10, 19].

nology should be one of the focal areas for research.

vation in general.

**3. Nutrient nitrogen**

Plant parts obtained from single or varied species are used to prepare medicinal products. Medicinal plant parts contain bioactive principles that are often referred to as secondary metabolites. Primary metabolites such as enzymes and proteins, lipids, chlorophyll and carbohydrates are fundamental to the life of the plant, while secondary metabolites (terpenoids, the alkaloids, the phenylpropanoid and some phenolic compounds) do not appear to be necessary to sustain life at a fundamental biochemical level. However, secondary metabolites play important defence and chemical ecological roles [7]. Medicinal properties can be obtained from the following plant parts: leaves, bulbs, essential oil, fatty acid, flowers, fruit, gum, stem, roots, rhizome, seed, tuber and wood. Plant secondary metabolites are thought to be responsible for the antimicrobial, antioxidant, anti-inflammatory and insecticidal activities of plant extracts [8]. These plant-derived extracts and compounds are exploited for the treatment of human and animal diseases. Large-scale production of secondary metabolites by plants is crucial in the medicinal plant industry. However, the production of secondary metabolites by plant depends on endogenous and exogenous factors [9]. Nitrogen is one of the most important nutrients needed by plants for growth. Information on the role of nitrogen in plant physiology is plentiful in literature. Nitrogen is involved in many physiological processes in plants including growth and photosynthesis. Consequently, nitrogenous fertilizers are among the most used fertilizers in the world. Nevertheless, excessive use of N can have negative economic and the environmental implications. Intensive N fertilization can lead to toxic N levels in plant tissues and herbivores. Thus, there are calls for implementation of better nitrogen use efficiency (NUE) [10].

Researchers have recognized the potential benefits of manipulating nutrient nitrogen supply for optimal plant growth and the need to minimize some of the setbacks associated with nitrogen fertilization. This has incentivized the quest for the development of precision fertilization and innovative plant cultivations methods. For examples, the use of sustainable, innovative and precision agronomic technologies such as hydroponics, aeroponics, aquaponics and organic farming can optimize the manufacturing of natural molecules of pharmaceutical and cosmetic significance. According to Masclaux-Daubresse et al. [10], increasing nitrogen use efficiency in the contexts of plant nutrition and limiting nitrogen fertilizer use is important. It is essential to preserve the environment, while promoting sustainable and productive agriculture. Therefore, knowledge on nitrogen availability and conservation in growth media, and nitrogen uptake, assimilation and translocation by plant are critically important to the development of efficient nitrogen fertilization strategies. This chapter discusses the potential risks, opportunities and setbacks associated with the use of N in cultivation of medicinal plants.

## **2. Demand for medicinal plants and rationale for commercial cultivation**

Until recently, the most commercial farmers have been focused on improving quality and quantity of agricultural and horticultural crops over medicinal plants. Medicinal plants are used in traditional practices worldwide and their use has been increasing steadily. Medicinal plants constitute an important component of health care systems, globally. The trade of medicinal plants is estimated to be worth R270 million annually [3]. According to Sobiecke [11], globally, products that are derived from traditional medicine are estimated to be worth R2.9 billion per year. On the demand for medicinal plants is increasing worldwide and it is estimated up to 700,000 tonnes of plant material are consumed annually to the value of about 150 million US dollars [4]. The World Health Organization estimates that 21,000 species are used for medicinal purposes around the world and in India 150 species are used commercially [12]. In Zimbabwe, herbal medicine is the most affordable and easily accessible form of treatment in primary health care and up to 93 medicinal plant species are used in the southcentral region of Zimbabwe [13]. In Pakistan, more than 500 species of plants are used in herbal medicine [14]. Street and Prinsloo [15] presented 10 highly used South African medicinal plants, such as *Agathosma betulina* (Rutaceae), *A. ferox* (Asphodelaceae), *Aspalathus linearis* (Fabaceae), *Harpagophytum procumbens* (Pedaliaceae), *Hypoxis hemerocallidea* (Hypoxidaceae), *Merwilla natalensis* (Hyacinthaceae), *Pelargonium sidoides* (Geraniaceae), *Siphonochilus aethiopicus* (Zingiberaceae) and *Sutherlandia frutescens* (Fabaceae) in a review paper. Although some critics have argued that traditionally it is not acceptable to use cultivated medicinal plants, a recent report on the perception of cultivation of medicinal plant species indicated that very high proportions (over 69%) of respondents are willing to buy and make use of cultivated medicinal plants [16]. This trend suggests that developing efficient and sustainable agro-technology should be one of the focal areas for research.

Cultivation of medicinal plants is gaining momentum among subsistence and commercial farmers [17]. Farming of medicinal plants has many advantages, for examples it can contribute to job creation and improvement of household earnings, and it can reduce overexploitation and harvesting of some wild and endangered species. Similar to the cultivation of food crops, medicinal plant cultivation programmes should have specific goals, which include to increase medicinal plant yield and plant growth rate, increase and standardized quality and quality of secondary metabolites produced and reduce toxicity to humans. It is worth noting that commercial cultivation may inadvertently lead to environmental degradation and loss of genetic diversity as well as loss of incentives to conserve wild populations [18]. However, Wiersum et al. [4] argued that the impact of the cultivation of medicinal plant can be beneficial if it is done within the context of protecting and strengthening the cultural values of biodiversity and creating a positive attitude towards biodiversity conservation in general.

## **3. Nutrient nitrogen**

In South Africa alone, there are some 27 million indigenous medicinal consumers [5]. The wellknown examples of plant species that are currently traded in South Africa include *Artemisia afra* (Asteraceae), *Melianthus comosus* (Melianthaceae), *Aloe ferox* (Asphodelaceae), *Aloe arborescens* (Asphodelaceae), *Salvia Africana-caerulea* (Lamiaceae) and *Helichrysum cymosum*

Plant parts obtained from single or varied species are used to prepare medicinal products. Medicinal plant parts contain bioactive principles that are often referred to as secondary metabolites. Primary metabolites such as enzymes and proteins, lipids, chlorophyll and carbohydrates are fundamental to the life of the plant, while secondary metabolites (terpenoids, the alkaloids, the phenylpropanoid and some phenolic compounds) do not appear to be necessary to sustain life at a fundamental biochemical level. However, secondary metabolites play important defence and chemical ecological roles [7]. Medicinal properties can be obtained from the following plant parts: leaves, bulbs, essential oil, fatty acid, flowers, fruit, gum, stem, roots, rhizome, seed, tuber and wood. Plant secondary metabolites are thought to be responsible for the antimicrobial, antioxidant, anti-inflammatory and insecticidal activities of plant extracts [8]. These plant-derived extracts and compounds are exploited for the treatment of human and animal diseases. Large-scale production of secondary metabolites by plants is crucial in the medicinal plant industry. However, the production of secondary metabolites by plant depends on endogenous and exogenous factors [9]. Nitrogen is one of the most important nutrients needed by plants for growth. Information on the role of nitrogen in plant physiology is plentiful in literature. Nitrogen is involved in many physiological processes in plants including growth and photosynthesis. Consequently, nitrogenous fertilizers are among the most used fertilizers in the world. Nevertheless, excessive use of N can have negative economic and the environmental implications. Intensive N fertilization can lead to toxic N levels in plant tissues and herbivores.

Thus, there are calls for implementation of better nitrogen use efficiency (NUE) [10].

Researchers have recognized the potential benefits of manipulating nutrient nitrogen supply for optimal plant growth and the need to minimize some of the setbacks associated with nitrogen fertilization. This has incentivized the quest for the development of precision fertilization and innovative plant cultivations methods. For examples, the use of sustainable, innovative and precision agronomic technologies such as hydroponics, aeroponics, aquaponics and organic farming can optimize the manufacturing of natural molecules of pharmaceutical and cosmetic significance. According to Masclaux-Daubresse et al. [10], increasing nitrogen use efficiency in the contexts of plant nutrition and limiting nitrogen fertilizer use is important. It is essential to preserve the environment, while promoting sustainable and productive agriculture. Therefore, knowledge on nitrogen availability and conservation in growth media, and nitrogen uptake, assimilation and translocation by plant are critically important to the development of efficient nitrogen fertilization strategies. This chapter discusses the potential risks, opportunities and setbacks associated with the use of N in cultivation of medicinal plants.

**2. Demand for medicinal plants and rationale for commercial cultivation**

Until recently, the most commercial farmers have been focused on improving quality and quantity of agricultural and horticultural crops over medicinal plants. Medicinal plants are

(Asteraceae) [6].

210 Nitrogen in Agriculture - Updates

Nitrogen is one of the most important nutrients needed by plants; it is an important element for the formation amino acids, it is essential for plant cell division, it is directly involved in photosynthesis, it is an important component of vitamins and it aids in the production of carbohydrates. Physiologically, N is mostly available to plants in the forms of ammonium and nitrate and preference for one of the two forms to be taken up by plants tend to be influenced by the plant species and soil conditions, including pH and soil temperatures [10, 19]. Nitrate uptake is followed by reduction to nitrite, which is then transported to the chloroplast wherein it is reduced to ammonium and is mostly assimilated in the plastid/chloroplast and finally undergoes nitrogen remobilization, whereby leaf proteins and especially photosynthetic proteins of plastids are extensively degraded during senescence, providing an enormous source of nitrogen that plants can tap to supplement the nutrition of growing organs such as new leaves and seeds [10]. Nitrogen is available to plants from varied sources and includes inorganic fertilizers (ammonium nitrate, ammonium sulphate, urea, calcium ammonium nitrate and diammonium phosphate and sodium nitrate), organic (compost, manure, seaweed, fish meal and fish emulsion and guano) sources. Although nitrogen occurs naturally in soils, generally, the quantity is quite low and varies geographically warranting external N inputs in the form of fertilizers.

**4. Physiological effect of nitrogen on medicinal plants**

bioactive medicinal principles.

**5. Nutrient nitrogen threshold**

and romaine lettuce plants and it was concluded that soil NO<sup>3</sup>

Fertilization programme in medicinal plants has two important objectives: high vegetative growth and high quantity and quality of secondary metabolites produced. Meeting these objectives could lead to high medicinal materials and increased medicinal value of a plant. Generally, N supply favour increased vegetative growth. Argyropoulou et al. [27] investigated the effect of nitrogen starvation on morphological, physiological and biochemical parameters of basil plants cultivated aeroponically. They observed that net photosynthesis rate, transpiration rate, the stomatal conductance and the concentration of total chlorophylls were strongly restricted by N deprivation rate and that total phenolic concentration significantly increased in N-starved plants indicating that biosynthesis of secondary plant metabolites is favoured in nitrogen-deficient plants. Periwinkle, a medicinal plant that is rich in terpenoid alkaloids, when exposed to mixture of nitrate and ammonium, produced the highest content of amino acids, proteins, total alkaloids, vincristine and vinblastine compared to each of the different N forms. It was also observed in the same study that increase in N level beyond 11 mM had an antagonistic effect on alkaloid content [28]. Previous studies have indicated that when plants have N deficiency they tend to have increased concentration of C-based secondary metabolites [29, 30]. Future studies that identify critical N levels for important medicinal plant species will guaranty both high production of medicinal material and quantity and quality of

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Nitrogen is a major constituent of enzymes, proteins, chlorophyll and is involved in many important biochemical processes in plants including photosynthesis. However, it has been shown in many studies that N effects on plant physiological processes like syntheses of amino acids and phenolics are dependent on tissue N concentration, plant species and other exogenous factors like water availability, temperature and light. Yañez-Mansilla et al. [31] hypothesized that there is an optimum N concentration threshold that ensure a high phenolic concentration and antioxidant capacity without detrimental effects on plant performance and proposed a threshold of 15 g N/kg DW as an optimum concentration for ensuring high antioxidant activity and quality in blueberry leaves, based on results obtained in their study. In order to meet requirements of new regulations in the coastal valleys of central California, USA, field trials were carried out by Bottoms [32] to identify commercial fields in which N application could be reduced or eliminated in order to improve nitrogen (N) fertilizer efficiency. Crop growth, N uptake and the value of soil and plant N diagnostic measures were evaluated in 24 iceberg

a reliable indicator that N application could be reduced or delayed. Many farmers, scientists, consumers and governments are becoming aware of the risks associated with excessive nitrogen fertilization and are seeking environmentally friendly and sustainable approaches of N fertilization. Medicinal crops farmers would have to take cognizance of the need to balance high yield, quality medicinal materials and minimum environmental toxicity. It is expected

–N greater than 20 mg/kg was

Both organic and inorganic N fertilizers have advantages and disadvantages. Inorganic fertilizers provide readily available nitrogen; however, they are easily lost by leaching, denitrification, volatilization and run-off. Furthermore, inorganic fertilizers have been frequently linked to cases of environmental contamination, soil acidification and salinity. On the other hand, organic fertilizers release of N to plant tends to be slower and depends on the mineralization rates. Nevertheless, organic fertilizers improve the soil physical and chemical properties. Some of the setbacks associated with the use of organic or inorganic fertilizers are predominant in plant cultivation whereby the growth medium is soil. Inherent variations in biophysicochemical properties of soils make it difficult to accurately determine the effects of fertilization on plant growth, yield and quality of produce. Factors such as seasonal changes, development stages, levels of pathogens, geographical differences and nutrient status of the soil affect the amount of secondary metabolites plants produce [20, 21]. These factors can potentially influence the standardization of the quality of medicinal materials. Consequently, more precise plant cultivation techniques are increasingly being used in crop cultivation.

According to Jehnson [22] and Hayden [23], hydroponics technology is a technique of growing plants in a nutrient solution (water and fertilizers) with or without the use of artificial medium (e.g. sand, rockwool, vermiculite, gravel, peat moss, coir and sawdust) to provide a mechanism of support. The advantages of using hydroponics include high-density maximum crop yield, crop production can be achieved in areas where good soil for production is not available, plants can be grown during off-season and temperature can be manipulated [22, 24]. In hydroponics, N is supplied to plants in the form of dissolved salts, which is usually prepared in small and precise quantities, and different nutrient recipes and combinations can be used. Hydroponic technology can be used to manipulate production of plant secondary metabolites [25]. It can favour plant vigour, decrease poisonous levels of plant toxins, increase uniformity and probability of obtaining bioactive extracts [26]. Other related technologies such as aquaponics and aeroponics can also be used to cultivate some medicinal plant species; however, they are still to be fully explored. Aquaponics is the combination of hydroponics and aquaculture in an integrated system to raise fish and grow plants, simultaneously, while aeroponics is a liquid hydroponics system with no other supporting medium for the roots of the plants [22]. In aeroponics plants are grown in misty environment.

## **4. Physiological effect of nitrogen on medicinal plants**

Fertilization programme in medicinal plants has two important objectives: high vegetative growth and high quantity and quality of secondary metabolites produced. Meeting these objectives could lead to high medicinal materials and increased medicinal value of a plant. Generally, N supply favour increased vegetative growth. Argyropoulou et al. [27] investigated the effect of nitrogen starvation on morphological, physiological and biochemical parameters of basil plants cultivated aeroponically. They observed that net photosynthesis rate, transpiration rate, the stomatal conductance and the concentration of total chlorophylls were strongly restricted by N deprivation rate and that total phenolic concentration significantly increased in N-starved plants indicating that biosynthesis of secondary plant metabolites is favoured in nitrogen-deficient plants. Periwinkle, a medicinal plant that is rich in terpenoid alkaloids, when exposed to mixture of nitrate and ammonium, produced the highest content of amino acids, proteins, total alkaloids, vincristine and vinblastine compared to each of the different N forms. It was also observed in the same study that increase in N level beyond 11 mM had an antagonistic effect on alkaloid content [28]. Previous studies have indicated that when plants have N deficiency they tend to have increased concentration of C-based secondary metabolites [29, 30]. Future studies that identify critical N levels for important medicinal plant species will guaranty both high production of medicinal material and quantity and quality of bioactive medicinal principles.

## **5. Nutrient nitrogen threshold**

Nitrate uptake is followed by reduction to nitrite, which is then transported to the chloroplast wherein it is reduced to ammonium and is mostly assimilated in the plastid/chloroplast and finally undergoes nitrogen remobilization, whereby leaf proteins and especially photosynthetic proteins of plastids are extensively degraded during senescence, providing an enormous source of nitrogen that plants can tap to supplement the nutrition of growing organs such as new leaves and seeds [10]. Nitrogen is available to plants from varied sources and includes inorganic fertilizers (ammonium nitrate, ammonium sulphate, urea, calcium ammonium nitrate and diammonium phosphate and sodium nitrate), organic (compost, manure, seaweed, fish meal and fish emulsion and guano) sources. Although nitrogen occurs naturally in soils, generally, the quantity is quite low and varies geographically warranting external N

Both organic and inorganic N fertilizers have advantages and disadvantages. Inorganic fertilizers provide readily available nitrogen; however, they are easily lost by leaching, denitrification, volatilization and run-off. Furthermore, inorganic fertilizers have been frequently linked to cases of environmental contamination, soil acidification and salinity. On the other hand, organic fertilizers release of N to plant tends to be slower and depends on the mineralization rates. Nevertheless, organic fertilizers improve the soil physical and chemical properties. Some of the setbacks associated with the use of organic or inorganic fertilizers are predominant in plant cultivation whereby the growth medium is soil. Inherent variations in biophysicochemical properties of soils make it difficult to accurately determine the effects of fertilization on plant growth, yield and quality of produce. Factors such as seasonal changes, development stages, levels of pathogens, geographical differences and nutrient status of the soil affect the amount of secondary metabolites plants produce [20, 21]. These factors can potentially influence the standardization of the quality of medicinal materials. Consequently, more precise plant cultivation techniques are increasingly being used in crop

According to Jehnson [22] and Hayden [23], hydroponics technology is a technique of growing plants in a nutrient solution (water and fertilizers) with or without the use of artificial medium (e.g. sand, rockwool, vermiculite, gravel, peat moss, coir and sawdust) to provide a mechanism of support. The advantages of using hydroponics include high-density maximum crop yield, crop production can be achieved in areas where good soil for production is not available, plants can be grown during off-season and temperature can be manipulated [22, 24]. In hydroponics, N is supplied to plants in the form of dissolved salts, which is usually prepared in small and precise quantities, and different nutrient recipes and combinations can be used. Hydroponic technology can be used to manipulate production of plant secondary metabolites [25]. It can favour plant vigour, decrease poisonous levels of plant toxins, increase uniformity and probability of obtaining bioactive extracts [26]. Other related technologies such as aquaponics and aeroponics can also be used to cultivate some medicinal plant species; however, they are still to be fully explored. Aquaponics is the combination of hydroponics and aquaculture in an integrated system to raise fish and grow plants, simultaneously, while aeroponics is a liquid hydroponics system with no other supporting medium for the roots of the plants [22]. In aeroponics plants are grown in misty

inputs in the form of fertilizers.

212 Nitrogen in Agriculture - Updates

cultivation.

environment.

Nitrogen is a major constituent of enzymes, proteins, chlorophyll and is involved in many important biochemical processes in plants including photosynthesis. However, it has been shown in many studies that N effects on plant physiological processes like syntheses of amino acids and phenolics are dependent on tissue N concentration, plant species and other exogenous factors like water availability, temperature and light. Yañez-Mansilla et al. [31] hypothesized that there is an optimum N concentration threshold that ensure a high phenolic concentration and antioxidant capacity without detrimental effects on plant performance and proposed a threshold of 15 g N/kg DW as an optimum concentration for ensuring high antioxidant activity and quality in blueberry leaves, based on results obtained in their study. In order to meet requirements of new regulations in the coastal valleys of central California, USA, field trials were carried out by Bottoms [32] to identify commercial fields in which N application could be reduced or eliminated in order to improve nitrogen (N) fertilizer efficiency. Crop growth, N uptake and the value of soil and plant N diagnostic measures were evaluated in 24 iceberg and romaine lettuce plants and it was concluded that soil NO<sup>3</sup> –N greater than 20 mg/kg was a reliable indicator that N application could be reduced or delayed. Many farmers, scientists, consumers and governments are becoming aware of the risks associated with excessive nitrogen fertilization and are seeking environmentally friendly and sustainable approaches of N fertilization. Medicinal crops farmers would have to take cognizance of the need to balance high yield, quality medicinal materials and minimum environmental toxicity. It is expected that indigenous plant species, especially those occurring in their natural habitats are adapted to their local conditions and may tend to have low critical levels for most of the nutrients. For example, medicinal plants occurring in the fynbos biome of South Africa are adapted to nutrient-poor and low pH soils. Therefore, exposing these species to high N concentration may have minimal effect on plant physiology and can even have detrimental effects on plant growth.

## **6. Economics of nitrogen fertilization**

Many studies have demonstrated that plant yield increases with N fertilization. The quest by farmers for high yield and high profit margins has encouraged the implementations of inappropriate N fertilization programmes. Excessive and inadequate N supply to plants could induce deleterious effects in plants and the environment. With increasing N fertilization costs, it is important to determine optimum N fertilization rates in order to achieve economically viable N fertilization in crop production. In a study carried out in Viçosa, Minas Gerais State, Brazil that aimed at determining the economic optimum N fertilization rates under cold and ambient conditions of four potato cultivars, it was found that economic optimum N fertilization rates ranged from 147 to 201 kg/ha depending upon cultivar and relative prices of N and potato tubers [33]. Farquharson et al. [34] recognized the importance of environmental effects such N<sup>2</sup> O emission of N fertilization in Australian wheat production and using an economic framework model, they predicted that the best fertilizer decision is reduced by about 4 kg N/ha (5%) when the Intergovernmental Panel on Climate Change (IPCC)-based environmental cost of N fertilizer is considered. Nyborg et al. [35] reported that economics of nitrogen fertilization of barley and rapeseed is influenced by nitrate-nitrogen level in the soil and suggested that soil testing to determine N2 O–N levels is essential for maximum economic returns from N fertilization. Based on the above-mentioned arguments, the use of precision N fertilization approach is encouraged, for example, in hydroponics it is possible to manipulate plants to produce higher yields of bioactive fractions [36].

**7.2. Materials and methods**

all microplates (105

Soil was collected from a commercial vegetable farm located in Kuilsriver, Western Cape, South Africa and the soil subsamples analysed (physico-chemical analysis) [38]. The field collected soil was used to prepare 3 kg potted soil samples. Ammonium nitrate salt was dissolved in 500 ml of sterile distilled water and the solution was poured into the potted soils to obtain a final soil concentration of nitrogen that was 136 ppm. Potted soils were placed in rows on a steel table. In the control treatment, only 500 ml of sterile distilled was added and the baseline N concentration was 32 ppm. Six weeks old rooted cuttings of *H. cymosum* were transplanted individually into each pot. A total of 16 pots, grouped into two treatments with eight replicates per treatment were used. Parameters such as plant height, nutrient concentration of leaves and leaf numbers were assessed in order to determine the effects of nitrogen and potassium on growth of *H. cymosum* at the end of the experiment, 13 weeks post-treatment. Leaf tissue analysis was carried out [39, 40]. Fresh foliage harvested at 13 weeks post-treatment was air dried at room temperature for 4 weeks. Dried plant materials were cut into smaller pieces and ground using a Jankel and Kunkel Model A 10 mill into fine powder. Powdered leaf material (5 g) was extracted with 100 ml of acetone in a glass beaker with the aid of a vortex mixer for 15 min and the supernatant filtered using Whatman No.1 filter paper. The extracted material was left to dry overnight. The micro-dilution method previously described by Eloff [41] was employed with slight modifications to determine the minimum inhibitory concentration (MIC) for the extracts. *Fusarium oxysporum* fungal culture was introduced to

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**Figure 1.** Hydroponics cultivation of the medicinal plant species *H. cymosum* in a greenhouse.

spores/ml). Mancozeb (60 mg/10 ml) was prepared using sterile distilled

water as a positive control and a mixture of sterile distilled water and acetone was used as a

negative control. Data were analysed using a one-way analysis of variance (ANOVA).

## **7. Case study**

Preliminary assessment of the effects of nutrient nitrogen on growth and antimicrobial activities of *H. cymosum* grown under greenhouse conditions.

#### **7.1. Introduction**

*H. cymosum* subsp. cymosum (Asteraceae) is an indigenous South African medicinal plant (**Figure 1**). It has high medicinal value and is heavily harvested from the wild. This species is distributed along the coastal areas of the Eastern and Western Cape Provinces. The soil of the coastal region of the Western Cape region is typically acidic and nutrient-poor and is derived from the weathering of granite [37]. The objective of this study was to assess the effect of N fertilization on growth, tissue nutrient content and antimicrobial activities of acetone leaf extracts of *H. cymosum* cultivated on field collected soil samples under greenhouse conditions.

**Figure 1.** Hydroponics cultivation of the medicinal plant species *H. cymosum* in a greenhouse.

#### **7.2. Materials and methods**

that indigenous plant species, especially those occurring in their natural habitats are adapted to their local conditions and may tend to have low critical levels for most of the nutrients. For example, medicinal plants occurring in the fynbos biome of South Africa are adapted to nutrient-poor and low pH soils. Therefore, exposing these species to high N concentration may have minimal effect on plant physiology and can even have detrimental effects on plant growth.

Many studies have demonstrated that plant yield increases with N fertilization. The quest by farmers for high yield and high profit margins has encouraged the implementations of inappropriate N fertilization programmes. Excessive and inadequate N supply to plants could induce deleterious effects in plants and the environment. With increasing N fertilization costs, it is important to determine optimum N fertilization rates in order to achieve economically viable N fertilization in crop production. In a study carried out in Viçosa, Minas Gerais State, Brazil that aimed at determining the economic optimum N fertilization rates under cold and ambient conditions of four potato cultivars, it was found that economic optimum N fertilization rates ranged from 147 to 201 kg/ha depending upon cultivar and relative prices of N and potato tubers [33]. Farquharson et al. [34] recognized the importance of environ-

an economic framework model, they predicted that the best fertilizer decision is reduced by about 4 kg N/ha (5%) when the Intergovernmental Panel on Climate Change (IPCC)-based environmental cost of N fertilizer is considered. Nyborg et al. [35] reported that economics of nitrogen fertilization of barley and rapeseed is influenced by nitrate-nitrogen level in the soil

returns from N fertilization. Based on the above-mentioned arguments, the use of precision N fertilization approach is encouraged, for example, in hydroponics it is possible to manipulate

Preliminary assessment of the effects of nutrient nitrogen on growth and antimicrobial activi-

*H. cymosum* subsp. cymosum (Asteraceae) is an indigenous South African medicinal plant (**Figure 1**). It has high medicinal value and is heavily harvested from the wild. This species is distributed along the coastal areas of the Eastern and Western Cape Provinces. The soil of the coastal region of the Western Cape region is typically acidic and nutrient-poor and is derived from the weathering of granite [37]. The objective of this study was to assess the effect of N fertilization on growth, tissue nutrient content and antimicrobial activities of acetone leaf extracts of *H. cymosum* cultivated on field collected soil samples under greenhouse conditions.

O emission of N fertilization in Australian wheat production and using

O–N levels is essential for maximum economic

**6. Economics of nitrogen fertilization**

and suggested that soil testing to determine N2

plants to produce higher yields of bioactive fractions [36].

ties of *H. cymosum* grown under greenhouse conditions.

mental effects such N<sup>2</sup>

214 Nitrogen in Agriculture - Updates

**7. Case study**

**7.1. Introduction**

Soil was collected from a commercial vegetable farm located in Kuilsriver, Western Cape, South Africa and the soil subsamples analysed (physico-chemical analysis) [38]. The field collected soil was used to prepare 3 kg potted soil samples. Ammonium nitrate salt was dissolved in 500 ml of sterile distilled water and the solution was poured into the potted soils to obtain a final soil concentration of nitrogen that was 136 ppm. Potted soils were placed in rows on a steel table. In the control treatment, only 500 ml of sterile distilled was added and the baseline N concentration was 32 ppm. Six weeks old rooted cuttings of *H. cymosum* were transplanted individually into each pot. A total of 16 pots, grouped into two treatments with eight replicates per treatment were used. Parameters such as plant height, nutrient concentration of leaves and leaf numbers were assessed in order to determine the effects of nitrogen and potassium on growth of *H. cymosum* at the end of the experiment, 13 weeks post-treatment. Leaf tissue analysis was carried out [39, 40]. Fresh foliage harvested at 13 weeks post-treatment was air dried at room temperature for 4 weeks. Dried plant materials were cut into smaller pieces and ground using a Jankel and Kunkel Model A 10 mill into fine powder. Powdered leaf material (5 g) was extracted with 100 ml of acetone in a glass beaker with the aid of a vortex mixer for 15 min and the supernatant filtered using Whatman No.1 filter paper. The extracted material was left to dry overnight. The micro-dilution method previously described by Eloff [41] was employed with slight modifications to determine the minimum inhibitory concentration (MIC) for the extracts. *Fusarium oxysporum* fungal culture was introduced to all microplates (105 spores/ml). Mancozeb (60 mg/10 ml) was prepared using sterile distilled water as a positive control and a mixture of sterile distilled water and acetone was used as a negative control. Data were analysed using a one-way analysis of variance (ANOVA).

#### **7.3. Results**

There was no significant difference (*P* > 0.05) in plant height exposed to higher level of N (51.4 ± 4.9 cm ) compared to those exposed to low level N (Control) (55.1 ± 5.1 cm) at 13 weeks post-treatment. Similarly, no significant difference (*P* < 0.05) was observed in the number of branches in plants exposed to the different N treatments. Comparatively, N-treated (1.9 ± 0.2 ppm) plants had a significantly high levels of tissue content N in the leaves (df 1,6; *F* = 7.8; *P* = 0.03) than those exposed to low nutrient N treatment (1.4 ± 0.1 ppm) at 13 weeks posttreatment (**Table 1**). MIC bioassay did not show a significant effect (*P* > 0.05) on antifungal activity following N treatment compared to control (0.187 mg/ml) (**Table 2**).

that genotypic variation in nitrate accumulation is associated with differences in water content for rape, Chinese cabbage and spinach. Vegetables account for over 70% of the total nitrogen intake of humans [43]. Increased concentration of nitrite and nitrates in diet are risk factors for many diseases in mammals [44]. Although nitrate intake from vegetables is receiving substantial attention, it is important that cultivated medicinal plants receive similar attention as the industry develops. Commercial cultivation of medicinal plants could lead to excessive N fertilization and high concentration of nitrites and nitrates in medicinal plant parts and subsequently in herbal decoctions and infusions. This can negate the beneficial effects of medicinal plants. Also, accumulation of unused nitrates in soils could have unfavourable effect on soil biological, physical and chemical properties. Furthermore, leached nitrates in water runoffs could lead to eutrophication of freshwater resources. Since plants have different N needs/ requirements, research on the N requirement of each plant in different growing conditions is important in order to achieve high yield, safe and good quality medicinal materials from

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217

The development of fertilization policies in many countries is an indication of recognition of the risk that is associated with the use of fertilizers including nitrogenous fertilizers. One of the main challenges facing regulation of the use of fertilizer inputs include high variations of rate of N fertilization across regions and crops and the stage of economic development [45]. The increasing demand for efficient fertilizer use has led the United Nations Economic Commission for Europe (UNECE) to review its so-called "Gothenburg Protocol to Abate Acidification, Eutrophication and Ground-level Ozone". Nitrogen use efficiency (NUE) and N balance will be used as two key indicators in this international convention in order to assess the efficacy of measures to decrease nitrogen (N) losses while maintaining agricultural productivity [46]. Recently, Pires et al. [47] demonstrated that increase in NUE would lead to reduced N fertilization in cereal production as well as improve agronomic, economic and environmental benefits. Considering that increase in global fertilizer consumption is expected to reach 69 million tons in 2030, and the increased use of nitrogen (N) fertilizers is responsible for 67% of this amount. Commercial medicinal plant cultivation will certainly exacerbate this problem in the future. Therefore, it is important for countries to develop efficient policies and

Nitrogen fertilization will be an important factor in commercial medicinal crop cultivation. In order to ensure sustainable commercial cultivation of medicinal plant, it is, therefore, necessary to develop efficient N fertilization management programmes as well protocols and policies. It is important that caution is exercised when implementing N fertilization in commercial farming of medicinal plants and the following important aspects should be addressed:

plants.

**9. Regulation of N fertilization**

guidelines for use of N fertilization going forward.

**10. Conclusion and recommendation**

#### **7.4. Discussion**

Nitrogen-treated plants had higher N content in the leaves compared to low N-exposed plants suggesting that the treatment with an increased level of N could have induced high uptake of nitrogen. The plant growth was not significantly different in plants treated with 136 ppm of nitrogen compared to control plants (32 ppm). This result suggests that higher nitrogen supply may not always result in high vegetative growth. A plausible explanation could be that plants occurring naturally in nutrient-poor area may have low optimum nutrient requirement and may not warrant excessive N treatment. Also, high N fertilization of medicinal plants may not necessarily reduce bioactivity of their extracts.


**Table 1.** Tissue nutrient content (ppm) in aerial parts of *H. cymosum* following exposure to control and N treated field collected soil samples after 13 weeks post-treatment.


**Table 2.** MIC antifungal activity of the acetone extract of *H. cymosum*.

## **8. Nitrogen toxicity**

Excessive nitrate fertilization can induce high accumulation of nitrates in plant tissues to levels that are potentially toxic to humans and livestock. However, Qiu et al. [42] showed that that genotypic variation in nitrate accumulation is associated with differences in water content for rape, Chinese cabbage and spinach. Vegetables account for over 70% of the total nitrogen intake of humans [43]. Increased concentration of nitrite and nitrates in diet are risk factors for many diseases in mammals [44]. Although nitrate intake from vegetables is receiving substantial attention, it is important that cultivated medicinal plants receive similar attention as the industry develops. Commercial cultivation of medicinal plants could lead to excessive N fertilization and high concentration of nitrites and nitrates in medicinal plant parts and subsequently in herbal decoctions and infusions. This can negate the beneficial effects of medicinal plants. Also, accumulation of unused nitrates in soils could have unfavourable effect on soil biological, physical and chemical properties. Furthermore, leached nitrates in water runoffs could lead to eutrophication of freshwater resources. Since plants have different N needs/ requirements, research on the N requirement of each plant in different growing conditions is important in order to achieve high yield, safe and good quality medicinal materials from plants.

## **9. Regulation of N fertilization**

**7.3. Results**

216 Nitrogen in Agriculture - Updates

**7.4. Discussion**

**8. Nitrogen toxicity**

There was no significant difference (*P* > 0.05) in plant height exposed to higher level of N (51.4 ± 4.9 cm ) compared to those exposed to low level N (Control) (55.1 ± 5.1 cm) at 13 weeks post-treatment. Similarly, no significant difference (*P* < 0.05) was observed in the number of branches in plants exposed to the different N treatments. Comparatively, N-treated (1.9 ± 0.2 ppm) plants had a significantly high levels of tissue content N in the leaves (df 1,6; *F* = 7.8; *P* = 0.03) than those exposed to low nutrient N treatment (1.4 ± 0.1 ppm) at 13 weeks posttreatment (**Table 1**). MIC bioassay did not show a significant effect (*P* > 0.05) on antifungal

Nitrogen-treated plants had higher N content in the leaves compared to low N-exposed plants suggesting that the treatment with an increased level of N could have induced high uptake of nitrogen. The plant growth was not significantly different in plants treated with 136 ppm of nitrogen compared to control plants (32 ppm). This result suggests that higher nitrogen supply may not always result in high vegetative growth. A plausible explanation could be that plants occurring naturally in nutrient-poor area may have low optimum nutrient requirement and may not warrant excessive N treatment. Also, high N fertilization of medicinal plants

Excessive nitrate fertilization can induce high accumulation of nitrates in plant tissues to levels that are potentially toxic to humans and livestock. However, Qiu et al. [42] showed that

**Acetone extracts Minimum inhibitory concentration (MIC mg/ml) of acetone extract of** *Helichrysum* 

**Table 1.** Tissue nutrient content (ppm) in aerial parts of *H. cymosum* following exposure to control and N treated field

*cymosum* **against** *Fusarium oxysporum*

N 0.82 ± 0.01 0.187 ± 0 Control 0.93 ± 0 0.187 ± 0

**Table 2.** MIC antifungal activity of the acetone extract of *H. cymosum*.

**24 h 48 h**

activity following N treatment compared to control (0.187 mg/ml) (**Table 2**).

may not necessarily reduce bioactivity of their extracts.

collected soil samples after 13 weeks post-treatment.

**Treatment N content ppm** N 1.4 ± 0.1 Control 1.9 ± 0.2

The development of fertilization policies in many countries is an indication of recognition of the risk that is associated with the use of fertilizers including nitrogenous fertilizers. One of the main challenges facing regulation of the use of fertilizer inputs include high variations of rate of N fertilization across regions and crops and the stage of economic development [45]. The increasing demand for efficient fertilizer use has led the United Nations Economic Commission for Europe (UNECE) to review its so-called "Gothenburg Protocol to Abate Acidification, Eutrophication and Ground-level Ozone". Nitrogen use efficiency (NUE) and N balance will be used as two key indicators in this international convention in order to assess the efficacy of measures to decrease nitrogen (N) losses while maintaining agricultural productivity [46]. Recently, Pires et al. [47] demonstrated that increase in NUE would lead to reduced N fertilization in cereal production as well as improve agronomic, economic and environmental benefits. Considering that increase in global fertilizer consumption is expected to reach 69 million tons in 2030, and the increased use of nitrogen (N) fertilizers is responsible for 67% of this amount. Commercial medicinal plant cultivation will certainly exacerbate this problem in the future. Therefore, it is important for countries to develop efficient policies and guidelines for use of N fertilization going forward.

## **10. Conclusion and recommendation**

Nitrogen fertilization will be an important factor in commercial medicinal crop cultivation. In order to ensure sustainable commercial cultivation of medicinal plant, it is, therefore, necessary to develop efficient N fertilization management programmes as well protocols and policies. It is important that caution is exercised when implementing N fertilization in commercial farming of medicinal plants and the following important aspects should be addressed:


[3] Dold, A. P. and Cocks, M.L. 2002. The trade in medicinal plants in the Eastern Cape

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## **Author details**

Felix Nchu\*, Yonela Matanzima and Charles P. Laubscher

\*Address all correspondence to: felixnchu@gmail.com

Department of Horticultural Sciences, Faculty of Applied Sciences, Cape Peninsula University of Technology, Bellville, Cape Town, South Africa

## **References**


[3] Dold, A. P. and Cocks, M.L. 2002. The trade in medicinal plants in the Eastern Cape Province of South Africa. South African Journal of Science **98**, 589-597.







Department of Horticultural Sciences, Faculty of Applied Sciences, Cape Peninsula University

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[2] Motaleb, M. A. 2010. Approaches to conservation of medicinal plants and traditional knowledge: A focus on the Chittagong Hill Tracts. IUCN (International Union for Conservation of Nature), Bangladesh Country Office, Dhaka, Bangladesh, pp. 8-30. https://www.iucn.org/sites/dev/files/import/downloads/medicinalplant11book.pdf

the United Nations. pp. 12-23. http://www.fao.org/3/a-w7261e.pdf

quality materials, and sustainable commercial cultivation of medicinal plant.

Felix Nchu\*, Yonela Matanzima and Charles P. Laubscher

\*Address all correspondence to: felixnchu@gmail.com

of Technology, Bellville, Cape Town, South Africa

and consumers.

218 Nitrogen in Agriculture - Updates

mental costs.

nutrients.

long-term.

**Author details**

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[38] The Non-Affiliated Soil Analysis Work Committee. 1990. Handbook of Standard Soil Testing Methods for Advisory Purposes. Soil Science Society of South Africa, Pretoria.

[39] Campbell, C. R. and Plank, C.O. 1998. Preparation of plant tissue for laboratory analysis. In: Kalra, Y.P. (ed.). Handbook of Reference Methods for Plant Analysis. CRC Press,

[40] Miller, F. C.1992. Composting as a process based on the control of ecologically selective factors. In: Blaine-Metting, F. (ed.). Soil Microbial Ecology: Applications in Agriculture

[41] Eloff, J.N. 1998. Which extractant should be used for the screening and isolation of antimicrobial components from plants? Journal of Ethnopharmacology, **60**, 1-8.

[37] Bargmann, C.J. 2005. Geology and wine in South Africa. Geoscientist, **15**, 4-8.

Environment Management, Marcel Dekker Inc., New York. p. 646.

cultivars. Potato Research, **53**, 167-179. doi: 10.1007/s11540-010-9160-3

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Medicine, **13**(3), 169-180.

Boca Raton, Fla, USA. pp. 37-49.


[29] Lou, Y. and Baldwin, I. 2004. Nitrogen supply influences herbivore-induced direct and indirect defences and transcriptional responses in *Nicotiana attenuata*. Plant Physiology, **135**, 496-506.

[16] Loundou, P.-M. and Watts, S. 2008. Medicinal Plant Trade and Opportunities for Sustainable Management in the Cape Peninsula, South Africa. Unpublished Thesis,

[17] Rashid, A.Z.M.M., Tunon, H., Khan, N.A. and Mukul, S.A. 2014. Commercial cultivation of medicinal plants in Northern Bangladesh. European Journal of Environmental

[18] Schippmann, U., Cunningham, A.B., Leaman, D.J 2002. Impact of Cultivation and Gathering of Medicinal Plants on Biodiversity: Global Trends and Issues. Case Study No. 7. Biodiversity and the Ecosystem Approach in Agriculture, Forestry and Fisheries, FAO, Rome, pp. 12-13. Visited 20th November 2014. http://www.fao.org/docrep/005/

[19] Boczulak, S.A., Hawkins, B.J., and Roy, R. 2014. Temperature effects on nitrogen form uptake by seedling roots of three contrasting conifers. Tree Physiology, **34**, 513-523. [20] Johanna, B.M. 2007. Variation of Active Constituents in *Euclea nataliensis* Based on Seedling Stages, Seasons and Fertilizers. Unpublished MSc dissertation, University of

[21] White, A.G., Davies-Coleman, M.T. and Ripley, B.S. 2008. Measuring and optimizing umckalin concentrations in wild harvested and cultivated *Pelargonium sidoides*

[23] Hayden, A.L. 2006. Aeroponic and hydroponic systems for medicinal herb, rhizome and

[24] Koohakan, P., Ikeda, H., Jeanaksorn, T., Tojo, M., Kusakari, S., Okada, K. and Sato, S. 2004. Evaluation of the indigenous micro-organisms in soilless culture: occurrence and quantitative characteristics in different growing systems. Scientia Horticulturae, **101**,

[25] Gontier, E., Clement, A., Tran, T.L.M., Gravot, A., Lie'vre, K., Guckert, A. and Bourgaud, A. 2002. Hydroponic combined with natural or forced root permeabilization: a promising technique for plant secondary metabolite production. Plant Science, **163**, 723-732. [26] Canter, M., Adeline, D. and Teadora, P. 2007. Researches concerning rooting technology

[27] Argyropoulou, K., Salahas, G, Hela, D. and Papasavvas, A. 2015. Impact of nitrogen deficiency on biomass production, morphological and biochemical characteristics of sweet basil (*Ocimum basilicum* L.) plants, cultivated aeroponically. Agriculture & Food,

[28] Abdolzadeh, A., Hosseinian, F., Aghdasi, M and Sadgipoor, H. 2006. Effects of nitrogen sources and levels on growth and alkaloid content of periwinkle. Asian Journal of Plant

(Geraniaceae). South African Journal of Botany, **74**, 260-267.

of *Pelargonium* genus. Bulletin USAMV-CV, **64**, 1-2.

[22] Jehnson, M.H. 1999. Hydroponics worldwide. Acta Horticulture, **48**, 719-730

University of Stellenbosch, South Africa. pp. 1-103.

Sciences, **4**, 60-68.

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y4586e/y4586e08.htm#TopOfPage

Pretoria, South Africa. pp. 1-93.

root crops. HortiScience, **41**, 536-538.

**3**, 32-42. ISSN: 1314-8591.

Sciences, **5**, 271-276.

179-188.


[42] Qiu, W., Wang, Z., Huang, C., Chen, B. and Yang, R. 2014. Nitrate accumulation in leafy vegetables and its relationship with water. Journal of Soil Science and Plant Nutrition, **14**(4), 761-768.

**Chapter 12**

**Provisional chapter**

**The Potential of Tree and Shrub Legumes in**

**The Potential of Tree and Shrub Legumes in** 

DOI: 10.5772/intechopen.69995

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee InTech. This chapter is 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.

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

**Keywords:** Africa, agroforestry, climate changes, sustainable agriculture, tree, shrub

Climate variability and changes are utmost important primary drivers of biological processes. They are intimately associated with a wide array of abiotic stresses, high‐ lighting the vulnerability of ecosystems and endangering biodiversity. Nitrogen‐fix‐ ing trees and shrubs (NFTSs) constitute a unique group of plants for their wide range of applications at the environmental, social and economic levels. In this chapter, we review and analyse the potential of this group of legumes in agroforestry towards sustainable agriculture in Africa. In the first part, the intertwined pillar of sustainable agriculture is brought forward under the context of growing population and climate changes. The second part addresses general aspects of legumes, including botany and the symbiosis with rhizobia. The third part includes the application of NFTS as N‐fertil‐ izers in agroforestry, highlighting the importance of an accurate choice of the crop(s)/ NFTS combination(s) and cropping type (intercropping, multistrata or fallows). The implementation of agroforestry systems with NFTS should be supported by funda‐ mental research strategies such as stable isotopes and systems biology and preceded by experimental assays, in order to identify the factors promoting N‐losses and to design appropriate management strategies that synchronize legume‐N availability with the

Ana I. Ribeiro‐Barros, Maria J. Silva, Isabel Moura, José C. Ramalho, Cristina Máguas‐Hanson and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69995

**Agroforestry Systems**

Ana I. Ribeiro-Barros, Maria J. Silva, Isabel Moura, José C. Ramalho, Cristina Máguas-Hanson and

**Agroforestry Systems**

Natasha S. Ribeiro

**Abstract**

crop demand.

legumes

Natasha S. Ribeiro


**Provisional chapter**

## **The Potential of Tree and Shrub Legumes in Agroforestry Systems Agroforestry Systems**

**The Potential of Tree and Shrub Legumes in** 

DOI: 10.5772/intechopen.69995

Ana I. Ribeiro‐Barros, Maria J. Silva, Isabel Moura, José C. Ramalho, Cristina Máguas‐Hanson and Natasha S. Ribeiro Isabel Moura, José C. Ramalho, Cristina Máguas-Hanson and Natasha S. Ribeiro Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69995

Ana I. Ribeiro-Barros, Maria J. Silva,

#### **Abstract**

[42] Qiu, W., Wang, Z., Huang, C., Chen, B. and Yang, R. 2014. Nitrate accumulation in leafy vegetables and its relationship with water. Journal of Soil Science and Plant Nutrition,

[43] Walker, R. 1990. Nitrates, nitrites and N-nitrosocompounds, a review of the occurrence in food and diet and the toxicological implications. Food Additives and Contaminants,

[44] Stanton, L.T. 2001. Nitrate Poisoning. Colorado State University Extension, No.1.610.

Journal of the Human Environment, **33**(6), 300-305.

[45] Ju, X., Liu, X., Zhang, F. and Roelcke, M. 2004. Nitrogen fertilization, soil nitrate accumulation, and policy recommendations in several agricultural regions of China. AMBIO: A

[46] Brentrup, F. and Palliere, C. 2010. Nitrogen Use Efficiency as an Agro-environmental Indicator. Accessed 19 June 2015. http://www.oecd.org/tad/sustainable-agri-

[47] Pires, M.V., da Cunha, D.A., de Matos Carlos, S. and Costa, M.H. 2015. Nitrogen-use efficiency, nitrous oxide emissions, and cereal production in Brazil: current trends and

forecasts. PLOS ONE, **10**(8), e0135234. doi: 10.1371/journal.pone.0135234.

**14**(4), 761-768.

222 Nitrogen in Agriculture - Updates

**7**, 717-768.

culture/44810433.pdf

Climate variability and changes are utmost important primary drivers of biological processes. They are intimately associated with a wide array of abiotic stresses, high‐ lighting the vulnerability of ecosystems and endangering biodiversity. Nitrogen‐fix‐ ing trees and shrubs (NFTSs) constitute a unique group of plants for their wide range of applications at the environmental, social and economic levels. In this chapter, we review and analyse the potential of this group of legumes in agroforestry towards sustainable agriculture in Africa. In the first part, the intertwined pillar of sustainable agriculture is brought forward under the context of growing population and climate changes. The second part addresses general aspects of legumes, including botany and the symbiosis with rhizobia. The third part includes the application of NFTS as N‐fertil‐ izers in agroforestry, highlighting the importance of an accurate choice of the crop(s)/ NFTS combination(s) and cropping type (intercropping, multistrata or fallows). The implementation of agroforestry systems with NFTS should be supported by funda‐ mental research strategies such as stable isotopes and systems biology and preceded by experimental assays, in order to identify the factors promoting N‐losses and to design appropriate management strategies that synchronize legume‐N availability with the crop demand.

**Keywords:** Africa, agroforestry, climate changes, sustainable agriculture, tree, shrub legumes

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. © 2018 The Author(s). Licensee InTech. This chapter is 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.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

## **1. Introduction**

Global agriculture is facing a series of challenges mainly related to growing population, climate changes and loss of biodiversity. Firstly, it is estimated that crop production must increase more than 60% by the year 2050 to fulfil the needs of the world's population [1]. Secondly, drought and soil salinization are expected to result in losses of up to 50% of arable lands by the middle of this century [1, 2]. Thirdly, the spreading of agriculture to arid and semi‐arid regions under intensive irrigation management will promote secondary soil sali‐ nization [3]. Thus, the future of agriculture must rely on the sustainable intensification of crop production to feed the increasingly growing population, as well as on the use of toler‐ ant cultivars that are able to cope with extreme environmental conditions, i.e. low fertility and saline soils, increasing water shortage periods as well as raising air temperatures and CO2 [4, 5].

**2. Description and functioning**

hosted and fix atmospheric N<sup>2</sup>

Includes the former sub‐family Mimosoideae.

\*

from LPWG [11]).

The Fabaceae or Leguminosae family is the third largest group of flowering plants and the second most important in agriculture [9]. According to recent molecular and morphological studies, Fabaceae is a single monophyletic family [9, 10], comprising more than 18,000 species distributed over ca. 800 genera and six sub‐families (**Table 1**) [11]: (i) Cercidoideae and (ii) Detarioideae, both comprising mainly tropical species; (iii) Duparquetioideae, a sub‐family from western and central Africa, with only one species identified; (iv) Dialioideae, widespread throughout the tropics; (v) the pantropical Caesalpinioideae, with more than 4000 species (including the former sub‐family Mimosoideae) and (vi) the cosmopolitan and largest legume sub‐family, Faboideae (Papilionoideae), with ca. 14,000 species, mainly herbs and small shrubs. Varying in habit from annual herbs to large trees, legumes are conspicuous and well rep‐ resented throughout temperate and tropical regions [9, 12, 13]. The family is particularly diverse in tropical forests and temperate shrub lands with a seasonally dry or arid climate. Such preference for semi‐arid to arid habitats seems to be related to a nitrogen‐demanding metabolism [9]. The vast majority of legume species (ca. 90%) is able to establish symbiosis with nitrogen‐fixing diazotrophic bacteria of the genera *Rhizobium* or *Bradirhizobium* (collec‐ tively called rhizobia) at the root and, in some cases, at the shoot level [14]. The symbiosis results in the formation of a new plant organ, i.e. the root‐ or stem‐nodule, where bacteria are

metabolism as well as the symbiotic process [15]. This type of symbiosis has around 58 million

Nitrogen is among the key elements for plant growth and production, being decisive to the adequate plant response to environmental stresses [17]. It is a major component of chlorophyll (photosynthesis), purines and pyrimidines (nucleic acids), amino acids (proteins) and ATP

Cercidoideae 12 ca. 335 Mainly tropical, e.g. *Bauhinia* spp.,

Detarioideae 84 ca. 760 Mainly tropical, e.g. *Amherstia* spp.,

Duparquetioideae 1 1 West and Central Africa, *Duparquetia* 

Dialioideae 17 ca. 85 Widespread throughout the tropics,

Caesalpinioideae\* 148 ca. 4400 Pantropical, e.g. *Caesalpinia* spp.,

Faboideae (Papilionoideae) 503 ca. 14,000 Cosmopolitan, e.g. *Astragalus* spp.,

**Table 1.** Sub‐families, number of genera and species, distribution and examples of tree and shrub legumes (adapted

years and arose from the genome duplication of the sub‐family Papilionoideae [16].

**Subfamily Genera (number) Species (number) Distribution**

, receiving in exchange energy and carbon to sustain their own

The Potential of Tree and Shrub Legumes in Agroforestry Systems

http://dx.doi.org/10.5772/intechopen.69995

225

*Cercis* spp.

*orchidaceae*

e.g. *Dialium* spp.

*Lupinus* spp., *Pisum* spp.

*Detarium* spp., *Tamarindus* spp.

*Senna* spp., *Mimosa* spp., *Acacia* spp.

These challenges will be particularly critical in the developing countries, which have the high‐ est rates of population growth and where most of the farmland is managed by smallholders [6]. It is estimated that in these countries one of five persons still live on less than \$1.25 a day [7]. In this context, intensive agriculture based on agro‐chemicals and mechanization is not sustainable and the systems must rely on appropriate cropping and post‐harvest practices, preferably based on local ecosystem‐based resources. Such practices include, for example, the implementation of integrated agroforestry systems, crop‐livestock integration and crop‐aqua‐ culture production, that concomitantly have the potential to promote the conservation and the rational use of biodiversity and other ecosystem services.

According to the Food and Agriculture Organization of the United Nations (FAO), sustainable agriculture lies at the core of the 2030 Agenda [7]. Indeed, 6 of the 17 sustainable development goals (SDGs) concentrate on this issue. These are as follows: (i) SDG 2—*End hunger, achieve food security and improved nutrition and promote sustainable agriculture*; (ii) SDG 6—*Ensure sustainable consumption and production patterns*; (iii) SDG 12—*Ensure sustainable consumption and production patterns*; (iv) SDG 13—*Take urgent action to combat climate change and its impacts*; (v) SDG 14—*Take urgent action to combat climate change and its impacts*; (vi) SDG 15—*Sustainably manage forests, combat desertification, halt and reverse land degradation, halt biodiversity loss*. Besides that, all the other 11 SDGs cross cut issues towards the end of hunger and poverty.

Since 2014, FAO has supported over 80 initiatives in Africa to promote sustainable agricul‐ tural production practices [8]. To achieve that, three intertwined pillars are considered essen‐ tial: (i) *efficient use of resources*, i.e. agriculture intensification to produce more with less impact on natural resources; (ii) *environment protection and conservation*, i.e. better management of natural resources in order to protect biodiversity (and ecosystem's stability), water, soil fertil‐ ity and reduce pollution and (iii) *resilient agriculture*, i.e. adopting approaches to adapt and mitigate the impact of climate change.

Legume fixing trees and shrubs play a crucial role in biodiversity dynamics. From the ecologi‐ cal point of view, their introduction in cropping systems may contribute to reduce the use of chemical fertilizers and to ecosystems stability.

## **2. Description and functioning**

**1. Introduction**

224 Nitrogen in Agriculture - Updates

CO2

[4, 5].

Global agriculture is facing a series of challenges mainly related to growing population, climate changes and loss of biodiversity. Firstly, it is estimated that crop production must increase more than 60% by the year 2050 to fulfil the needs of the world's population [1]. Secondly, drought and soil salinization are expected to result in losses of up to 50% of arable lands by the middle of this century [1, 2]. Thirdly, the spreading of agriculture to arid and semi‐arid regions under intensive irrigation management will promote secondary soil sali‐ nization [3]. Thus, the future of agriculture must rely on the sustainable intensification of crop production to feed the increasingly growing population, as well as on the use of toler‐ ant cultivars that are able to cope with extreme environmental conditions, i.e. low fertility and saline soils, increasing water shortage periods as well as raising air temperatures and

These challenges will be particularly critical in the developing countries, which have the high‐ est rates of population growth and where most of the farmland is managed by smallholders [6]. It is estimated that in these countries one of five persons still live on less than \$1.25 a day [7]. In this context, intensive agriculture based on agro‐chemicals and mechanization is not sustainable and the systems must rely on appropriate cropping and post‐harvest practices, preferably based on local ecosystem‐based resources. Such practices include, for example, the implementation of integrated agroforestry systems, crop‐livestock integration and crop‐aqua‐ culture production, that concomitantly have the potential to promote the conservation and

According to the Food and Agriculture Organization of the United Nations (FAO), sustainable agriculture lies at the core of the 2030 Agenda [7]. Indeed, 6 of the 17 sustainable development goals (SDGs) concentrate on this issue. These are as follows: (i) SDG 2—*End hunger, achieve food security and improved nutrition and promote sustainable agriculture*; (ii) SDG 6—*Ensure sustainable consumption and production patterns*; (iii) SDG 12—*Ensure sustainable consumption and production patterns*; (iv) SDG 13—*Take urgent action to combat climate change and its impacts*; (v) SDG 14—*Take urgent action to combat climate change and its impacts*; (vi) SDG 15—*Sustainably manage forests, combat desertification, halt and reverse land degradation, halt biodiversity loss*. Besides that,

Since 2014, FAO has supported over 80 initiatives in Africa to promote sustainable agricul‐ tural production practices [8]. To achieve that, three intertwined pillars are considered essen‐ tial: (i) *efficient use of resources*, i.e. agriculture intensification to produce more with less impact on natural resources; (ii) *environment protection and conservation*, i.e. better management of natural resources in order to protect biodiversity (and ecosystem's stability), water, soil fertil‐ ity and reduce pollution and (iii) *resilient agriculture*, i.e. adopting approaches to adapt and

Legume fixing trees and shrubs play a crucial role in biodiversity dynamics. From the ecologi‐ cal point of view, their introduction in cropping systems may contribute to reduce the use of

all the other 11 SDGs cross cut issues towards the end of hunger and poverty.

the rational use of biodiversity and other ecosystem services.

mitigate the impact of climate change.

chemical fertilizers and to ecosystems stability.

The Fabaceae or Leguminosae family is the third largest group of flowering plants and the second most important in agriculture [9]. According to recent molecular and morphological studies, Fabaceae is a single monophyletic family [9, 10], comprising more than 18,000 species distributed over ca. 800 genera and six sub‐families (**Table 1**) [11]: (i) Cercidoideae and (ii) Detarioideae, both comprising mainly tropical species; (iii) Duparquetioideae, a sub‐family from western and central Africa, with only one species identified; (iv) Dialioideae, widespread throughout the tropics; (v) the pantropical Caesalpinioideae, with more than 4000 species (including the former sub‐family Mimosoideae) and (vi) the cosmopolitan and largest legume sub‐family, Faboideae (Papilionoideae), with ca. 14,000 species, mainly herbs and small shrubs.

Varying in habit from annual herbs to large trees, legumes are conspicuous and well rep‐ resented throughout temperate and tropical regions [9, 12, 13]. The family is particularly diverse in tropical forests and temperate shrub lands with a seasonally dry or arid climate. Such preference for semi‐arid to arid habitats seems to be related to a nitrogen‐demanding metabolism [9]. The vast majority of legume species (ca. 90%) is able to establish symbiosis with nitrogen‐fixing diazotrophic bacteria of the genera *Rhizobium* or *Bradirhizobium* (collec‐ tively called rhizobia) at the root and, in some cases, at the shoot level [14]. The symbiosis results in the formation of a new plant organ, i.e. the root‐ or stem‐nodule, where bacteria are hosted and fix atmospheric N<sup>2</sup> , receiving in exchange energy and carbon to sustain their own metabolism as well as the symbiotic process [15]. This type of symbiosis has around 58 million years and arose from the genome duplication of the sub‐family Papilionoideae [16].

Nitrogen is among the key elements for plant growth and production, being decisive to the adequate plant response to environmental stresses [17]. It is a major component of chlorophyll (photosynthesis), purines and pyrimidines (nucleic acids), amino acids (proteins) and ATP


**Table 1.** Sub‐families, number of genera and species, distribution and examples of tree and shrub legumes (adapted from LPWG [11]).

(energy). Although it is one of the most abundant elements in the Earth, its predominant form, i.e. N2 (g), cannot be directly assimilated by the plants, which need reduced forms of this ele‐ ment (NH4 + , NO2 − and NO3 − ) [18, 19]. This conversion can be achieved chemically through the Harber‐Bosch process, or biologically through bacterial nitrogen fixation [20]. While chemical nitrogen fixation is cost intensive and 40–50% of the nitrogen applied as fertilizer is lost via denitrification, runoff or leaching, only 10–20% of the biologically fixed nitrogen is lost that way [21]. Besides that, the use of chemical fertilizers has a series of ecological impacts, such as air, soil and water pollution [22]. Thus, there is a strong interest in symbiotic N2 fixation between legumes and rhizobia towards the improvement of agricultural systems, i.e. better productivity with the least ecological impact [23, 24].

**Species Applications Origin kg.N.ha−1.**

Building, furniture and handicrafts (stem); fish poison (bark); body anointment (root bark); tannin, dyestuff (Sap); forage (foliage); honey (flowers); medicine (various); erosion

Firewood, charcoal (Stem and branches); tannin, ropes (Bark); forage (leaves and young branches); food (flowers), ropes, fishnets (fiber); gum (seeds and bark); medicine (various); shading; fencing; fertilizer; nitrogen fixation; intercropping [91]

Firewood (stem and branches); forage and insecticide (leaves); erosion control, shading; land reclamation; nitrogen fixation; fertilizer;

(leaves); medicine (various); shading; fencing; nitrogen fixation; fertilizer [91]

**Table 2.** Examples of tree and shrub legumes and their applications in formal and informal economies.

**Figure 1.** Details from *Brachystegia boehmii* leaves (A); *Brachystegia spiciformis* leaves and flowers (B); *Cajanus cajan* leaves, flowers and pods (C); *Pterocarpus angolensis* young leaves and mature fruit (D). Credits to Moura (A, D) and Catarino (B, C).

control; nitrogen fixation [91]

fencing; intercropping [91]

*Tephrosia vogelii* Hook.f Fish poison, insecticide and molluscicide

*Pterocarpus angolensis*

*Sesbania sesban* (L.)

*Tephrosia candida* (Roxb.)

DC. (**Figure 1D**)

Merr.

DC.

**yr−1**

227

Not available

100 [103]

available

150 [103]

Angola, Mozambique, Namibia, South Africa, Swaziland, Tanzania, Zaire,

The Potential of Tree and Shrub Legumes in Agroforestry Systems

Africa, Asia, Australia [13] 84 [102]

http://dx.doi.org/10.5772/intechopen.69995

India, SE Asia [13] Not

Tropical Africa, SE Asia

[13, 91]

Zambia [13]

In Africa, tree and shrub legumes provide a wealth of goods and services (e.g. wood, food, medicines, energy and housing) to millions of rural and urban dwellers (**Table 2** and **Figure 1**) [25, 26]. The interest on this group of legumes has increased tremendously in the last decades, particularly regarding soil erosion control [27, 28] and farming systems (e.g. windbreaks, shade trees, nitrogen fertilizers, forage, fruits and vegetables) [29–32].



(energy). Although it is one of the most abundant elements in the Earth, its predominant form,

Harber‐Bosch process, or biologically through bacterial nitrogen fixation [20]. While chemical nitrogen fixation is cost intensive and 40–50% of the nitrogen applied as fertilizer is lost via denitrification, runoff or leaching, only 10–20% of the biologically fixed nitrogen is lost that way [21]. Besides that, the use of chemical fertilizers has a series of ecological impacts, such

between legumes and rhizobia towards the improvement of agricultural systems, i.e. better

In Africa, tree and shrub legumes provide a wealth of goods and services (e.g. wood, food, medicines, energy and housing) to millions of rural and urban dwellers (**Table 2** and **Figure 1**) [25, 26]. The interest on this group of legumes has increased tremendously in the last decades, particularly regarding soil erosion control [27, 28] and farming systems (e.g. windbreaks,

**Species Applications Origin kg.N.ha−1.**

as air, soil and water pollution [22]. Thus, there is a strong interest in symbiotic N2

(g), cannot be directly assimilated by the plants, which need reduced forms of this ele‐

) [18, 19]. This conversion can be achieved chemically through the

fixation

**yr−1**

Not available

Not available

260 [91] 86 [94] 96 [102] 142 [103]

210 [104] 35–38 [105] 108 [106]

Not available

28.7–46.7 [33] 7–12 [93] less than 20 [94]

Drier tropical Africa, from Senegal and Mauritania (west) to Eritrea and Ethiopia (north‐east) and South Africa (south); Oman, Pakistan and

Angola, Botswana, Malawi, Mozambique, Tanzania, Zaire, Zambia, Zimbabwe

Angola, Kenya, Malawi, Mozambique, Tanzania, Zaire, Zambia, Zimbabwe

Unknown origin, probably Indian and African [13, 101];

Central America, Caribbean, South America, Asia (Java and Peninsular Malaysia) [13]

Mexico and Guatemala

[13, 91]

India [92]

[13]

[13]

India [100]

i.e. N2

ment (NH4

*Brachystegia boehmii* Taub. (**Figure 1A**)

*Brachystegia spiciformis* Benth. (**Figure 1B**)

*Cajanus cajan* (L.) Millsp. (**Figure 1C**)

*Gliricidia sepium* (Jacq.)

*Leucaena collinsii* Britton

Walp.

& Rose1

+ , NO2 −

226 Nitrogen in Agriculture - Updates

and NO3

−

productivity with the least ecological impact [23, 24].

*Acacia senegal* (L.) Willd. Poles, household, agriculture crafts,

shade trees, nitrogen fertilizers, forage, fruits and vegetables) [29–32].

firewood, charcoal (stem and branches); tannin, ropes (bark); food (pods and seeds); forage (foliage and pods); honey (flowers); gum Arabic (Gum), medicine (various); erosion control; nitrogen fixation; fertilizer;

Small articles, firewood (stem and branches); ropes, twine, cloth and fishing nets, tanning, beehives (bark); food for edible larvae (leaves); medicinal (various) [25, 95–97]

Construction, furniture, household items, firewood, charcoal (stem and branches); tannin, beehives, ropes, sacks (bark); forage (foliage and pods); honey (flowers); medicinal (various); nitrogen fixation,

Light construction, baskets, fuel (stems and branches); forage (vegetative parts); honey (flowers); food (seeds and pods); medicinal (various); erosion control; shading, sheltering, nitrogen fixation; fertilizer;

fencing; intercropping [90, 91]

shading [91, 98, 99]

intercropping [91, 100]

Farm implements, furniture, posts, firewood, charcoal (stem and branches); forage (foliage and pods); honey and food (flowers); medicine and rodenticide (various); erosion control; shading; nitrogen

fixation; fertilizer; fencing [91]

Timber, firewood (stems and branches); forage (leaves); food (seeds); similar to gum Arabic (Gum); shading; nitrogen fixation; fertilizer; fencing; intercropping [91]

**Table 2.** Examples of tree and shrub legumes and their applications in formal and informal economies.

**Figure 1.** Details from *Brachystegia boehmii* leaves (A); *Brachystegia spiciformis* leaves and flowers (B); *Cajanus cajan* leaves, flowers and pods (C); *Pterocarpus angolensis* young leaves and mature fruit (D). Credits to Moura (A, D) and Catarino (B, C).

## **3. Importance and role in agroforestry tropical systems**

Most tree and shrub legumes are resilient to extreme environments, e.g. erosion, low fertil‐ ity, salinity, drought, fire and other adverse conditions [33–36]. Such abilities seem to be innate and enhanced by the symbiosis with N2 ‐fixing rhizobia [30, 37]. According to Diabate et al. [30] and Sprent [38], the use of nitrogen‐fixing tree and shrubs (NFTSs) constitute a promising strategy to recover soil fertility, representing a sustainable agricultural approach to smallholder farmers. This is particularly important in sub‐Saharan Africa where 80% of the farmland is managed by smallholders whose livelihoods depend strongly on the agricultural sector [6]. Most of these households live below the poverty line and therefore cannot afford the use of fertilizers. For example, smallholders from Niger, Namibia and Mozambique use less than 1 kg.N.ha−1.yr−1, i.e., 100 times less than the average fertilizer needs for most crops [1, 34].

The rates of N2 fixation by NFTS depend on the species, climate and soil type, ranging from 0.1 to 700 kg.N.ha−1.yr−1 (**Table 2**) [33, 39, 40]. Despite the fact that many genera from the sub‐ families Mimosoideae and Caesalpinioideae do not always establish root‐nodule symbiosis, under proper environmental conditions, many species nodulate and fix atmospheric N at rates closer to those obtained with the traditional legumes belonging to the Papilionoideae [33]. Additionally there is also evidence that NTFS are also able to increase P availability in the soil, mostly due to mycorrhizal associations [41].

coffee plantations were above 40 kg.N.ha−1.yr−1, corresponding to 53% of the average amount of fertilizer applied annually. This observation reinforces the importance of the use of non‐ crop legumes in coffee agro‐ecosystems [48–50]. According to the literature [49, 50], *Inga* spp. is the most popular choice from Mexico to Nicaragua. *G. sepium* and *Erythrina poeppigiana* are often the common choice in the low‐lying areas of Honduras and Nicaragua and Costa Rica, respectively [51]. In Africa, similar systems may constitute a promising and sustainable solu‐

The Potential of Tree and Shrub Legumes in Agroforestry Systems

http://dx.doi.org/10.5772/intechopen.69995

229

**Figure 2.** Agroforestry system with *Albizia* sp., coffee and maize in Gorongoza, Mozambique. Credits to Stalmans.

Legume research has been mainly focused on annual grain crops [34, 52–54]. Instead, a lim‐ ited amount of knowledge has been produced in perennials. In this section, we will discuss the potential of the two promising strategies to analyse nitrogen mineralization and metabo‐

The use of stable isotopes at natural abundance levels has brought a new dimension to our under‐ standing of plant physiology and ecology. Analyses of the relative natural abundances of stable isotopes of carbon (13C/12C), oxygen (18O/16O), nitrogen (15N/14N) and deuterium (D/H) have been used across a wide range of scales, from cell to community and ecosystem level, contributing much to our understanding of the interactions between biosphere, pedosphere and atmosphere. In general terms, processes such as diffusion and enzymatic incorporation favour the lighter isotope and lead to depletion of the heavier isotope as compared to source material. Natural abundance of 15N can provide valuable information about N sources used by plants and fluxes of N in the ecosystems [e.g. 55–57]. There has been some debate on the interrelationship between nitrogen natural abundance (15N/14N) in soils and plants, and the use of a tracer or indicator of fractionation during N‐uptake, assimilation and transport [58, 59]. Indeed, a vari‐ ety of fractionations may occur during processes related to nitrogen transformation in soils

tion to improve coffee (or fruits) productivity in the region.

lism in tree legumes, i.e. stable isotopes and, briefly, systems biology.

**4. Towards scientific knowledge**

**4.1. Stable isotopes**

The use of fertilizer tree legumes (*Acacia anguistissima, Cajanus cajan, Gliricidia sepium*, *Leucaena collinsii, Sesbania sesban, Tephrosia candida* and *Tephrosia vogelii*) for sustainable maize (*Zea mays*) production has been analysed by Akinnifesi and collaborators [42] in East and Southern Africa (Zambia, Zimbabwe, Malawi and Tanzania). The authors reported a contribution of more than 60 kg.N.ha−1.yr−1 through biological nitrogen fixation (BNF), reducing the need of chemical N fertilizers in 75%. Besides that, N‐fertilizer trees substantially increased crop yield, providing evidence that together with good management practices, maize yields can double as compared with traditional practices (without mineral fertilization). In Zambia, Mafongoya and Jiri [43] have analysed the use of *G. sepium* as green manure for cabbage (*Brassica oleracea*) and onion (*Allium cepa*) production. This practice produced higher crop yields than the unfertilized and full rate fertilized controls: ca. 16 (unfertilized), 43 (full rate fertilized), 48 (gliricidia) and 65 ton. ha−1 (half rate fertilizer and gliricidia) for cabbage and 22 (unfertilized), 43 (full rate fertilized), 65 (gliricidia) and 85 ton.ha−1 (half rate fertilizer and gliricidia) for onion. In addition, gliricidia biomass replenished the soil with residual N, which was used by a subsequent crop, maize. In this case, the yields obtained (ca. 3–5 ton.ha−1) were similar or slightly higher than those obtained with full rate fertilizer (ca. 2.5 to 4 ton.ha−1) and unfertilized crop (ca. 1.5–3 ton.ha−1). Nevertheless, caution should be paid to the potential environmental hazard of NO3 leaching and resultant eutrophication [43–45], as well as to the choice of the best crop(s)/NFTS combination(s) and cropping type (intercropping, multistrata or fallows) [46–47].

Another interesting NFTS‐based agroforestry system is the tree cropping system, like those used for coffee production (**Figure 2**). Such system is very popular in Latin America [48–51] and less exploited in Africa. In Mexico, the rates of N<sup>2</sup> fixation obtained by *Inga jinicuil* in

**Figure 2.** Agroforestry system with *Albizia* sp., coffee and maize in Gorongoza, Mozambique. Credits to Stalmans.

coffee plantations were above 40 kg.N.ha−1.yr−1, corresponding to 53% of the average amount of fertilizer applied annually. This observation reinforces the importance of the use of non‐ crop legumes in coffee agro‐ecosystems [48–50]. According to the literature [49, 50], *Inga* spp. is the most popular choice from Mexico to Nicaragua. *G. sepium* and *Erythrina poeppigiana* are often the common choice in the low‐lying areas of Honduras and Nicaragua and Costa Rica, respectively [51]. In Africa, similar systems may constitute a promising and sustainable solu‐ tion to improve coffee (or fruits) productivity in the region.

## **4. Towards scientific knowledge**

Legume research has been mainly focused on annual grain crops [34, 52–54]. Instead, a lim‐ ited amount of knowledge has been produced in perennials. In this section, we will discuss the potential of the two promising strategies to analyse nitrogen mineralization and metabo‐ lism in tree legumes, i.e. stable isotopes and, briefly, systems biology.

#### **4.1. Stable isotopes**

**3. Importance and role in agroforestry tropical systems**

innate and enhanced by the symbiosis with N2

the soil, mostly due to mycorrhizal associations [41].

needs for most crops [1, 34].

228 Nitrogen in Agriculture - Updates

The rates of N2

Most tree and shrub legumes are resilient to extreme environments, e.g. erosion, low fertil‐ ity, salinity, drought, fire and other adverse conditions [33–36]. Such abilities seem to be

et al. [30] and Sprent [38], the use of nitrogen‐fixing tree and shrubs (NFTSs) constitute a promising strategy to recover soil fertility, representing a sustainable agricultural approach to smallholder farmers. This is particularly important in sub‐Saharan Africa where 80% of the farmland is managed by smallholders whose livelihoods depend strongly on the agricultural sector [6]. Most of these households live below the poverty line and therefore cannot afford the use of fertilizers. For example, smallholders from Niger, Namibia and Mozambique use less than 1 kg.N.ha−1.yr−1, i.e., 100 times less than the average fertilizer

0.1 to 700 kg.N.ha−1.yr−1 (**Table 2**) [33, 39, 40]. Despite the fact that many genera from the sub‐ families Mimosoideae and Caesalpinioideae do not always establish root‐nodule symbiosis, under proper environmental conditions, many species nodulate and fix atmospheric N at rates closer to those obtained with the traditional legumes belonging to the Papilionoideae [33]. Additionally there is also evidence that NTFS are also able to increase P availability in

The use of fertilizer tree legumes (*Acacia anguistissima, Cajanus cajan, Gliricidia sepium*, *Leucaena collinsii, Sesbania sesban, Tephrosia candida* and *Tephrosia vogelii*) for sustainable maize (*Zea mays*) production has been analysed by Akinnifesi and collaborators [42] in East and Southern Africa (Zambia, Zimbabwe, Malawi and Tanzania). The authors reported a contribution of more than 60 kg.N.ha−1.yr−1 through biological nitrogen fixation (BNF), reducing the need of chemical N fertilizers in 75%. Besides that, N‐fertilizer trees substantially increased crop yield, providing evidence that together with good management practices, maize yields can double as compared with traditional practices (without mineral fertilization). In Zambia, Mafongoya and Jiri [43] have analysed the use of *G. sepium* as green manure for cabbage (*Brassica oleracea*) and onion (*Allium cepa*) production. This practice produced higher crop yields than the unfertilized and full rate fertilized controls: ca. 16 (unfertilized), 43 (full rate fertilized), 48 (gliricidia) and 65 ton. ha−1 (half rate fertilizer and gliricidia) for cabbage and 22 (unfertilized), 43 (full rate fertilized), 65 (gliricidia) and 85 ton.ha−1 (half rate fertilizer and gliricidia) for onion. In addition, gliricidia biomass replenished the soil with residual N, which was used by a subsequent crop, maize. In this case, the yields obtained (ca. 3–5 ton.ha−1) were similar or slightly higher than those obtained with full rate fertilizer (ca. 2.5 to 4 ton.ha−1) and unfertilized crop (ca. 1.5–3 ton.ha−1).

Nevertheless, caution should be paid to the potential environmental hazard of NO3

and cropping type (intercropping, multistrata or fallows) [46–47].

and less exploited in Africa. In Mexico, the rates of N<sup>2</sup>

resultant eutrophication [43–45], as well as to the choice of the best crop(s)/NFTS combination(s)

Another interesting NFTS‐based agroforestry system is the tree cropping system, like those used for coffee production (**Figure 2**). Such system is very popular in Latin America [48–51]

fixation by NFTS depend on the species, climate and soil type, ranging from

‐fixing rhizobia [30, 37]. According to Diabate

leaching and

fixation obtained by *Inga jinicuil* in

The use of stable isotopes at natural abundance levels has brought a new dimension to our under‐ standing of plant physiology and ecology. Analyses of the relative natural abundances of stable isotopes of carbon (13C/12C), oxygen (18O/16O), nitrogen (15N/14N) and deuterium (D/H) have been used across a wide range of scales, from cell to community and ecosystem level, contributing much to our understanding of the interactions between biosphere, pedosphere and atmosphere.

In general terms, processes such as diffusion and enzymatic incorporation favour the lighter isotope and lead to depletion of the heavier isotope as compared to source material. Natural abundance of 15N can provide valuable information about N sources used by plants and fluxes of N in the ecosystems [e.g. 55–57]. There has been some debate on the interrelationship between nitrogen natural abundance (15N/14N) in soils and plants, and the use of a tracer or indicator of fractionation during N‐uptake, assimilation and transport [58, 59]. Indeed, a vari‐ ety of fractionations may occur during processes related to nitrogen transformation in soils [60, 61] and plants [59], which may complicate source‐sink relationships. For example, nitrifi‐ cation discriminates against 15N more than N mineralization, which makes NH4 + isotopically heavier than the organic N from which it is derived [60]. Additionally, the δ15N of a particular compound may change and, together with the complexity of the N geochemical cycle, the use of δ15N should be carefully evaluated when applied to natural ecosystems.

*Colophospermum mopane*) to abiotic stresses, namely high temperatures, drought and low soil fer‐ tility. Preliminary results indicate that these plants have an innate ability to cope with extreme environments and that such capacity is linked to an enhanced water and mineral use efficiency [88], reinforcement of the photosynthetic machinery and the antioxidant system as well as an

The Potential of Tree and Shrub Legumes in Agroforestry Systems

http://dx.doi.org/10.5772/intechopen.69995

231

Agriculture has a primordial role to fight poverty and hunger and increase crop resilience to climate changes. The introduction of tree and shrub nitrogen‐fixing trees into cropping systems is the most straightforward approach to reduce the use of chemical fertilizers, improving the soil ecosystem and the livelihoods of smallholder farmers in southern Africa. Additionally, agrofor‐ estry may improve ecosystem services such as, soil organic matter, biodiversity and N‐retention. However, it is not devoid of environmental consequences, specifically N‐leaching. Therefore, the implementation of agroforestry systems with NFTS should be preceded by experimental assays, in order to identify the factors promoting N‐losses and design appropriate management

The authors like to acknowledge Fundação para a Ciência e a Tecnologia through the research units UID/AGR/04129/2013 (LEAF), UID/GEO/04035/2013 (GeoBioTec), CE3C, Fundo para a Investigação Aplicada e Multissectorial. The authors thank Luís Catarino (CE3C), and Mark Stalmans (E.O. Wilson Biodiversity, Lab. Gorongoza National Park) for kindly providing the

, José C. Ramalho1,2,

strategies that synchronize legume‐N availability with the crop demand.

Ana I. Ribeiro‐Barros1,2\*, Maria J. Silva1,2, Isabel Moura1

\*Address all correspondence to: aribeiro@itqb.unl.pt

and Natasha S. Ribeiro4

(LEAF), School of Agriculture (ISA), University of Lisbon (ULisboa), Portugal

1 Plant Stress & Biodiversity Lab, Linking Landscape, Environment, Agriculture and Food

2 GeoBioTec, Faculdade de Ciências e Tecnologia (FCT), Universidade NOVA de Lisboa

3 Center for Ecology, Evolution and Environmental Changes (cE3c), Faculdade de Ciências,

4 Faculty of Agronomy and Forest Engineering, Eduardo Mondlane University, Maputo,

elevated osmoprotection state [unpublished data; 89].

**5. Concluding remarks**

**Acknowledgements**

photographs.

**Author details**

(UNL), Portugal

Mozambique

Cristina Máguas‐Hanson3

Universidade de Lisboa, Portugal

However, there are substantial evidence that the natural abundance ratios for 15N/14N in soil and plants are useful integrators of the types and turnover rates of N cycling [62–64]. These ratios can indicate whether a range of plants have access to the same N source [59]. For instance, differences in leaf δ15N can indicate differences in rooting depth or root characteristics, such as mycorrhizal or N‐fixing root associations [59, 65]. Also, nitrification and plant uptake properties (such as timing and type of uptake) can be determined by the leaf δ15N signatures [66, 67]. Robinson [59] developed a mixing model to account for contrasting N sources which provided useful insights on the quantification of biological N fixation in tree legumes [68, 69]. Since N2 ‐fixing species typically have δ15N signatures close to the atmospheric value (0%), which strongly differ from the δ15N signature of non‐fixing species, δ15N can be used as a sensitive tracer of N flow within an ecosystem. This approach was successfully used in the oligotrophic Portuguese primary dunes utilizing foliar δ15N of the non‐leguminous native shrub *Corema album* [70–72]. As the invasive *Acacia longifolia* and the native *Stauracanthus spectabilis* were the only legumes co‐occurring with *C. album*, with no further sources of organic matter, this system represents an ideal model to quantify the impact of *A. longifolia* invasion. Similar to other ericoid δ15N mycorrhizal plants [73], *C. album* exhibited depleted foliar values without legume influence which, together with its high abundance in this system, may function as a good monitoring plant for legume influence [71].

#### **4.2. Systems biology**

Systems biology is an emerging approach applied to biological scientific research that focuses on the complex interactions within biological systems, frequently associated with the envi‐ ronmental conditions. The best known example is the *Human Genome Project* which allowed major advances in human genetics and in the development of new medical therapies [74, 75]. Systems biology, commonly called '*Omics'* is associated with high‐throughput analysis of e.g. genomes (DNA, *genomics*), transcriptomes (RNA, *transcriptomics*), proteomes (proteins, *proteomics*), metabolomes (metabolites, *metabolomics*), lipidomes (lipids, *lipidomics*) and inter‐ actomes (interactions between molecules, *interactomics*) coupled with bioinformatics, which integrates computational, statistical and mathematical modelling [76, 77]. In plants, systems biology has been essentially focused on models, such as arabidopsis and annual crops (e.g. rice, wheat, tomato, soybean, maize, sorghum, chickpea or groundnut) [78–85]. Systems biol‐ ogy research in perennial plants is still restricted to a small group of trees, namely eucalyptus, poplar, abies and pine (reviewed in Refs. [86, 87]). Among others, such studies led to sig‐ nificant advances on the global knowledge of plant biology (development and functioning), genomics‐assisted breeding towards the production of crops tolerant to extreme temperatures, salinity, drought, pests and diseases, or the discovery of new bio‐compounds with application in agriculture, medicine and in a wide range of industries [78, 79, 86, 87].

In our laboratory, we have recently initiated an integrated approach, combining eco‐physiology and system's biology to understand the responses of two tree legumes (*Brachystegia boehmii* and *Colophospermum mopane*) to abiotic stresses, namely high temperatures, drought and low soil fer‐ tility. Preliminary results indicate that these plants have an innate ability to cope with extreme environments and that such capacity is linked to an enhanced water and mineral use efficiency [88], reinforcement of the photosynthetic machinery and the antioxidant system as well as an elevated osmoprotection state [unpublished data; 89].

## **5. Concluding remarks**

[60, 61] and plants [59], which may complicate source‐sink relationships. For example, nitrifi‐

heavier than the organic N from which it is derived [60]. Additionally, the δ15N of a particular compound may change and, together with the complexity of the N geochemical cycle, the use

However, there are substantial evidence that the natural abundance ratios for 15N/14N in soil and plants are useful integrators of the types and turnover rates of N cycling [62–64]. These ratios can indicate whether a range of plants have access to the same N source [59]. For instance, differences in leaf δ15N can indicate differences in rooting depth or root characteristics, such as mycorrhizal or N‐fixing root associations [59, 65]. Also, nitrification and plant uptake properties (such as timing and type of uptake) can be determined by the leaf δ15N signatures [66, 67]. Robinson [59] developed a mixing model to account for contrasting N sources which provided useful insights

typically have δ15N signatures close to the atmospheric value (0%), which strongly differ from the δ15N signature of non‐fixing species, δ15N can be used as a sensitive tracer of N flow within an ecosystem. This approach was successfully used in the oligotrophic Portuguese primary dunes utilizing foliar δ15N of the non‐leguminous native shrub *Corema album* [70–72]. As the invasive *Acacia longifolia* and the native *Stauracanthus spectabilis* were the only legumes co‐occurring with *C. album*, with no further sources of organic matter, this system represents an ideal model to quantify the impact of *A. longifolia* invasion. Similar to other ericoid δ15N mycorrhizal plants [73], *C. album* exhibited depleted foliar values without legume influence which, together with its high abundance in this system, may function as a good monitoring plant for legume influence [71].

Systems biology is an emerging approach applied to biological scientific research that focuses on the complex interactions within biological systems, frequently associated with the envi‐ ronmental conditions. The best known example is the *Human Genome Project* which allowed major advances in human genetics and in the development of new medical therapies [74, 75]. Systems biology, commonly called '*Omics'* is associated with high‐throughput analysis of e.g. genomes (DNA, *genomics*), transcriptomes (RNA, *transcriptomics*), proteomes (proteins, *proteomics*), metabolomes (metabolites, *metabolomics*), lipidomes (lipids, *lipidomics*) and inter‐ actomes (interactions between molecules, *interactomics*) coupled with bioinformatics, which integrates computational, statistical and mathematical modelling [76, 77]. In plants, systems biology has been essentially focused on models, such as arabidopsis and annual crops (e.g. rice, wheat, tomato, soybean, maize, sorghum, chickpea or groundnut) [78–85]. Systems biol‐ ogy research in perennial plants is still restricted to a small group of trees, namely eucalyptus, poplar, abies and pine (reviewed in Refs. [86, 87]). Among others, such studies led to sig‐ nificant advances on the global knowledge of plant biology (development and functioning), genomics‐assisted breeding towards the production of crops tolerant to extreme temperatures, salinity, drought, pests and diseases, or the discovery of new bio‐compounds with application

In our laboratory, we have recently initiated an integrated approach, combining eco‐physiology and system's biology to understand the responses of two tree legumes (*Brachystegia boehmii* and

in agriculture, medicine and in a wide range of industries [78, 79, 86, 87].

+

isotopically

‐fixing species

cation discriminates against 15N more than N mineralization, which makes NH4

on the quantification of biological N fixation in tree legumes [68, 69]. Since N2

**4.2. Systems biology**

230 Nitrogen in Agriculture - Updates

of δ15N should be carefully evaluated when applied to natural ecosystems.

Agriculture has a primordial role to fight poverty and hunger and increase crop resilience to climate changes. The introduction of tree and shrub nitrogen‐fixing trees into cropping systems is the most straightforward approach to reduce the use of chemical fertilizers, improving the soil ecosystem and the livelihoods of smallholder farmers in southern Africa. Additionally, agrofor‐ estry may improve ecosystem services such as, soil organic matter, biodiversity and N‐retention. However, it is not devoid of environmental consequences, specifically N‐leaching. Therefore, the implementation of agroforestry systems with NFTS should be preceded by experimental assays, in order to identify the factors promoting N‐losses and design appropriate management strategies that synchronize legume‐N availability with the crop demand.

## **Acknowledgements**

The authors like to acknowledge Fundação para a Ciência e a Tecnologia through the research units UID/AGR/04129/2013 (LEAF), UID/GEO/04035/2013 (GeoBioTec), CE3C, Fundo para a Investigação Aplicada e Multissectorial. The authors thank Luís Catarino (CE3C), and Mark Stalmans (E.O. Wilson Biodiversity, Lab. Gorongoza National Park) for kindly providing the photographs.

## **Author details**

Ana I. Ribeiro‐Barros1,2\*, Maria J. Silva1,2, Isabel Moura1 , José C. Ramalho1,2, Cristina Máguas‐Hanson3 and Natasha S. Ribeiro4

\*Address all correspondence to: aribeiro@itqb.unl.pt

1 Plant Stress & Biodiversity Lab, Linking Landscape, Environment, Agriculture and Food (LEAF), School of Agriculture (ISA), University of Lisbon (ULisboa), Portugal

2 GeoBioTec, Faculdade de Ciências e Tecnologia (FCT), Universidade NOVA de Lisboa (UNL), Portugal

3 Center for Ecology, Evolution and Environmental Changes (cE3c), Faculdade de Ciências, Universidade de Lisboa, Portugal

4 Faculty of Agronomy and Forest Engineering, Eduardo Mondlane University, Maputo, Mozambique

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233

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## *Edited by Amanullah and Shah Fahad*

Nitrogen is the most yield-restraining nutrient in crop production globally. Efficient nitrogen management is one of the most important factor for improving nitrogen use efficiency, field crops productivity and profitability. Efficient use of nitrogen for crop production is therefore very important for increasing grain yield, maximizing economic return and minimizing nitrous oxide (N2 O) emission from the fields and nitrate (NO3 ) leaching to ground water. Integrated nitrogen management is a good strategy to improve plant growth, increase yield and yield components, grain quality and reduce environmental problems. Integrated nitrogen management (combined use of chemical + organic + bio-fertilizers) in field crop production is more resilient to climate change.

Nitrogen in Agriculture - Updates

Nitrogen in Agriculture

Updates

*Edited by Amanullah and Shah Fahad*

Photo by phanasitti / iStock