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The growth and development of maize (Zea mays L.) largely depends on its nutrient uptake through the root. Hence, studying its growth, response, and associated metabolic reprogramming to stress conditions is becoming an important research direction. A genome-scale metabolic model (GSM) for the maize root was developed to study its metabolic reprogramming under nitrogen stress conditions. The model was reconstructed based on the available information from KEGG, UniProt, and MaizeCyc. Transcriptomics data derived from the roots of hydroponically grown maize plants were used to incorporate regulatory constraints in the model and simulate nitrogen-non-limiting (N+) and nitrogen-deficient (N−) condition. Model-predicted flux-sum variability analysis achieved 70% accuracy compared with the experimental change of metabolite levels. In addition to predicting important metabolic reprogramming in central carbon, fatty acid, amino acid, and other secondary metabolism, maize root GSM predicted several metabolites (l-methionine, l-asparagine, l-lysine, cholesterol, and l-pipecolate) playing a regulatory role in the root biomass growth. Furthermore, this study revealed eight phosphatidylcholine and phosphatidylglycerol metabolites which, even though not coupled with biomass production, played a key role in the increased biomass production under N-deficient conditions. Overall, the omics-integrated GSM provides a promising tool to facilitate stress condition analysis for maize root and engineer better stress-tolerant maize genotypes.

Drought is one of the major abiotic stresses with a detrimental impact on plant growth and development irrespective of the developmental stage. Thus, identifying the physiological mechanisms driving drought resistance in crops remains challenging. Drought tolerance was evaluated in nine durum wheat cultivars (Triticum durum Desf.) at an early stage of plant development using plants grown under hydroponic conditions. Young wheat plants were subjected to three polyethylene glycol (PEG) treatments (0%, 1.8%, and 2.6%) over 21 days. Nineteen morpho-physiological parameters were then measured to monitor the impact of drought stress caused by the presence of PEG. An integrative analysis allowed the identification of genotypes exhibiting various levels of tolerance to drought, also revealing the impact of water deficiency on key phenotypic and physiological markers. Among the nine wheat genotypes, the landrace INRAT 69 was the most tolerant, whereas the commercial cultivar Ben Bechir was the most sensitive. We also found that amino acid, total soluble sugar, proline, and starch contents were the physiological parameters that were the most representative of drought stress. The use of these parameters as marker traits to select drought-tolerant wheat genotypes at early stages of plant development is discussed.

Glutamine synthetase (GS) is a key enzyme responsible for the incorporation of inorganic nitrogen in the form of ammonium into the amino acid glutamine. In plants, two groups of functional GS enzymes are found: eubacterial GSIIb (GLN2) and eukaryotic GSIIe (GLN1/GS). Only GLN1/GS genes are found in vascular plants, which suggests that they are involved in the final adaptation of plants to terrestrial life. The present phylogenetic study reclassifies the different GS of seed plants into three clusters: GS1a, GS1b and GS2. The presence of genes encoding GS2 has been expanded to Cycadopsida gymnosperms, which suggests the origin of this gene in a common ancestor of Cycadopsida, Ginkgoopsida and angiosperms. GS1a genes have been identified in all gymnosperms, basal angiosperms and some Magnoliidae species. Previous studies in conifers and the gene expression profiles obtained in ginkgo and magnolia in the present work could explain the absence of GS1a in more recent angiosperm species (e.g., monocots and eudicots) due to the redundant roles of GS1a and GS2 in photosynthetic cells. Altogether, the results provide a better understanding of the evolution of plant GS isoenzymes and their physiological roles, which is valuable for improving crop nitrogen use efficiency and productivity. This work was supported by Spanish Ministerio de Ciencia e Inno-vación grant numbers: BIO2015-73512-JIN MINECO/AEI/FEDER,UE; and RTI2018-094041-B-I00 and EQC2018-004346-P. This work was also supported by Junta de Andalucía, grant number P20_00036 PAIDI 2020/FEDER, UE. Funding for open access charge: Universidad de Málaga/CBUA. JMVM was supported by a grant from the Spanish Ministerio de Educación y Formación Profesional (FPU17/03517). FO was supported by grants from the Universidad de Málaga (Programa Operativo de Empleo Juvenil vía SNJG, UMAJI11, FEDER, FSE, Junta de Andalucía) and BIO-114, Junta de Andalucía.

Maize plants overexpressing NADH-GOGAT were produced in order to determine if boosting 2-Oxoglurate production used as a carbon skeleton for the biosynthesis of amino acids will improve plant biomass and kernel production. The NADH-GOGAT enzyme recycles glutamate and incorporates carbon skeletons into the ammonium assimilation pathway using the organic acid 2-Oxoglutarate as a substrate. Gene pyramiding was then conducted with NAD-IDH and NADH-GDH, two enzymes also involved in the synthesis of 2-Oxoglurate. NADH-GOGAT overexpression was detrimental for shoot biomass production but did not markedly affect kernel yield. Additional NAD-IDH and NADH-GDH activity did not improve plant performance. A decrease in kernel production was observed when NADH-GDH was pyramided to NADH-GOGAT and NAD-IDH. This decrease could not be restored even when additional cytosolic GS activity was present in the plants overexpressing the three enzymes producing 2-Oxoglutarate. Detailed leaf metabolic profiling of the different transgenic plants revealed that the NADH-GOGAT over-expressors were characterized by an accumulation of amino acids derived from glutamate and a decrease in the amount of carbohydrates further used to provide carbon skeletons for its synthesis. The study suggests that 2-Oxoglutarate synthesis is a key element acting at the interface of carbohydrate and amino acid metabolism and that its accumulation induces an imbalance of primary carbon and nitrogen metabolism that is detrimental for maize productivity.

To evaluate the combined effect of different agricultural practices on photosynthetic nitrogen and water-use efficiency, winter wheat was grown in the field under tillage and no-till conditions, with and without cover crops under low and high nitrogen fertilization inputs. Leaf physiological traits, such as the rate of photosynthesis, stomatal conductance, the rate of transpiration, the chlorophyll content index, the leaf area ratio, and specific leaf area were used as indicators representative of nitrogen and water-use efficiency. Six years after conversion to no-till, in the presence and in the absence of cover crops, significant increases in photosynthetic water-use efficiency and soil water content were observed both under low and high nitrogen fertilization input. Moreover, we observed that photosynthetic nitrogen-use efficiency, the rate of photosynthesis and specific leaf area were higher in the absence of tilling than in the presence of tilling. Thus, agronomic practices based on continuous no-till appear to be promising for increasing both photosynthetic nitrogen- and water-use efficiency in winter wheat.

An updated review of how plant-associated bacterial diazotrophs and fungal endophytes may improve plant N nutrition, with an emphasis on the underlying mechanisms and their exploitation for reducing the use of chemically synthesized N fertilizers., Nitrogen (N) is an essential element for plant productivity, thus, it is abundantly applied to the soil in the form of organic or chemical fertilizers that have negative impacts on the environment. Exploiting the potential of beneficial microbes and identifying crop genotypes that can capitalize on symbiotic associations may be possible ways to significantly reduce the use of N fertilizers. The best-known example of symbiotic association that can reduce the use of N fertilizers is the N2-fixing rhizobial bacteria and legumes. Bacterial taxa other than rhizobial species can develop associative symbiotic interactions with plants and also fix N. These include bacteria of the genera Azospirillum, Azotobacter, and Bacillus, some of which are commercialized as bio-inoculants. Arbuscular mycorrhizal fungi are other microorganisms that can develop symbiotic associations with most terrestrial plants, favoring access to nutrients in a larger soil volume through their extraradical mycelium. Using combinations of different beneficial microbial species is a promising strategy to boost plant N acquisition and foster a synergistic beneficial effect between symbiotic microorganisms. Complex biological mechanisms including molecular, metabolic, and physiological processes dictate the establishment and efficiency of such multipartite symbiotic associations. In this review, we present an overview of the current knowledge and future prospects regarding plant N nutrition improvement through the use of beneficial bacteria and fungi associated with plants, individually or in combination.

A metabolomic, biochemical, fluxomic, and metabolic modeling approach developed using 19 genetically distant maize lines links leaf physiology to kernel yield.

International audience; Nitrogen cycling in agroecosystems is heavily dependent upon arbuscular mycorrhizal fungi (AMF) present in the soil microbiome. These fungi develop obligate symbioses with various host plant species, thus increasing their ability to acquire nutrients. However, AMF are particularly sensitive to physical, chemical and biological disturbances caused by human actions that limit their establishment. For a more sustainable agriculture, it will be necessary to further investigate which agricultural practices could be favorable to maximize the benefits of AMF to improve crop nitrogen use efficiency (NUE), thus reducing nitrogen (N) fertilizer usage. Direct seeding, mulch-based cropping systems prevent soil mycelium disruption and increase AMF propagule abundance. Such cropping systems lead to more efficient root colonization by AMF and thus a better establishment of the plant/fungal symbiosis. In addition, the use of continuous cover cropping systems can also enhance the formation of more efficient interconnected hyphal networks between mycorrhizae colonized plants. Taking into account both fundamental and agronomic aspects of mineral nutrition by plant/AMF symbioses, we have critically described, how improving fungal colonization through the reduction of soil perturbation and maintenance of an ecological balance could be helpful for increasing crop NUE.

UMR 1332- Equipe Virologie; A combined metabolomic, biochemical, fluxomic, and metabolic modeling approach was developed using 19 genetically distant maize (Zea mays) lines from Europe and America. Considerable differences were detected between the lines when leaf metabolic profiles and activities of the main enzymes involved in primary metabolism were compared. During grain filling, the leaf metabolic composition appeared to be a reliable marker, allowing a classification matching the genetic diversity of the lines. During the same period, there was a significant correlation between the genetic distance of the lines and the activities of enzymes involved in carbon metabolism, notably glycolysis. Although large differences were observed in terms of leaf metabolic fluxes, these variations were not tightly linked to the genome structure of the lines. Both correlation studies and metabolic network analyses allowed the description of a maize ideotype with a high grain yield potential. Such an ideotype is characterized by low accumulation of soluble amino acids and carbohydrates in the leaves and high activity of enzymes involved in the C4 photosynthetic pathway and in the biosynthesis of amino acids derived from glutamate. Chlorogenates appear to be important markers that can be used to select for maize lines that produce larger kernels.

Using a metabolomic approach, we have quantified the metabolite composition of the phloem sap exudate of seventeen European and American lines of maize that had been previously classified into five main groups on the basis of molecular marker polymorphisms. In addition to sucrose, glutamate and aspartate, which are abundant in the phloem sap of many plant species, large quantities of aconitate and alanine were also found in the phloem sap exudates of maize. Genetic variability of the phloem sap composition was observed in the different maize lines, although there was no obvious relationship between the phloem sap composition and the five previously classified groups. However, following hierarchical clustering analysis there was a clear relationship between two of the subclusters of lines defined on the basis of the composition of the phloem sap exudate and the earliness of silking date. A comparison between the metabolite contents of the ear leaves and the phloem sap exudates of each genotype, revealed that the relative content of most of the carbon- and nitrogen-containing metabolites was similar. Correlation studies performed between the metabolite content of the phloem sap exudates and yield-related traits also revealed that for some carbohydrates such as arabitol and sucrose there was a negative or positive correlation with kernel yield and kernel weight respectively. A posititive correlation was also found between kernel number and soluble histidine.

In higher plants, NAD(H)-glutamate dehydrogenase (GDH; EC 1.4.1.2) is an abundant enzyme that exists in different isoenzymic forms. In Arabidopsis thaliana, three genes (Gdh1, Gdh2 and Gdh3) encode three different GDH subunits (beta, alpha and gamma) that randomly associate to form a complex array of homo- and heterohexamers. The modification of the GDH isoenzyme pattern and its regulation was studied during the development of A. thaliana in the gdh1, gdh2 single mutants and the gdh1-2 double mutant, with particular emphasis on GDH3. Investigations showed that the GDH3 isoenzyme could not be detected in closely related Arabidopsis species. The induction and regulation of GDH3 activity in the leaves and roots was investigated following nitrogen deprivation in the presence or absence of sucrose or kinetin. These experiments indicate that GDH3 is likely to play an important role during senescence and nutrient remobilization. (C) 2013 Elsevier Masson SAS. All rights reserved.

Nitrogen fertilizer is the most used nutrient source in modern agriculture and represents significant environmental and production costs. In the meantime, the demand for grain increases and production per area has to increase as new cultivated areas are scarce. In this context, breeding for an efficient use of nitrogen became a major objective. In wheat, nitrogen is required to maintain a photosynthetically active canopy ensuring grain yield and to produce grain storage proteins that are generally needed to maintain a high end-use quality. This review presents current knowledge of physiological, metabolic and genetic factors influencing nitrogen uptake and utilization in the context of different nitrogen management systems. This includes the role of root system and its interactions with microorganisms, nitrate assimilation and its relationship with photosynthesis as postanthesis remobilization and nitrogen partitioning. Regarding nitrogen-use efficiency complexity, several physiological avenues for increasing it were discussed and their phenotyping methods were reviewed. Phenotypic and molecular breeding strategies were also reviewed and discussed regarding nitrogen regimes and genetic diversity.

Nitrogen (N) metabolism was characterized in the developing ear of glutamine synthetase deficient mutants (gln1-3, gln1-4 and gln1-3/gln1-4) of maize exhibiting a reduction in kernel yield. During the grain-filling period, the metabolite contents, enzyme activities and steady-state levels of transcripts for marker genes of amino acid synthesis and interconversion were monitored in the cob and kernels. The ear of gln1-3 and gln1-3/gln1-4 had a higher free amino acid content and a lower C/N ratio, when compared to the wild type. The free ammonium concentrations were also much higher in gln1-3/gln1-4, and Asn accumulation was higher in gln1-3 and gln1-3/gln1-4. The level of transcripts of ZmAS3 and ZmAS4, two genes encoding asparagine synthetase, increased in the 'aborted kernels' of gln1-3 and gln1-3/gln1-4. The results show that N metabolism is clearly different in developing and 'aborted kernels'. The data support the hypothesis that N accumulated in 'aborted kernels' is remobilized via the cob to developing kernels using Asn as a transport molecule. The two genes ZmAS3 and ZmAS4 are likely to play an important role during this process.

Introduction - Maize mutants deficient for the expression of two genes encoding cytosolic glutamine syntehtase (GS) isoenzymes GS1.3 and GS1.4 displayed reduced kernel number and kernel size, respectively, the effect of the mutation being cumulative in the double mutant. However, at maturity, shoot biomass production was not modified in all the mutants, indicating that the reaction catalysed by the enzyme is specifically involved in the control of grain yield. Objective - To examine the physiological impact of the GS mutations on the leaf metabolic profile during the kernel filling period, during which nitrogen is remobilised from the shoots to be further exported to the kernels. Methodology - An (1)H-NMR spectroscopy metabolomic was applied to the investigation of metabolic change of the gln1.3, gln1.4 and gln1.3/1.4 double mutant. Results - In the three GS mutants, an increase in the amount of several N-containing metabolites such as asparagine, alanine, threonine and phophatidylcholine was observed whatever the level of nitrogen fertilisation. In addition, we found an accumulation of phenylalanine and tyrosine, two metabolites involved the primary steps of the phenylpropanoid pathway. Conclusion - Changes in the metabolic profile of the GS mutants suggest that, when cytosolic GS activity is strongly reduced, either alternative metabolic pathways participate in the reassimilation of ammonium released during leaf protein remobilisation or that premature leaf senescence is induced when kernel set and kernel filling are affected. The accumulation of phenylalanine and tyrosine in the mutant plants indicates that lignin biosynthesis is altered, thus possibly affecting ear development.

International audience; In higher plants the glutamate dehydrogenase (GDH) enzyme catalyzes the reversible amination of 2-oxoglutarate to form glutamate, using ammonium as a substrate. For a better understanding of the physiological function of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate, we used transgenic tobacco (Nicotiana tabacum L.) plants overexpressing the two genes encoding the enzyme. An in vivo real time (15)N-nuclear magnetic resonance (NMR) spectroscopy approach allowed the demonstration that, when the two GDH genes were overexpressed individually or simultaneously, the transgenic plant leaves did not synthesize glutamate in the presence of ammonium when glutamine synthetase (GS) was inhibited. In contrast we confirmed that the primary function of GDH is to deaminate Glu. When the two GDH unlabeled substrates ammonium and Glu were provided simultaneously with either [(15)N]Glu or (15)NH(4)(+) respectively, we found that the ammonium released from the deamination of Glu was reassimilated by the enzyme GS, suggesting the occurrence of a futile cycle recycling both ammonium and Glu. Taken together, these results strongly suggest that the GDH enzyme, in conjunction with NADH-GOGAT, contributes to the control of leaf Glu homeostasis, an amino acid that plays a central signaling and metabolic role at the interface of the carbon and nitrogen assimilatory pathways. Thus, in vivo NMR spectroscopy appears to be an attractive technique to follow the flux of metabolites in both normal and genetically modified plants.

NAD-dependent glutamate dehydrogenase (NAD-GDH) of higher plants has a central position at the interface between carbon and nitrogen metabolism due to its ability to carry out the deamination of glutamate. In order to obtain a better understanding of the physiological function of NAD-GDH under salt stress conditions, transgenic tobacco (Nicotiana tabacum L.) plants that overexpress two genes from Nicotiana plumbaginifolia individually (GDHA and GDHB) or simultaneously (GDHA/B) were grown in the presence of 50 mM NaCl. In the different GDH overexpressors, the NaCl treatment induced an additional increase in GDH enzyme activity, indicating that a post-transcriptional mechanism regulates the final enzyme activity under salt stress conditions. A greater shoot and root biomass production was observed in the three types of GDH overexpressors following growth in 50 mM NaCl, when compared with the untransformed plants subjected to the same salinity stress. Changes in metabolites representative of the plant carbon and nitrogen status were also observed. They were mainly characterized by an increased amount of starch present in the leaves of the GDH overexpressors as compared with the wild type when plants were grown in 50 mM NaCl. Metabolomic analysis revealed that overexpressing the two genes GDHA and GDHB, individually or simultaneously, induced a differential accumulation of several carbon- and nitrogen-containing molecules involved in a variety of metabolic, developmental and stress-responsive processes. An accumulation of digalactosylglycerol, erythronate and porphyrin was found in the GDHA, GDHB and GDHA/B overexpressors, suggesting that these molecules could contribute to the improved performance of the transgenic plants under salinity stress conditions.

Summary Improved nitrogen use efficiency (NUE: capacity to produce a supplement of yield for each added unit of N fertilizer) has the potential to enhance yield under low nitrogen (N) and thereby improve crop nutritional quality while reducing ground water contamination by nitrates. In order to realize potential benefits with respect to sustainable agriculture, we need to identify the physiological, biochemical and molecular mechanisms controlling component processes, including inorganic N uptake, N reduction, N partitioning between roots and shoots and N-incorporation into organic molecules as a function of carbon availability. Moreover, sustained decreases in fertilizer input and improved or stabilized yield require an improved understanding of the transition between N assimilation and N remobilization during plant development. Recent advances in plant molecular biotechnology, combined with modern physiological and biochemical studies, have expanded our understanding of the regulatory mechanisms control...

Maize (Zea mays) is an important C4 plant due to its widespread use as a cereal and energy crop. A second-generation genome-scale metabolic model for the maize leaf was created to capture C4 carbon fixation and investigate nitrogen (N) assimilation by modeling the interactions between the bundle sheath and mesophyll cells. The model contains gene-protein-reaction relationships, elemental and charge-balanced reactions, and incorporates experimental evidence pertaining to the biomass composition, compartmentalization, and flux constraints. Condition-specific biomass descriptions were introduced that account for amino acids, fatty acids, soluble sugars, proteins, chlorophyll, lignocellulose, and nucleic acids as experimentally measured biomass constituents. Compartmentalization of the model is based on proteomic/transcriptomic data and literature evidence. With the incorporation of information from the MetaCrop and MaizeCyc databases, this updated model spans 5,824 genes, 8,525 reactions, and 9,153 metabolites, an increase of approximately 4 times the size of the earlier iRS1563 model. Transcriptomic and proteomic data have also been used to introduce regulatory constraints in the model to simulate an N-limited condition and mutants deficient in glutamine synthetase, gln1-3 and gln1-4. Model-predicted results achieved 90% accuracy when comparing the wild type grown under an N-complete condition with the wild type grown under an N-deficient condition.

Summary • In wheat the period of grain filling is characterized by a transition for all vegetative organs from sink to source status. • To study this transition, the progression of physiological markers and enzyme activities representative of nitrogen metabolism was monitored from the vegetative stage to maturity in different leaf stages and stem sections of two wheat (Triticum aestivum) cultivars grown at high and low levels of N fertilization. • In the two cultivars examined, we found a general decrease of the metabolic and enzyme markers occurred during leaf ageing, and that this decrease was enhanced when plants were N-limited. Both correlation studies and principal components analysis (PCA) showed that there was a strong relationship among total N, chlorophyll, soluble protein, ammonium, amino acids and glutamine synthetase (GS) activity. • The use of a marker such as GS activity to predict the N status of wheat, as a function of both plant development and N availability, is discussed with the aim of selecting wheat genotypes with better N-use efficiency.

In order to improve our understanding of the regulation of nitrogen assimilation and recycling in wheat (Triticum aestivum L.), we studied the localization of plastidic (GS2) and cytosolic (GS1) glutamine synthetase isoenzymes and of glutamate dehydrogenase (GDH) during natural senescence of the flag leaf and in the stem. In mature flag leaves, large amounts of GS1 were detected in the connections between the mestome sheath cells and the vascular cells, suggesting an active transfer of nitrogen organic molecules within the vascular system in the mature flag leaf. Parallel to leaf senescence, an increase of a GS1 polypeptide (GS1b) was detected in the mesophyll cytosol of senescing leaves, while the GS protein content represented by another polypetide (GS1a) in the phloem companion cells remained practically constant in both leaves and stems. Both GDH aminating activity and protein content were strongly induced in senescing flag leaves. The induction occurred both in the mitochondria and in the cytosol of phloem companion cells, suggesting that the shift in GDH cellular compartmentation is important during leaf nitrogen remobilization although the metabolic or sensing role of the enzyme remains to be elucidated. Taken together, our results suggest that in wheat, nitrogen assimilation and recycling are compartmentalized between the mesophyll and the vasculature, and are shifted in different cellular compartments within these two tissues during the transition of sink leaves to source leaves.

Summary • Here, nitrogen management within the plant was compared in an early-senescing maize hybrid and in a late-senescing maize hybrid, both grown under field conditions with a high fertilisation input involving large quantities of fertiliser. •W e monitored, in representative leaf stages, the changes in metabolite content, enzyme activities and steady-state levels of transcripts for marker genes of N primary assimilation, N recycling and leaf senescence. • The hybrids differed in terms of persistence of leaf greenness, the expression of marker genes and the concentration of enzymes used to describe the transition from N assimilation to N recycling. The transcription of leaf-senescence marker genes did not differ. Agronomic studies confirmed the ability of the late-senescing hybrid to absorb and store more N in shoots. • Despite the differences in the mode of N management adopted by the two hybrids, we conclude that leaf senescence occurs independently of the source-to-sink transition at the high level of fertilisation used involving large quantities of fertiliser. The possibility of improving N metabolic efficiency in the latest maize hybrids is discussed.

A large genetic variability was observed in the shoot NO(3) (-) content of recombinant inbred lines (RILs) of Lotus japonicus. To determine the cause of this variability, we have studied some aspects of nitrate uptake and assimilation in the two parental ecotypes (Gifu and Funakura) and four representatives of the RILs population differing both in their shoot biomass and shoot NO(3) (-) content. Higher shoot NO(3) (-)content was mainly due to an increase in the uptake of the ion regardless of the plant biomass production. The positive correlation observed between the shoot NO(3) (-) content and the steady state level of mRNA encoding high affinity NO(3) (-) transporters suggests that the higher NO(3) (-) influx is due to enhanced expression of the transporters. In contrast, neither the level of nitrate reductase mRNA, nor the potential enzyme activity in vivo in the different lines was correlated with the shoot NO(3) (-) content. This indicates that NO(3) (-) transport in Lotus is one of the main checkpoints controlling shoot NO(3) (-) accumulation. In addition, this study shows that at least in Lotus, it is possible, through breeding strategies, to lower the NO(3) (-) content without affecting biomass production.

Although good progress has been made to dissect and better understand both the main steps and the regulation of inorganic nitrogen assimilation in higher plants, the role of alternative metabolic pathways which are potentially able to incorporate ammonium into organic molecules is still not fully understood. One of them is the reaction catalysed by the mitochondrial enzyme glutamate dehydrogenase (NAD(H)-GDH, EC 1.4.1.2) which is either able to incorporate ammonium into 2-oxoglutarate to form glutamate or to function in the opposite direction to oxidise glutamate. Although it has been clearly demonstrated by the means of 15 N- or 13 C-labelling experiments that the later reaction occurs in the cell, it has been argued that under certain physiological conditions, when the ammonium concentration reaches a certain threshold, the enzyme is able to function in the aminating direction. More recently, it has been found that in grapes, a high proportion of the protein is located in the mitochondria of the phloem companion cells and that a significant amount of enzyme is present in the cytosolic fraction of senescing flowers. Using cytoimmunochemistry, we confirmed in the present study that, in other higher plant species, GDH protein is localised in the mitochondria of the phloem companion cells and in the cytosol of senescing organs or tissues. These findings open, therefore, new perspectives toward a better understanding of the function of GDH, particularly in relation to stress and plant development. Both transgenic studies performed in the past and the quantitative genetic approach presented in this paper strongly suggest that the reaction catalysed by NAD(H)-GDH is of major importance in the control of plant growth and productivity.

In higher plants, recent advances in plant molecular genetics, combined with modern physiological and biochemical studies, have expanded our understanding of the regulatory mechanisms controlling the primary steps of inorganic nitrogen assimilation and the subsequent biochemical pathways involved in nitrogen supply and recycling for higher plant metabolism, growth and development. In this presentation, we describe improvements in our understanding of the molecular controls of nitrogen assimilation through the use of transgenic plants and the study of genetic variability in model and crop species. To illustrate this research programme, the physiological impact of modified gene expression, using either transgenic plants or different genotypes, was studied using 15N-labelling experiments in order to monitor the influx of nitrate or ammonia and its subsequent incorporation into amino acids.

A glutamine synthetase gene (GS15) coding for soybean cytosolic glutamine synthetase (GS1) fused to a constitutive promoter (CaMV 35S), a putative nodule-specific promoter (LBC3) and a putative root-specific promoter (rolD) was transformed into Pisum sativum L. cv. Greenfeast. Four lines with single copies of GS15 (one 35S-GS15 line, one LBC 3 -GS15 line, and two rolD-GS15 lines) were tested for the expression of GS15, levels of GS1, GS activity, N accumulation, N2 fixation, and plant growth at different levels of nitrate. Enhanced levels of GS1 were detected in leaves of three transformed lines (the 35S-GS15 and rolD-GS15 transformants), in nodules of three lines (the LBC 3 -GS15 and rolD-GS15 transformants), and in roots of all four transformants. Despite increased levels of GS1 in leaves and nodules, there were no differences in GS activity in these tissues or in whole-plant N content, N2 fixation, or biomass accumulation among all the transgenic lines and the wild-type control. However, the rolD-GS15 transformants, which displayed the highest levels of GS1 in the roots of all the transformants, had significantly higher GS activity in roots than the wild type. In one of the rolD-GS15 transformed lines (Line 8), increased root GS activity resulted in a lower N content and biomass accumulation, supporting the findings of earlier studies with Lotus japonicus (Limami et al. 1999 ). However, N content and biomass accumulation was not negatively affected in the other rolD-GS15 transformant (Line 9) and, in fact, these parameters were positively affected in the 0.1 mM $$ {\rm NO}_3^ - $$ treatment. These findings indicate that overexpression of GS15 in various tissues of pea does not consistently result in increases in GS activity. The current study also indicates that the increase in root GS activity is not always consistent with decreases in plant N and biomass accumulation and that further investigation of the relationship between root GS activity and growth responses is warranted.

A novel cDNA encoding glutamate dehydrogenase (GDH) from tobacco ( Nicotiana tabacum ), named gdh1, was characterized. The gdh1 mRNA was detected in roots, stems and source/senescent leaves. In order to investigate diurnal regulation of gdh1 in leaves, the content in gdh1 mRNA was measured every 3 h over a 48 h period and compared to nia and gs2 mRNA levels, encoding, respectively, nitrate reductase (NR) and chloroplastic glutamine synthetase (GS2). In source leaves, gdh1 mRNA levels exhibit diurnal fluctuations. A 12 h shift was observed between the day‐ night rhythms of gdh1 and nia expression. Metabolite contents were also measured and a shift in the day‐night fluctuations of both glutamate (GLU) and g -aminobutyric acid (GABA) was observed between sink and source leaves, whereas the diurnal rhythm of a -ketoglutarate showed no change. A possible role of GDH in the shift of GLU and GABA contents is discussed. Leaf disc experiments showed that gdh1 expression is enhanced in conditions of continuous darkness. This trend is inhibited by sucrose feeding. The opposite was observed for nia expression. An important outcome of this work is the reverse regulation of gdh1 and nia genes. A possible role of sugars and amino acids in the co-regulation of gdh1 and nia genes is suggested.

The diurnal changes in carbon and nitrogen metabolite concentrations were studied in transgenic tobacco plants ( Nicotiana tabacum L. cv. Xanthi) expressing an antisense ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) mRNA. In parallel, enzyme activities, and abundance of the proteins and transcripts involved in ammonia assimilation were monitored during the day/night cycle in both transgenic and untransformed control plants. When the transgenic plants were transferred from non-photorespiratory (high CO 2 ) to photorespiratory conditions (air), photorespiratory NH 4 + accumulated in the leaves during the second half of the light period; it decreased progressively through the night until the beginning of the next light period. Glutamine and 2-oxoglutarate (2-OG) also accumulated during the second part of the light period; this was followed by a decrease at the end of the night. A concomitant increase in asparagine was observed during the night, suggesting that this amino acid serves as a temporary storage compound for the partial elimination of excess photorespiratory ammonia. We also observed that the direction of the glutamate reaction varied during the day/night cycle such that a higher ratio of aminating/deaminating activity occurred in the first half of the light period. This was correlated with the decline in NH 4 + and 2-OG concentrations. Both of these observations suggest that alternative metabolic pathways may be transiently activated during a day/night cycle in plants with low Fd-GOGAT activity.

In this review, we will present the latest developments in systems biology with particular emphasis on improving nitrogen-use efficiency (NUE) in crops such as maize and demonstrating the application of metabolic models. The review highlights the importance of improving NUE in crops and provides an overview of the transcriptome, proteome, and metabolome datasets available, focusing on a comprehensive understanding of nitrogen regulation. 'Omics' data are hard to interpret in the absence of metabolic flux information within genome-scale models. These models, when integrated with 'omics' data, can serve as a basis for generating predictions that focus and guide further experimental studies. By simulating different nitrogen (N) conditions at a pseudo-steady state, the reactions affecting NUE and additional gene regulations can be determined. Such models thus provide a framework for improving our understanding of the metabolic processes underlying the more efficient use of N-based fertilizers.

To enhance our understanding of the genetic basis of nitrogen use efficiency in maize (Zea mays), we have developed a quantitative genetic approach by associating metabolic functions and agronomic traits to DNA markers. In this study, leaves of vegetative recombinant inbred lines of maize, already assessed for their agronomic performance, were analyzed for physiological traits such as nitrate content, nitrate reductase (NR), and glutamine synthetase (GS) activities. A significant genotypic variation was found for these traits and a positive correlation was observed between nitrate content, GS activity and yield, and its components. NR activity, on the other hand, was negatively correlated. These results suggest that increased productivity in maize genotypes was due to their ability to accumulate nitrate in their leaves during vegetative growth and to efficiently remobilize this stored nitrogen during grain filling. Quantitative trait loci (QTL) for various agronomic and physiological traits were searched for and located on the genetic map of maize. Coincidences of QTL for yield and its components with genes encoding cytosolic GS and the corresponding enzyme activity were detected. In particular, it appears that the GS locus on chromosome 5 is a good candidate gene that can, at least partially, explain variations in yield or kernel weight. Because at this locus coincidences of QTLs for grain yield, GS, NR activity, and nitrate content were also observed, we hypothesize that leaf nitrate accumulation and the reactions catalyzed by NR and GS are coregulated and represent key elements controlling nitrogen use efficiency in maize.

Mesophyll cells (MCs) and bundle-sheath cells (BSCs) of leaves of the C4 plant maize (Zea mays L.) were separated by cellulase digestion to determine the relative proportion of the glutamine synthetase (GS; EC 6.3.1.2) or the NADH-glutamate dehydrogenase (GDH; EC 1.4.1.2) isoforms in each cell type. The degree of cross-contamination between our MC and BSC preparations was checked by the analysis of marker proteins in each fraction. Nitrate reductase (EC 1.6.6.1) proteins (110 kDa) were found only in the MC fraction. In contrast, ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) proteins (160 kDa) were almost exclusively present in the BSC fraction. These results are consistent with the known intercellular distribution of nitrate reductase and Fd-GOGAT proteins in maize leaves and show that the cross-contamination between our MC and BSC fractions was very low. Proteins corresponding to cytosolic GS (GS-1) or plastidic GS (GS-2) were found in both the MC and BSC fractions. While equal levels of GS-1 (40 kDa) and GS-2 (44 kDa) polypeptides were present in the BSC fraction, the GS-1 protein level in the MC fraction was 1.8-fold higher than the GS-2 protein pool. Following separation of the GS isoforms by anion-exchange chromatography of MC or BSC soluble protein extracts, the relative GS-1 activity in the MC fraction was found to be higher than the relative GS-2 activity. In the BSC fraction, the relative GS-1 activity was very similar to the relative GS-2 activity. Two isoforms of GDH with apparent molecular weights of 41 kDa and 42 kDa, respectively, were detected in the BSC fraction of maize leaves. Both GDH isoenzymes appear to be absent from the MC fraction. In the BSCs, the level of the 42-kDa GDH isoform was 1.7-fold higher than the level of the 41-kDa GDH isoform. A possible role for GS-1 and GDH co-acting in the synthesis of glutamine for the transport of nitrogen is discussed.

Tobacco (Nicotiana tabacum) plants expressing a partial ferredoxin-dependent glutamine-2-oxoglutarate aminotransferase (Fd-GOGAT) cDNA in the antisense orientation under the control of the 35S promoter, were used to study the metabolism of amino acids, 2-oxoglutarate and ammonium following the transition from CO2 enrichment (where photorespiration is inhibited) to air (where photorespiration is a major process of ammonium production in leaves). The leaves of the lowest Fd-GOGAT expressors accumulated more foliar glutamine (Gln) and α-ketoglutarate (α-KG) than the untransformed controls in both growth conditions. Photorespiration-dependent increases in foliar ammonium, glutamine, α-KG and total amino acids were proportional to the decreases in foliar Fd-GOGAT activity. No change in endoprotease activity was observed following transfer to air in the Fd-GOGAT transformants or the untransformed controls which has similar activities over a broad range of pH values. We conclude that several pathways of amino acid biosynthesis are modified when NH3 + and Gln accumulate in leaves.

In this article we discuss the ways in which our understanding of the nature of the molecular controls of nitrogen assimilation have been increased by the use of leguminous and non-leguminous plants with modified capacities for ammonium assimilation. These modifications have been achieved through genetic engineering and breeding. An improved understanding of nitrogen assimilation will be vital if improvements in crop nitrogen use efficiency are to be made to reduce the need for excessive input of fertilisers. In this review we present an overall view of past work and more recent studies on this topic. In our work, using tobacco and Lotus as model plants, glutamine synthetase and glutamate synthase activites have been altered by stimulating or inhibiting in an organ- or tissue-specific manner the expression of the corresponding genes. The physiological impact of these genetic manipulations has been studied on plants grown under different nitrogen regimes.

To investigate the contribution of root cytosolic glutamine synthetase (GS) activity in plant biomass production, two different approaches were conducted using the model legume Lotus japonicus. In the first series of experiments, it was found that overexpressing GS activity in roots of transgenic plants leads to a decrease in plant biomass production. Using (15)N labelling it was shown that this decrease is likely to be due to a lower nitrate uptake accompanied by a redistribution to the shoots of the newly absorbed nitrogen which cannot be reduced due to the lack of nitrate reductase activity in this organ. In the second series of experiments, the relationship between plant growth and root GS activity was analysed using a series of recombinant inbred lines issued from the crossing of two different Lotus ecotypes, Gifu and Funakura. It was confirmed that a negative relationship exists between root GS expression and plant biomass production in both the two parental lines and their progeny. Statistical analysis allowed it to be estimated that at least 13% of plant growth variation can be accounted for by variation in GS activity.

Compared to other enzymatic systems, the regulation of GS isoenzyme expression shows a unique diversity. Considering that GS is one of the oldest existing and functioning genes found in all extant life forms, we can hypothesise that the evolution of metabolic pathways from primitive pre-procaryotes to lower and then higher plants might have gradually refined the function of GS to provide reduced nitrogen forms for the rest of the metabolism (Kumada et al. 1993). This refinement might explain the genetic and biological diversity encountered in the various modes of expression and regulation of higher plant GS isoenzymes both at the cellular and intracellular levels (Fig. 1). Although model plants are valuable sources of information helping to decipher fine regulatory control mechanisms (Lam et al. 1996), the study of this genetic diversity appears to be one of the most promising areas of research, necessary to better understand ammonia assimilation in plants and more generally improve nitrogen use efficiency.

In order to identify important promoter elements controlling the ammonium-regulated expression of the soybean gene GS15 encoding cytosolic glutamine synthetase, a series of 5' promoter deletions were fused to the GUS reporter gene. To allow the detection of positive and negative regulatory elements, a series of 3' deletions were fused to a -90 CaMV 35S promoter fragment placed upstream of the GUS gene. Both types of construct were introduced into Lotus corniculatus plants and soybean roots via Agrobacterium rhizogenes-mediated transformation. Both spectrophotometric enzymatic analysis and histochemical localization of GUS activity in roots, root nodules and shoots of transgenic plants revealed that a strong constitutive positive element (SCPE) of 400 bp, located in the promoter distal region is indispensable for the ammonium-regulated expression of GS15. Interestingly, this SCPE was able to direct constitutive expression in both a legume and non-legume background to a level similar to that driven by the CaMV 35S full-length promoter. In addition, results showed that separate proximal elements, located in the first 727 bp relative to the transcription start site, are essential for root- and root nodule-specific expression. This proximal region contains an AAAGAT and two TATTTAT consensus sequences characteristic of nodulin or nodule-enhanced gene promoters. A putative silencer region containing the same TATTTAT consensus sequence was identified between the SCPE and the organ-specific elements. The presence of positive, negative and organ-specific elements together with the three TATTTAT consensus sequences within the promoter strongly suggest that these multiple promoter fragments act in a cooperative manner, depending on the spatial conformation of the DNA for trans-acting factor accessibility.

The regulation by UV-A or UV-B light of the nuclear gene(s) encoding the plastidic enzyme nitrite reductase (NiR; EC 1.7.7.1) was examined in the cotyledons of tomato (Lycopersicon esculentum L.). Two NiR isoforms designated NiR1 and NiR2 with apparent molecular masses of 63 kDa and 62 kDa, respectively, were detected by immunoblot analysis in total soluble protein extracts derived from tomato seedling cotyledons. Genomic Southern blot analysis indicated the presence of two NiR genes per haploid tomato genome. In etiolated tomato cotyledons, the total NiR protein pool was almost exclusively constituted by NiR1. In contrast, NiR2 was the predominant NiR isoform in the cotyledons of tomato seedlings grown in white light. Illumination of etiolated tomato cotyledons with UV-A or UV-B light resulted in an increase in both the total NiR transcript level and the NiR2 protein abundance. Blue light stimulated the NiR2 protein pool above the level obtained with red light of equal photon fluence rate. These results show that NiR2 protein expression is light-inducible and that the light-stimulation of NiR2 protein accumulation involves the action of both phytochrome and a specific blue-light photoreceptor. The NiR1 protein level remained virtually unaffected by the light treatments. The change in the relative proportion of the NiR isoforms during greening of etiolated tomato cotyledons is, therefore, due to the different light-responsiveness of the genes corresponding to NiR1 or NiR2. The physiological significance of the presence of NiR isoforms that are regulated differently by light in tomato cotyledons is discussed.

The influence of ultraviolet A (UV-A) or B (UV-B) light and of the nitrogen source on the induction of ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was examined in etiolated cotyledons of tomato (Lycopersicon esculentum L.). The Fd-GOGAT activity increased upon illumination of etiolated tomato cotyledons with UV-A or UV-B light. This stimulation of Fd-GOGAT activity was correlated with an increase in both the Fd-GOGAT transcript level and the Fd-GOGAT protein abundance. These results suggest that UV-A or UV-B light stimulates the de novo synthesis of Fd-GOGAT in etiolated tomato cotyledons. Both UV-A and UV-B light failed to influence the activity of NADH-GOGAT (EC 1.4.1.14) in etiolated tomato cotyledons. Taken together, our data indicate that the tomato genes encoding Fd- or NADH-dependent glutamate synthase are regulated differently by UV-A or UV-B light. No difference with respect to both the Fd-GOGAT transcript and protein abundance was found between cotyledons of tomato seedlings grown with either nitrate or ammonium as the sole N-source in the dark or in white light. In addition, the increase in the Fd-GOGAT protein pool induced by white light in etiolated nitrate-grown tomato seedling cotyledons was similar to that induced by white light in etiolated ammonium-grown tomato seedling cotyledons. These results show that the tomato Fd-GOGAT protein level does not depend strongly on the nature of the nitrogen source and that there appears to be no major stimulatory effect on the Fd-GOGAT protein pool produced by nitrate during the illumination of etiolated tomato cotyledons.

A soybean cytosolic glutamine synthetase gene (GS15) was fused with the constitutive 35S cauliflower mosaic virus (CaMV) promoter in order to direct overexpression in Lotus corniculatus L. plants. Following transformation with Agrobacterium rhizogenes, eight independent Lotus transformants were obtained which synthesized additional cytosolic glutamine synthetase (GS) in the shoots. To eliminate any interference caused by the T-DNA from the Ri plasmid, three primary transformants were crossed with untransformed plants and progeny devoid of TL- and TR-DNA sequences were chosen for further analyses. These plants had a 50-80% increase in total leaf GS activity. Plants were grown under different nitrogen regimes (4 or 12 mM NH4+) and aspects of carbon and nitrogen metabolism were examined. In roots, an increase in free amino acids and ammonium was accompanied by a decrease in soluble carbohydrates in the transgenic plants cultivated with 12 mM NH4+ in comparison to the wild type grown under the same conditions. Labelling experiments using 15NH4+ were carried out in order to monitor the influx of ammonium and its subsequent incorporation into amino acids. This experiment showed that both ammonium uptake in the roots and the subsequent translocation of amino acids to the shoots was lower in plants overexpressing GS. It was concluded that the build up of ammonium and the increase in amino acid concentration in the roots was the result of shoot protein degradation. Moreover, following three weeks of hydroponic culture early floral development was observed in the transformed plants. As all these properties are characteristic of senescent plants, these findings suggest that expression of cytosolic GS in the shoots may accelerate plant development, leading to early senescence and premature flowering when plants are grown on an ammonium-rich medium.

Glutamate dehydrogenase (GDH; EC 1.4.1.2) is able to carry out the deamination of glutamate in higher plants. In order to obtain a better understanding of the physiological function of GDH in leaves, transgenic tobacco (Nicotiana tabacum L.) plants were constructed that overexpress two genes from Nicotiana plumbaginifolia (GDHA and GDHB under the control of the Cauliflower mosiac virus 35S promoter), which encode the α- and β-subunits of GDH individually or simultaneously. In the transgenic plants, the GDH protein accumulated in the mitochondria of mesophyll cells and in the mitochondria of the phloem companion cells (CCs), where the native enzyme is normally expressed. Such a shift in the cellular location of the GDH enzyme induced major changes in carbon and nitrogen metabolite accumulation and a reduction in growth. These changes were mainly characterized by a decrease in the amount of sucrose, starch and glutamine in the leaves, which was accompanied by an increase in the amount of nitrate and Chl. In addition, there was an increase in the content of asparagine and a decrease in proline. Such changes may explain the lower plant biomass determined in the GDH-overexpressing lines. Overexpressing the two genes GDHA and GDHB individually or simultaneously induced a differential accumulation of glutamate and glutamine and a modification of the glutamate to glutamine ratio. The impact of the metabolic changes occurring in the different types of GDH-overexpressing plants is discussed in relation to the possible physiological function of each subunit when present in the form of homohexamers or heterohexamers.

L'article original est publié par The American Society of Plant Biologists; The role of NADH-dependent glutamate dehydrogenase (GDH) was investigated by studying the physiological impact of a complete lack of enzyme activity in an Arabidopsis thaliana plant deficient in three genes encoding the enzyme. This study was conducted following the discovery that a third GDH gene is expressed in the mitochondria of the root companion cells, where all three active GDH enzyme proteins were shown to be present. A gdh1-2-3 triple mutant was constructed and exhibited major differences from the wild type in gene transcription and metabolite concentrations, and these differences appeared to originate in the roots. By placing the gdh triple mutant under continuous darkness for several days and comparing it to the wild type, the evidence strongly suggested that the main physiological function of NADH-GDH is to provide 2-oxoglutarate for the tricarboxylic acid cycle. The differences in key metabolites of the tricarboxylic acid cycle in the triple mutant versus the wild type indicated that, through metabolic processes operating mainly in roots, there was a strong impact on amino acid accumulation, in particular alanine, gamma-aminobutyrate, and aspartate in both roots and leaves. These results are discussed in relation to the possible signaling and physiological functions of the enzyme at the interface of carbon and nitrogen metabolism.

Equipe : GAPV, pôle : APE; Linking plant phenotype to gene and protein expression and also to metabolite synthesis and accumulation is one of the main challenges for improving agricultural production worldwide. Such a challenge is particularly relevant to crop nitrogen use efficiency (NUE). Here, the differences in leaf gene transcript, protein, and metabolite accumulation in maize subjected to long-term nitrogen (N)-deficient growth conditions at two important stages of plant development have been studied. The impact of N deficiency was examined at the transcriptomic, proteomic, and metabolomic levels. It was found that a number of key plant biological functions were either up- or down-regulated when N was limiting, including major alterations to photosynthesis, carbon (C) metabolism, and, to a lesser extent, downstream metabolic pathways. It was also found that the impact of the N deficiency stress resembled the response of plants to a number of other biotic and abiotic stresses, in terms of transcript, protein, and metabolite accumulation. The genetic and metabolic alterations were different during the N assimilation and the grain-filling period, indicating that plant development is an important component for identifying the key elements involved in the control of plant NUE. It was also found that integration of the three 'omics' studies is not straightforward, since different levels of regulation seem to occur in a stepwise manner from gene expression to metabolite accumulation. The potential use of these 'omics' studies is discussed with a view to improve our understanding of whole plant nitrogen economics, which should have applications in breeding and agronomy.

Quantitative trait loci (QTLs) for the main steps of nitrogen (N) metabolism in the developing ear of maize (Zea mays L.) and their co-localization with QTLs for kernel yield and putative candidate genes were searched in order to identify chromosomal regions putatively involved in the determination of yield. During the grain-filling period, the changes in physiological traits were monitored in the cob and in the developing kernels, representative of carbon and N metabolism in the developing ear. The correlations between these physiological traits and traits related to yield were examined and localized with the corresponding QTLs on a genetic map. Glycine and serine metabolism in developing kernels and the cognate genes appeared to be of major importance for kernel production. The importance of kernel glutamine synthesis in the determination of yield was also confirmed. The genetic and physiological bases of N metabolism in the developing ear can be studied in an integrated manner by means of a quantitative genetic approach using molecular markers and genomics, and combining agronomic, physiological and correlation studies. Such an approach leads to the identification of possible new regulatory metabolic and developmental networks specific to the ear that may be of major importance for maize productivity.

At least two genes encoding cytosolic glutamine synthetase (GS) are actively transcribed in soybean (Glycine max. L. cv. Prize) plants. The expression of the first gene, GS15, is regulated by the availability of ammonia provided externally to non-inoculated roots or formed as the result of nitrogen fixation in nodulated plants. Moreover, in addition to transcription in roots and root nodules, GS15 is also actively transcribed in other organs such as anthers and pulvini. Separate promoter elements are able to control either the organ- or ammonia-regulated expression of GS15 independently. Another cytosolic GS gene, GS21, has also been identified and is specifically expressed in nodules

During the grain-filling period of maize, the changes in metabolite content, enzyme activities, and transcript abundance of marker genes of amino acid synthesis and interconversion and carbon metabolism in three lines F2, Io, and B73 have been monitored in the cob and in the kernels. An integrative statistical approach using principal component analysis (PCA) and hierarchical clustering of physiological and transcript abundance data in the three maize lines was performed to determine if it was possible to link the expression of a physiological trait and a molecular biomarker to grain yield and its components. In this study, it was confirmed that, in maize, there was a genetic and organ-specific control of the main steps of nitrogen (N) and carbon metabolism in reproductive sink organs during the grain-filling period. PCA analysis allowed the identification of groups of physiological and molecular markers linked to either a genotype, an organ or to both biological parameters. A hierarchical clustering analysis was then performed to identify correlative relationships existing between these markers and agronomic traits related to yield. Such a clustering approach provided new information on putative marker traits that could be used to improve yield in a given genetic background. This can be achieved using either genetic manipulation or breeding to increase transcript abundance for the genes encoding the enzymes glutamine synthetase (GS), alanine amino transferase (AlaAT), aspartate amino transferase (AspAT), and delta 1-pyrroline-5-carboxylate synthetase (P5CS).

*The main steps of nitrogen (N) metabolism were characterized in the developing ear of the two maize (Zea mays) lines F2 and Io, which were previously used to investigate the genetic basis of nitrogen use efficiency (NUE) in relation to yield. *During the grain-filling period, we monitored changes in metabolite content, enzyme activities and steady-state levels of transcripts for marker genes of amino acid synthesis and interconversion in the cob and the kernels. *Under low N fertilization conditions, line Io accumulated glutamine, asparagine and alanine preferentially in the developing kernels, whereas in line F2, glutamine and proline were the predominant amino acids. Quantification of the mRNA-encoding enzymes involved in asparagine, alanine and proline biosynthesis confirmed that the differences observed between the two lines at the physiological level are likely to be attributable to enhanced expression of the cognate genes. *Integrative analysis of physiological and gene expression data indicated that the developing ear of line Io had higher N use and transport capacities than line F2. Thus, in maize there is genetic and environmental control of N metabolism not only in vegetative source organs but also in reproductive sink organs.

A full-length cDNA clone encoding cytosolic glutamine synthetase (GS), expressed in roots and root nodules of soybean, was isolated by direct complementation of an Escherichia coli gln A- mutant. This sequence is induced in roots by the availability of ammonia. A 3.5-kilobase promoter fragment of a genomic clone (lambda GS15) corresponding to this cDNA was isolated and fused with a reporter [beta-glucuronidase (GUS)] gene. The GS-GUS fusion was introduced into a legume (Lotus corniculatus) and a nonlegume (tobacco) plant by way of Agrobacterium-mediated transformations. This chimeric gene was found to be expressed in a root-specific manner in both tobacco and L. corniculatus, the expression being restricted to the growing root apices and the vascular bundles of the mature root. Treatment with ammonia increased the expression of this chimeric gene in the legume background (i.e., L. corniculatus); however, no induction was observed in tobacco roots. Histochemical localization of GUS activity in ammonia-treated transgenic L. corniculatus roots showed a uniform distribution across all cell types. These data suggest that the tissue specificity of the soybean cytosolic GS gene is conserved in both tobacco and L. corniculatus; however, in the latter case, this gene is ammonia inducible. Furthermore, the ammonia-enhanced GS gene expression in L. corniculatus is due to an increase in transcription. That this gene is directly regulated by externally supplied or symbiotically fixed nitrogen is also evident from the expression of GS-GUS in the infection zone, including the uninfected cells, and the inner cortex of transgenic L. corniculatus nodules, where a flux of ammonia is encountered by this tissue. The lack of expression of GS-GUS in the outer cortex of the nodules suggests that ammonia may not be able to diffuse outside the endodermis.

The in vivo and vitro nitrate reductase (NR; EC 1.6.6.1) activities of suspension cell cultures were investigated in two strains of Nicotiana tabacum : a photomixotrophic green wild type (WT) and non-photosynthetic green type RubisCo − (SP25). During a batch culture cycle of 21 days, two peaks of in vivo NR activity (NRA) were observed: an early peak at 7 h and a second peak which reached a maximum at 6–8 days. At the beginning of the culture, in vitro activity was detected early for the WT cells compared to SP25 cells for which the activity was very low or absent. During the second peak, in vitro activity was present in the two strains and correlated very well with in vivo activity. Immunoblot experiment suggested that NR 120 kDa polypeptide did not accumulate in the cytosol at the beginning of the first phase of activity and the NR protein seemed to be labile. In contrast, the NR protein was stable during the second peak. On the other hand, it was observed that the dividing cells at 5–8 days seemed to accumulate nitrate, while the non-dividing cells, at the beginning of the culture, are unable to do that. This feature might be related to a change of cell permeability for nitrate because NR activity causing nitrate reduction is better in the second peak than in the first one. In correlation with this likely permeability change, external nitrate concentration modulated the first peak NRA and not the second one. The differential sensitivity of NRA of the two peaks to nitrate, led us to rediscuss the localization site of NR and to focus our experiments on a membrane-bound form of this enzyme.

International audience; In this review, recent developments and future prospects of obtaining a better understanding of the regulation of nitrogen use efficiency in the main crop species cultivated in the world are presented. In these crops, an increased knowledge of the regulatory mechanisms controlling plant nitrogen economy is vital for improving nitrogen use efficiency and for reducing excessive input of fertilizers, while maintaining an acceptable yield. Using plants grown under agronomic conditions at low and high nitrogen fertilization regimes, it is now possible to develop wholeplant physiological studies combined with gene, protein, and metabolite profiling to build up a comprehensive picture depicting the different steps of nitrogen uptake, assimilation, and recycling to the final deposition in the seed. A critical overview is provided on how understanding of the physiological and molecular controls of N assimilation under varying environmental conditions in crops has been improved through the use of combined approaches, mainly based on whole-plant physiology, quantitative genetics, and forward and reverse genetics approaches. Current knowledge and prospects for future agronomic development and application for reeding crops adapted to lower fertilizer input are explored, taking into account the world economic and environmental constraints in the next century.