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From the human nutrition point of view rice and wheat are the most important cereals and their production in north-west India in rice-wheat cropping system, which covers about 10 million hectares, is the backbone of the India's food security (Prasad 2005). Rice-Wheat cropping system produces 5-14 t/ha/yr grain and this depends heavily on nitrogen fertilization which ranges from 100-150 kg N/ha/crop or even more, especially in rice. From the animal nutrition point of view maize, sorghum and pearl millet stovers which contain 27 to 51% of nitrogen harvested by the crop in stover are more important both for milch as well draught cattle. On the contrary rice and wheat straw is low in nitrogen content and is a poor protein source. Nevertheless they meet majority calorie requirements of the cattle. Also most sorghum and pearlmillet is grown in rainfed areas where nitrogen application rates are low and even response to N application is low. Nitrogen removal per metric ton as well as its percentage in grain in pulses depends very much upon the plant stature and its vegetative growth. For example Prasad et al. (2004) reported a removal of 50.6 kg/t in chickpea and 92.1 kg/t in pigeon pea; these are the two major pulse crops in India. Most of this nitrogen is obtained by N-fixation by Rhizobia as very little fertilizer N is applied to pulses. Again depending upon the plant stature and vegetative growth 63.3% of total N removed by chickpea was contained in its grain, while the values for pigeon pea a tall and heavily fertilized plant was 31.6%. The protein rich pulse foliage is widely used for enriching rice or wheat straw fed to cattle. Before the mechanization of Indian agriculture which is even now limited mostly to north-western India, draught animals were the major source of farm power and the Indian agriculture provided a characteristic `humans-animals-crops' ecosystem where man survived on the grains and the animals on the straw/stover. Taking an average N contribution by grain legumes at 30 kg N/ha about 0.66 million metric tons of N is annually added to soil on 22 million hectares occupied by them. Another 0.34 million metric tons N may be added by leguminous trees and plants in forests and grasslands and by leguminous oilseed crops such as groundnut. Thus the N contribution of legumes in Indian soils can be roughly estimated at least at 1 million metric tons, it is likely to be much more. In addition some N is added by rains and use of N-fixing biofertilizer such as Azotobacter, Azospirillum, Acetobacter, Blue-green algae and Azolla.
Concept of Nitrogen Use Efficiency (NUE)
Nitrogen use efficiency (NUE) at the plant level is its ability to utilize the available nitrogen (N) resources to optimize its productivity (Raghuram et al. 2006). As a concept, NUE includes N uptake, utilization or acquisition efficiency, expressed as a ratio of the total plant N, grain N, biomass yield , grain yield (output) and total N, soil N or N-fertilizer applied (input) (Pathak et al. 2008). NUE is quantified based on apparent nitrogen recovery using physiological and agronomic parameters. Agronomic efficiency is an integrative index of total economic outputs relative to the available soil N (native and applied). Apparent nitrogen recovery is related to the efficiency of N uptake; physiological NUE deals with N utilization to produce grain or total plant dry matter. Nitrogen use efficiency (NUE) in the context of photosynthesis is called as photosynthetic nitrogen use efficiency (PNUE), which is determined by the rate of carbon assimilation per unit leaf nitrogen (Kumar et al. 2002). The most suitable way to estimate NUE depends on the crop, its harvest product and the processes involved in it.
Strategies for minimizing N-pollution in Agriculture
Various strategies were adopted to minimize the N-loss from the agricultural fields. Split application of N, use of slow-release fertilizers, nitrification inhibitors and the use of organic manures are some agronomic techniques used. Bulk of the fertilizer nitrogen in India is broadcast on surface and both surface run-off (on slopy lands) and ammonia volatilization lead to N losses. This can be easily overcome by deep placement of N a few centimetres below soil surface. For example, Sarkar et al. (2005) showed that in wheat surface broadcast application of urea as band or top dressing caused 15-20% loss of N due to agriculture volatilization. Surface broadcast application followed by its mixing with top soil reduced the volatilization loss to 10%, while side band placement of urea reduced it further to only 5%. Thus the farmers need to be told about the advantage of incorporation in surface soil or if possible its placement using a ferti-drill or a pora in upland crops. Split application is a well established technique for increasing NUE. In wheat and maize, studies with 15N showed that application of 40 kg N/ha as basal followed by 60 kg N/ha at crown root initiation (CRI) gave significantly higher yield than all basal application and other split application combinations (Sachdev et al. 2000; Narang et al. 2000). Havangi and Hegde (1983) showed in pearlmillet also two or three split applications were found to be better than a single application. In rice two split applications are recommended for short and medium duration varieties, while three split applications are recommended for long duration varieties (Prasad 1999). Another way is nitrification inhibitors (NI's), these are a group of chemicals that are toxic to Nitrosomonas sp. and Nitrosomonas sp. involved in the conversion of NH4 to NO2- as well as to Nitrobacter sp. involved in the conversion of NO2 to NO3 and therefore, inhibits nitrification, which reduces losses due to leaching and denitrification. The most widely tested NI's are N-serve (2-chloro-6-trichloromethyl pyridine), AM (2 amino-4-chloro-6 methyl pyrimidine), DCD (Dicyandiamide) and ST (sulphathiazole) (Prasad and Power 1995). Research on the use of NI's for reducing N losses and increasing NUE from the soil was initiated in India by Prasad (1999) at the Indian Agricultural Research Institute (IARI), New Delhi with a field experiment on rice. Treatment of ammonium sulphate with N-Serve significantly increased rice yield and nitrogen uptake by the rice crop Prasad (2005) showed from a laboratory experiment that N losses due to denitrification could be considerably reduced by treating ammonium sulphate with NI's N-Serve and AM. Prasad and Prasad (1996) showed through field experiments that treatment of urea with NI's, N-Serve and AM significantly increased rice yield and N uptake. Das et al. (2004) showed the effect of N-serve and AM on nitrification under field capacity moisture (upland) and water-logged (low-land paddy) conditions at New Delhi. Both the NI's were effective in retarding nitrification. The nitrification rate (nitrates expressed as percentage of total mineral N) after 40 days of incubation was 78% with N-Serve at 2 ppm and 76% with AM at 10 ppm (mg/kg) as against 100% with untreated urea. Slow-release N fertilizers (SRF's) were developed with an aim to slow-down the dissolution of applied N so that most of it is taken up by crop plants rather than be subjected to N loss mechanisms. There are two kinds of slow-release N fertilizers, namely, coated fertilizers and inherently slow dissolution rate materials. The examples of coated slow-release N fertilizers are sulphur coated urea (developed by TVA, USA), lac coated urea (developed by Indian Lac Research Institute), polymer coated urea and to some extent neem cake coated urea. The other kind of slow-release fertilizers are generally urea-aldehyde condensates e.g. urea-form (urea and formaldehyde products developed in USA), isobutylidene diurea or IBDU (urea and isobutyraldehyde product developed in Japan and USA) and CD-urea (urea and crotonaldehyde product developed in Germany) (Prasad 2005). After 20 days of incubation under field capacity conditions the mineral-N (NH4+ NO3-) in soil was 67, 43, 31 and 27 ppm (mg/kg soil) with urea, oxamide, isobutylidene diurea (IBDU) and sulphur coated urea (SCU), respectively. As would be expected under submerged conditions, NO3--N was not detected and the NH4+-N content in soil after 20 days of incubation was 67, 61, 46 and 15 ppm with urea, oxamide, IBDU and SCU, respectively. Thus of the 3 SRF's oxamide released the N the fastest and SCU the slowest.
Physiological and Molecular aspects for improving Nitrogen use efficiency
Nitrogen use efficiency (NUE) at the plant level is its ability to utilize the available nitrogen (N) resources to optimize its productivity. In terms of agriculture, it is the optimal utilisation of nitrogenous manures or fertilisers for plant growth, yield and protein content, as atmospheric nitrogen gas is not utilised by higher plants, except symbiotic legumes. The inherent efficiency of the plant to utilize available N for higher productivity needs to be tackled biologically (Abrol et al. 1999, Abdin et al. 2005). This includes uptake, assimilation and redistribution of nitrogen within the cell and balance storage and current use at the cellular and whole plant level. Moreover, since N demand and its actual availability tend to vary in time, space and environmental conditions, the regulation of plant nitrogen metabolism must be responsive to nutritional, metabolic and environmental cues.
Regulation of nitrate uptake
Plants have evolved an active, regulated and multiphasic transport system making their NO3- uptake scheme efficient enough to transport sufficient NO3- to satisfy total nitrogen demand of the plant in face of varying external NO3- concentrations. Plants can also take up other forms of nitrogen, such as amino acids and ammonium ions. Root NH4+ uptake is carried out by both high affinity and low affinity NH4+ transporters that are encoded by a multigene family (Glass et al. 2002). However, nitrate is the most abundant form of nitrogen available to the plant roots in aerated soils. Nitrate influx is an active process driven by the H+ gradient and can work against an electrochemical potential gradient (Vidmar et al. 2000). The uptake involves high and low affinity transport systems, also known as HATS and LATS respectively (Forde 2000). One of the high affinity systems is strongly induced in presence of NO3- and is known as inducible high affinity transport system (or iHATS,), while the second high affinity system (the cHATS) and LATS are constitutively expressed (Aslam et al. 1993; Glass et al. 1995; Forde 2002). The Km values of iHATS, cHATS and LATS for nitrate are in the ranges of 13-79 æM, 6-20 æM and >1mM respectively.
The iHATS is a multicomponent system encoded partly by genes of the NRT2 family or nitrate - nitrite porter family of transporters. Recently, two dual affinity transporters have been identified in Arabidopsis, AtKUP1 and AtNRT1.1, of which the latter is induced as HATS by phosphorylation at threonine residue 101. This family of transporters is recognized as being exceptional in both the variety of different substrates which its members can mobilise (oligopeptides, amino acids, NO3-, chlorate) and in the ability of individual transporters to handle substrates of very different sizes and charges. Nitrate acts as a regulator for its own uptake, a specific property which is not seen in other ion transport systems such as phosphate, sulphate etc. On exposure of the cells to external NO3- the uptake capacity increases after a lag period of 0.5 to 1.5 hours and reaches a new steady state after 4 to 6 hrs. Use of RNA and protein synthesis inhibitors provided early evidence that induction of the iHATS involves gene expression and the synthesis of new transporter protein (Aslam et al. 1993). The evidence that the inducer of iHATS is indeed nitrate ion and not its downstream metabolite came from NR-deficient mutants of Arabidopsis and N. plumbaginifolia (Krapp et al. 1998; Lejay et al. 1999). Studies in the last decade have shown that enhancing the uptake of N by overexpressing transporters may not necessarily improve NUE. For example, transgenic overexpression of a CHL1 cDNA (representing the constitutive HATS) driven by the cauliflower mosaic virus 35S promoter in a chl1 mutant, recovered the phenotype for the constitutive phase but not for the induced phase (Liu et al. 2003). Similarly, the NO3- contents in transgenic tobacco plants overexpressing the NpNRT2.1 gene (encoding HATS), were remarkably similar to their wild-type levels, despite an increase in the NO3- influx. These findings indicate that genetic manipulation of nitrate uptake may not necessarily lead to associated improvement in nitrate retention, utilization or NUE, though it remains to be seen whether different plants respond differently to the overexpression of different transporters (Pathak et al. 2008). Light as an important abiotic factor is known to enhance NO3- uptake in a number of plant species (C rdenas-Navarro et al. 1999), and diurnal changes in nitrate uptake have been observed (Anjana et al., 2007). These changes seem to be linked to the imbalance between nitrate uptake and reduction due to the light regime and as well as to the rate of photosynthesis in shoots. Reduced nitrate uptake during darkness could be reversed by exogenous supply of sugars (Raghuram and Sopory 1995). Recent evidence on the up-regulation of AtNRT1.1 gene expression by auxin (Li et al. 2007) suggests that nitrate transporters may also be regulated by hormones.
Physiology of nitrate reduction in crops
A portion of the nitrate taken up is utilized/stored in the root cells, while the rest is transported to other parts of the plant. Due to the abundant availability of photosynthetic reductants, leaf mesophyll cells are the main sites of nitrate reduction. This is initiated by the NAD/NADP-dependent NR enzyme, which converts nitrate to nitrite by catalytic reaction in the cytosol. Nitrite is transported into the chloroplast, where it is further reduced into ammonium ion by a ferredoxin-dependent NiR. Being the first, irreversible and often rate-determining step of the N-assimilatory pathway, nitrate reduction has been a favorite step for physiological and biochemical approaches to optimize fertilizer N use.
Developing plants with transport gene systems using genetic engineering tools.
Plants receive N from the soil in the form of nitrate or ammonia, however, some may utilize amino acid as an important sources of N. Specific transporters located in the root cell membrane are responsible for uptake of N from the soil. Subsequent to its uptake, NO3- is assimilated via a series of enzymatic steps. Nitrate reductase being the first enzyme in nitrate assimilatory pathway and thus an important gene for manipulation. NR activity in leaf blades, express either as seasonal average or converted into seasonal input of reduced N, has been related to total reduced N, grain N and grain yield of cereals. The pattern of nitrate assimilation from different plant parts, viz. the main shoot of wheat, developing ear of wheat plants grown at different soil N levels and in the leaf blades at different stages of growth has revealed a direct positive correlation between increasing NR activity and increasing rates of nitrogenous fertilization. Most plant tissues have the capacity to assimilate nitrate, though their NR activity varies widely. Several endogenous as well as exogenous factors have been found to influence the expression of NR genes at both translational as well as transcriptional levels.
Andrews et al. (2004) reported that overexpression of either the NR or the NiR gene often affects N uptake by increasing mRNA levels in the plants. However, this does not seem to increase the growth or yield of plants, irrespective of N source. It is believed to be due, in part, to the complex regulation of both NR and the pathway as a whole. Transcriptional regulation of NR has only minor influence on the levels of free amino acids, ammonium, and nitrate whereas, post-translational regulation of NR strongly affects these compounds (Lea et al. 2006).
The light/dark conditions affect NR activity; heterotrophic nitrate assimilation in darkness is closely linked to the oxidative pentose phosphate pathway and the supply of glucose-6-phosphate. Under photoautotrophic conditions, glucose-6-phosphate dehydrogenase is inhibited by reduction with thioredoxin in light, thus replacing the heterotrophic dark nitrate assimilatory pathway with regulatory reactions functioning in light. These studies as well as bioenergetic calculations have indicated that both yield and N-harvest or protein can be increased to some extent with adequate nitrogen supply by altered management practices, thus improving the fertilizer NUE. Genotypic differences in the NR levels also provide insight in the relation of varietal differences in N assimilation. The genotypic differences in NR expression have been reported in corn, wheat, sorghum and barley. In sorghum, a positive relationship between decline in the height of the plant and enhancement of NR activity was observed, though no such relationship was evident in tall and dwarf cultivars of wheat, T. aestivum. Wheat genotypes revealed over twofold variability in NR activity, which supports genetic findings that the enzyme level is highly heritable, its differences are reflected in N harvest and that hybrids could be bred with predictable NR levels by selecting parents appropriately. In the high NR genotypes, higher levels of NR activity were found under low N levels, often with significantly higher N concentration in the grains. They also have sustained activity at later stages of growth, such as flag leaf emergence and anthesis. The reasons for these genetic differences are not fully understood, except that the regulation operated at the level of gene expression and that low levels of NADH might limit NR activity in low NR genotypes. Similarly, overexpressing NiR genes in Arabidopsis and tobacco resulted in increased NiR transcript levels but decreased enzyme activity levels, which were attributed to post-translational modifications.