Glutamine synthetase (GS) catalyzes the critical incorporation of inorganic ammonium into glutamine. In higher plants, it is represented by two groups of protein-the cystolic and plastidic forms (Miffin and Habash 2002). Cystosolic GS (GS1) is known to be encoded by a complex multigene family, whereas plastidic Gs (GS2) is encoded by a single gene. Glutamate synthase (Glutamine (amide): 2-oxoglutarate aminotransferase, GOGAT) catalyses the reductive transfer of the amide group of glutamine (produced by GS) to 2-oxoglutarate (α-keto glutarate) to form two glutamate molecules (Ireland and Lea, 1999).GS/GOGAT pathway is of crucial importance since the glutamine and glutamate produced are donors of amino groups for the biosynthesis of major N-containing compounds, including amino acids, nucleotides, chlorophylls, polyamines and alkaloids (Lea and Ireland, 1999; Hirel and Lea, 2001). A direct correlation was reported between an enhanced GS activity in transgenic plants in some cases, which is depicted by an increase in biomass or yield by transforming novel gs1 construct. Similarly, Kozaki and Takeba (1996) constructed transgenic tobacco plants enriched or reduced in plastidic glutamine synthetase (GS2, a key enzyme in photorespiration). Ectopic expression of GS1 has been shown to alter plant growth (Fuentes et al. 2001; Oliveira et al. 2002) and the over expression of GS1 in transgenic plants could cause the enhancement of photosynthetic rates, higher rates of photorespiration and enhanced resistance to water stress (Fuentes et al. 2001). The overexpression of soybean cytosolic GS1 in the shoots of Lotus corniculatus was reported to accelerate plant development, leading to early senescence and premature flowering, particularly when plants were grown under conditions of high ammonium (Vincentz et al. 1993). Man et al. (2005) provided additional empirical evidence for enhanced nitrogen-assimilation efficiency in GS1 transgenic lines. However, differences in the degree of ectopic GS1 expression have been reported (Fuentes et al. 2001) and attributed to positional effects, effectiveness of chimeric constructs, or differences in growth conditions. This may be due to lack of correlation between the enhanced expression of GS1 and concomitant growth (Vincentz et al., 1993; Ortega et al. 2001). A significant increase in leaf area, plant area, plant height and dry weight has been recorded in poplar ttrees transformed with conifer gs1a gene. Striking differences were observed at low nitrate concentration. Furthermore, higher rates at 15N incorporation into the transgenic plants demonstrate that the transformed plants have increased NUE (Man et al. 2005).
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Transgenic overexpression and antisense technology have been employed recently to modulate the expression of NADH-GOGAT in alfalfa and rice plants (Yamaya et al. 2002). The studies on transgenic rice plants expressing antisense RNA for either GS1 or NADH - GOGAT point towards the possible involvement of GS1 in the export of N via phloem in senescing leaves. On the other hand, in case of developing leaf blades and spikelets, NADH-GOGAT was implicated in the utilization of glutamine transported from senescing organs (Yamaya et al. 2003). While these genes appear to be good candidates for improving NUE in the short run, the degree of improvement may vary with the crop and cropping conditions. Therefore, the utility of transgenic overexpression of N-assimilatory genes for major improvements of NUE remains uncertain, though the possibility that different crops respond differently cannot be ruled out yet.
Other gene systems regulating N metabolism and their manipulation
Enzymes like asparagine synthetase (AS), that catalyzes the formation of asparagine (Asn) and glutamate from glutamine (Gln) and aspartate. In higher plants, AS is encoded by a small gene family (Lam et al. 1998). Together with GS, AS is believed to play a crucial role in primary N metabolism. The observation made by Carvalho et al. (2003) that the levels of AS transcripts and polypeptides in the transgenic nodules of Medicago truncatula increase when GS is reduced suggests that AS can compensate for the reduced GS ammonium assimilatory activity. However, it was also demonstrated that GS activity is essential for maintaining the higher level of AS. Thus, GS is required to synthesize enough Gln to support Asp biosynthesis via NADH-GOGAT and AspAT (Carvalho et al. (2003). A reduction in GS activity in transgenic Lotus japonicus is also correlated with an increase in asparagine content (Harrison et al. 2007), supporting the hypothesis that when GS becomes limiting, AS may be important in controlling the flux of reduced N into plants. With the aim of increasing Asn production in plants and to study the role of AS, several researches have attempted to clone AS genes and to examine the corresponding gene expression in plants. Lam et al. 2009 showed overexpression of the ASN1 gene in Arabidopsis and demonstrated that the transgenic plants have enhanced soluble seed protein content, enhanced total protein content, and better growth on N-limiting medium. Arabidopsis plants overexpressing the ASN2 gene accumulate less endogenous ammonium than wild-type plants when grown on medium containing 50-mM ammonium. This study indicates that signaling processes may provide an attractive route for metabolic engineering. In comparison to GS/GOGAT enzymes, the physiological role of glutamate dehydrogenase (GDH) has been less clear (Dubois et al. 2003). In an attempt to investigate the role of GDH by expressing a bacterial gdhA gene from E. coli in tobacco, Ameziane et al. 2000 found that biomass production is consistently increased in gdhA transgenics, regardless of whether they are grown under controlled conditions or in the field.
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Signaling and regulation of nitrogen metabolism
It is a well known concept in signal transduction that whenever multiple genes are subject to transcriptional regulation by a common signal, it is mediated through a regulatory sequence that exists in all the genes that respond to the signal. These signature sequences, commonly known as response elements, are identified by mutations that abolish their function, and their conserved nature as revealed by homology comparisons. Early experiments in transgenic Nicotiana plants using GUS gene fused to NR and NiR promoter sequences clearly demonstrated for the first time that nitrate induction of gene expression requires some sequence(s) associated with the NR and NiR promoters (Raghuram et al. 2006). Subsequent studies in transgenic tobacco incorporating the 5' flanking regions of the nitrate reductase genes NR1 and NR2 (designated NP1 and NP2) , in case of Arabidopsis thaliana, demonstrated that 238 and 330 bp of NP1and NP2 respectively are sufficient for nitrate-dependent transcription (Lin et al. 2006). These nitrate-responsive elements (NREs) are composed of several copies of a core A[G/C]TCA sequence motif preceded by an ~7-bp AT-rich sequence present in the 5'flanking regions of nitrate reductase (NR1 and NR2) genes. This particular sequence motif was also found to be very well conserved in the 5' flanking regions of NR and NiR genes from eight other plants. Sarkar (2003) compared the flanking sequences of all available plant nitrate responsive genes and found that the NRE core sequence (A[C/G]TCA) was present in multiple copies on both strands in all the known nitrate-responsive genes in many dicots, monocots and cyanobacteria. Though most of the NREs examined contained both the core sequence and a proceeding AT rich sequence, there were some cases which had GC rich regions or did not reveal any AT/GC bias. A more detailed bioinformatic analysis of the entire Arabidopsis genome in our lab revealed that the proposed NREs are randomly distributed, with no difference between nitrate responsive genes and the presumably nonresponsive genes and intergenic regions in the rest of the genome (Raghuram et al. 2006). These findings raise doubts on the validity of the proposed NRE as comprising of (A[C/G] TCA) elements preceded by AT-rich sequence. Further work in this area will need a combination of bioinformatic and experimental approaches to redefine the NREs that mediate the expression of all nitrate responsive genes in all plants. The discovery of NREs is important, as it provides an end point for nitrate signal transduction.
QTL approach to NUE
Nitrogen use efficiency in plants is a complex quantitative trait that depends on a number of internal and external factors in addition to soil nitrogen availability, such as photosynthetic carbon fixation to provide precursors required for amino acid biosynthesis or respiration to provide energy. Although this trait is controlled by a large number of loci acting individually or together, depending on nutritional, environmental and plant developmental conditions, it is possible to find enough phenotypic and genotypic variability to partially understand the genetic basis of NUE and thus identify some of the key components of yield for marker assisted breeding. Thus the development of molecular markers has facilitated the evaluation of the inheritance of NUE using specific quantitative trait loci (QTLs) that could be identified. In maize, Hirel et al. (2001) and Masclaux et al. (2001) analyzed recombinant inbred lines for physiological traits such as nitrate content, NR and GS activities. When the variation in these traits and yield components were compared, it was found that there was a positive correlation between nitrate content, GS activity and yield. When the loci that govern quantitative traits were determined on the map of the maize genome, the positions of QTLs for yield components and the locations of the genes for cytosolic GS (GS1) coincided. . In maize, studies on different genotypes or populations of recombinant inbred lines based on NUE components, chromosomal regions and putative candidate genes have hinted at some factors that might control yield and its components directly or indirectly, when the amount of N fertilizers provided to the plant is varied (Hirel et al. 2007).
Similar results were obtained in rice by Obara et al. (2001), confirming the earlier indications that the GS1 enzymatic activity in the leaf cytosol is one of the major steps controlling organic matter reallocation from source to sink organs during senescence and for grain-filling in cereals. Previous studies have already demonstrated that when GS1 is over expressed in Lotus, nitrogen remobilization was prematurely induced leading to early senescence of the plant (Vincentz et al. 1993). In rice (Yamaya 2002) and wheat (Habash et al. 2001), preliminary investigations with enhanced or decreased GS1 activity indicated that grain yield and grain nitrogen content were modified. In other species such as tobacco (Migge et al. 2000) or poplar (Gallardo et al. 1999), overexpression of GS2 or GS1 significantly increased plant biomass production at early stages of plant development. With these experiments, two out of seven QTLs for GS1 protein content were detected in different regions from other physiological and biological traits. In maize, QTLs for the activities of acid-soluble invertase and sucrose-phosphate-synthase were detected in the regions were each structural gene was mapped (Ishimaru et al. 2001).
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Thus, quantitative studies of genetic variability for NUE using molecular markers and combining agronomic and physiological studies will be increasingly used in the future to identify new genes or loci involved in the regulation of these metabolic pathways and their interconnection with carbon assimilation and recycling and to select genotypes that assimilate or remobilize nitrogen more efficiently.
Proteomics approach to NUE
The ability of crop plants to cope up with the variety of environmental stresses depends upon a number of changes in their proteins, which may be up- and down- regulated as a result of altered gene expression. Under a stressful condition, the modifications in the expression levels of these proteins could provide us valuable information about the nature of stress factor as well as the physiological and molecular state of a biological system. Hence, provides us some clues to understand the nature of defensive mechanism and adaptability, besides stress monitoring in these biological systems.
Proteomic based technologies have been recently applied for the systematic analysis of the induced gene products in a number of plant species subjugated to a wide range of abiotic and biotic challenges. Proteome analysis is becoming a powerful tool in the functional characterization of plants. Due to the availability of vast nucleotide sequence information and based on the progress achieved in sensitive and rapid protein identification by mass spectrometry, proteome approaches open up new perspectives to analyze the complex functions of model plants and crop species at different levels. Improvements in proteomic technology regarding protein separation and detection, as well as mass spectrometry based protein identification, have an increasing impact on the study of plant responses to salinity stress (Parker et al. 2006; Qureshi et al. 2007; Caruso et al. 2008). Proteomics has provided valuable information in various fields of plant biology. Construction of several plant protein databases is in progress for A. thaliana, rice, trees and maize, where different genetic, cellular and physiological information is available, such as expression in various organs or tissues, response to treatments, cellular localization and genetic bases (Thiellement et al. 1999). Recent advances in MS techniques will facilitate protein identification so that in the future this will not be a limiting factor in the interpretation of variations detected on 2D gels. By providing information on affected and unaffected proteins, large scale protein identification will simplify determining the consequences of mutations, plant transformation or natural polymorphism for plant metabolism, as well as interpreting the effects of protein changes on development, or in response to biotic and abiotic stress. Studies in Saccharomyces cerevisiae, for which hundreds of proteins have been identified, show the power of the proteomic approach in the study of the regulation of metabolic pathways. Schiltz et al. (2005) studied that during seed filling, the accumulation of proteins in the seeds relies on the nitrogen supply from the mother plant, and a proteomic approach was used to study the mobilization of proteins from the leaves to the filling seeds in pea. Two contrasting N-responsive wheat varieties have differential expressions of root as well as leaf proteins when grown under controlled conditions at different N-levels (Bahrman et al. 2004, 2005). These proteins were grouped into two categories, one involved in carbon metabolism and the other associated with other pathways and functions like thiol-specific antioxidant proteins etc. This study revealed that levels of gene expressions are modified with the varying levels of nitrate supply, even if only a few polypeptides appear, disappear or change. Sarry et al. (2006) have demonstrated the protein level changes associated with nitrogen and sulphur metabolism, and their interaction. With the help of high throughput proteomic tools, they were able to detect various enzymes including ATP sulphurylase, sulphite reductase, cysteine synthase, S adenosylmethionine synthase, glutamine synthase, aspartate aminotransferase, glutamate dehydrogenase etc, involved directly or indirectly in S and N metabolism. Recently a study for the detection of low nitrogen responsive proteins in cultivated rice species was done by Kim et al. (2009). Studies at constructing 2-D gel reference map for use in comparative proteomics among cultivars for N responsive proteins might provide an insight for precise identification of potential molecular protein markers to assist the breeders for screening N-efficient genotypes and help in understanding how crop adapts to low N availability. Correlations between the level of expression and N use efficiency might bring information on the possible role of the genes involved in nitrogen metabolism.
Present review provides an overview of plant nutriomics, which is still at a conceptual stage. Although considerable efforts are in progress with the aim at enhancing plant nutrient efficiency through molecular and genetic approaches. We have focused here largely on nitrogen with which we have been working on along molecular biology lines. Crop response to N and NUE is very low in developing countries including India. Use of nitrification inhibitors and slow release nitrogen fertilizers and efficient crop and fertilizer management can significantly increase NUE. It is clearly evident that optimizing the plants NUE goes beyond the primary process of uptake and reduction of nitrate, involving quality of events, including metabolite partitioning, secondary remobilization, C-N interactions, as well as signalling pathways and regulatory controls outside the metabolic cascades. Despite the various attempts to manipulate each of the above steps in some plant or the other, we are far from finding a universal switch that controls NUE in all plants. However, transgenic studies, QTL and proteomics approaches seem to increasingly suggest that the enzymes of secondary ammonia remobilization are better targets for manipulation, followed by regulatory processes that control N-C flux, rather than the individual genes/enzymes of primary nitrate assimilation. There is an urgent need of large scale, co-ordinated research on plant nutriomics, involving sincere efforts from both national and international researchers to develop the nutrient efficient, high yielding and stress tolerant genotypes/varieties that will contribute to both environmental safety as well as food security world-wide.