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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.

The growth and development of maize (Zea mays L.) largely depends on its nutrient uptake through 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 condition. 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 was used to incorporate regulatory constraints in the model and simulate nitrogen-non-limiting (N−) and nitrogen-deficient (N−) conditions. Model-predicted result achieved 70% accuracy comparing to the experimental direction 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 (e.g., L-methionine, L-asparagine, L-lysine, cholesterol, and L-pipecolate) playing critical regulatory role in the root biomass growth. Furthermore, this study revealed eight phosphatidyl-choline and phosphatidyl-glycerol metabolites which even though not coupled with biomass production played a key role in the increased biomass production under N-. Overall, the omics-integrated-GSM provides a promising tool to facilitate stress-condition analysis for maize root and ultimately engineer better stress-tolerant maize genotypes.SummaryThe growth and development of maize (Zea mays L.) largely depends on its nutrient uptake through 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 condition. 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 was used to incorporate regulatory constraints in the model and simulate nitrogen-non-limiting (N+) and nitrogen-deficient (N−) conditions. Model-predicted result achieved 70% accuracy comparing to the experimental direction 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 (e.g., L-methionine, L-asparagine, L-lysine, cholesterol, and L-pipecolate) playing critical regulatory role in the root biomass growth. Furthermore, this study revealed eight phosphatidyl-choline and phosphatidyl-glycerol metabolites which even though not coupled with biomass production played a key role in the increased biomass production under N−.Overall, the omics-integrated-GSM provides a promising tool to facilitate stress-condition analysis for maize root and ultimately engineer better stress-tolerant maize genotypes.

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.

Salinity is one of the most important stresses that reduces plant growth and productivity in several parts of the world. Nine Tunisian durum wheat genotypes grown under hydroponic conditions were subjected to two levels of salt stress (100 and 170 mM NaCl) for 21 days. An integrative analysis revealing the impact of salinity on key phenotypic and physiological marker traits was then conducted. Principal component analysis grouped these traits into three different clusters corresponding to the absence of salt stress and the two levels of salt stress. This analysis also allowed the identification of genotypes exhibiting various levels of tolerance to NaCl. Among the nine genotypes of Triticum durum Desf., cultivar Om Rabiaa was the most tolerant whereas cultivar Mahmoudi genotype was the most sensitive. Following the multivariate analysis of the examined phenotypic and physiological traits, we found that shoot length, shoot fresh weight, leaf area, the whole-plant stable isotope ratios of nitrogen (&delta, 15N), shoot ammonium and proline contents, and shoot glutamine synthetase activity could be used as markers for the selection of salt-tolerant wheat genotypes.

International audience; The impact of nitrogen (N)-limiting conditions after silking on kernel yield (KY)-related traits and whole plant N management was investigated using fifteen maize lines representative of plant genetic diversity in Europe and America. A large level of genetic variability of these traits was observed in the different lines when post-silking fertilization of N was strongly reduced. Under such N-fertilization conditions, four different groups of lines were identified on the basis of KY and kernel N content. Although the pattern of N management, including N uptake and N use was variable in the four groups of lines, a number of them were able to maintain both a high yield and a high kernel N content by increasing shoot N remobilization. No obvious relationship between the genetic background of the lines and their mode of N management was found. When N was limiting after silking, N remobilization appeared to be a good predictive marker for identifying maize lines that were able to maintain a high yield and a high kernel N content irrespective of their female flowering date. The use of N remobilization as a trait to select maize genotypes adapted to low N input is discussed.

Maize farming requires high amounts of nitrogen (N) fertilizer, which can have detrimental effects on agronomic sustainability and the environment. Thus, irrespective of the mode of N fertilization, an increased knowledge of the mechanisms controlling plant N metabolism is essential for improving nitrogen use efficiency (NUE) in maize. This new knowledge will reduce the excessive input of fertilizers, while maintaining an acceptable yield and a sufficient profit margin for the farmers. It is now possible to further develop whole-plant agronomic and physiological studies. These can be combined with gene, protein, and metabolite profiling to build up a comprehensive picture depicting the different steps of N uptake, assimilation, and recycling to produce either biomass in vegetative organs or proteins in storage organs. We provide an overview describing how our understanding of the physiological and molecular controls of N assimilation in maize has been advanced using combined approaches. These are based on agronomic, whole-plant physiology, genetic, modeling, and systems biology approaches. Current knowledge and prospects for selecting high-yielding maize genotypes adapted to lower N fertilizer input and for identifying biological markers representative of the plant N status for breeding and agronomic purposes are reviewed.

International audience; A field study was conducted in northern France over two consecutive years to evaluate the combined effect of conventional tillage (CT) vs no till (NT) with or without cover crops (cc) and nitrogen (N) fertilization on various agronomic traits related to N use efficiency in winter wheat. Five years after conversion of CT to NT, significant increases in N use efficiency, N utilization efficiency, N agronomic efficiency, N partial factor productivity, N apparent recovery fraction and N remobilization were observed under three N fertilization regimes (0, 161, 215 kg ha(-1)). It was also observed that grain yield and grain N content were similar under CT and NT. The N nutrition index was higher under NT at the three rates of N fertilization. Moreover, N use efficiency related traits were increased in the presence of cc both under NT and CT. Thus, agronomic practices based on continuous NT in the presence of cc, appear to be promising strategies to increase N use efficiency in wheat, while reducing both the use and the loss of N-based fertilizers.

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.

A two-year experiment was conducted in the field to measure the combined impact of tilling and N fertilization on various agronomic traits related to nitrogen (N) use efficiency and to grain yield in maize cultivated in the presence of a cover crop. Four years after conversion to no-till, a significant increase in N use efficiency N harvest index, N remobilization and N remobilization efficiency was observed both under no and high N fertilization conditions. Moreover, we observed that grain yield and grain N content were higher under no-till conditions only when N fertilizers were applied. Thus, agronomic practices based on continuous no-till appear to be a promising for increasing N use efficiency in maize.

The doubling of the world’s agricultural production for the past four decades has been associated with a seven-fold increase in nitrogen (N) fertilization [1] which has caused major detrimental impacts onthediversityandfunctioningofthenon-agriculturalbacterial,animalandplantecosystems,notably through the process of freshwater and marine ecosystem eutrophication [2].[...]

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.

The human body contains approximately 3.2% nitrogen (N), mainly present as protein and amino acids. Although N exists at a high concentration (78%) in the air, it is not readily available to animals and most plants. Plants are however able to take up both nitrate (NO3- ) and ammonium (NH4+ ) ions from the soil and convert them to amino acids and proteins, which are excellent sources for all animals. Most N is available as the stable isotope 14 N, but a second form, 15 N, is present in very low concentrations. 15 N can be detected in extracts of plants by gas chromatography followed by mass spectrometry (GC/MS). In this protocol, the methods are described for tracing the pathway by which plants are able to take up 15 N-labeled nitrate and ammonium and convert them into amino acids and proteins. A protocol for extracting and quantifying amino acids and 15 N enrichment in maize (Zea mays L.) leaves labeled with 15 NH4+ is described. Following amino acid extraction, purification, and separation by GC/MS, a calculation of the 15 N enrichment of each amino acid is carried out on a relative basis to identify any differences in the dynamics of amino acid accumulation. This will allow a study of the impact of genetic modifications or mutations on key reactions involved in primary nitrogen and carbon metabolism. © 2018 by John Wiley & Sons, Inc.

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.

Estimating the proportion of N remobilization and postsilking N uptake allocated to kernels may help to improve N-use efficiency in maize (Zea mays L.). In this study, we show theoretically and experimentally, that 15 N labeling at the beginning of stem elongation can be used in the field to estimate the proportion of N remobilized from the vegetative parts to the kernels of maize by measuring 15 N distribution only at maturity. In the same way, 15 N labeling at silking allows a determination of the proportion of postsilking N uptake allocated to the kernels by measuring 15 N distribution only at maturity. Two 1-yr experiments with three and four genotypes and one 2-yr experiment with testcross progenies from 66 recombinant inbred lines were developed. Nitrogen-15 labeling during vegetative growth provided an estimate of the proportion of N remobilized with a greater accuracy compared with the "balance" method, which computes the difference between the amount of N present in the stover at silking and the amount of N present in the stover at maturity. The validity of our 15 N method is mainly based on the assumption that less than 15 to 20% of 15 N is taken up after silking. The determination of the proportion of postsilking N uptake allocated to kernels requires assumptions that are more difficult to fulfill. Nitrogen-15 labeling in the field at the beginning of rapid stem growth appears to be a useful tool for studying the genetic variability of N remobilization using a large number of genotypes.

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.

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 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.

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.

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.

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.

It is now well established that the GS/GOGAT cycle is the major route for ammonium assimilation in higher plants. However, it has often been argued that other enzymes, such as glutamate dehydrogenase, have the capacity to assimilate ammonium, leading to the hypothesis that alternative ammonium assimilatory pathways could operate under particular physiological conditions. The GDH enzyme is encoded by two distinct genes, GDH1 and GDH2. A third gene, GDH3, potentially encoding GDH has recently been identified by in silico studies performed on Arabidopsis thaliana. In order to precise its function, the metabolic profile of gdh3 knock out mutants were compared to wild type plants using the 1H-NMR technique. 1H-NMR spectra coupled with principal component analysis and partial least square-discriminant analysis were applied to identify changes of the metabolic profiles. These experiments were performed on roots, leaves and stems. In the gdh3 mutant, metabolic variations were observed for carboxylic acids, amino acids and carbohydrates content.

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.

Nitrogen (N) plays the most important role of all soil mineral nutrients in plant growth and development. In grapevine, nitrogen is most likely to be deficient al-though it is the main fertlizer commonly applied to vineyards to increase produc-tivity (Keller et al. 1998) and to influence grape juice composition (Ough and Bell 1980). Considerable new information has been obtained over the past twenty years on the uptake, translocation, distribution, partitioning, and storage of ni-trogenous compounds in grapevines as well as new insights toward a better un-derstanding on the regulation of the synthesis and degradation of amino acids and other nitrogenous compounds and of the enzymes associated with these reactions. However, in spite of the widespread usage of nitrogen fertilization in vineyards, the physiological and biochemical effects of nitrogen on shoot and fruit growth, fruit bud initiation, flowering, fruit set, and crop yield are still poorly understood. Grapevines differ from herbaceous plants and many other woody plants in that they do not form terminal buds at the end of shoots, but they can continue to grow late into the season. The individual flower parts are formed after bud break, unlike most deciduous fruit trees. Nitrate and ammonium ions are the most common forms of nitrogen avail-able to plants in soils. The relative importance and concentration of each of these two ions depends on the genetic, developmental, and physiological status of each

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.

Although the physiological role of the enzyme glutamate dehydrogenase which catalyses in vitro the reversible amination of 2-oxoglutarate to glutamate remains to be elucidated, it is now well established that in higher plants the enzyme preferentially occurs in the mitochondria of phloem companion cells. The Nicotiana plumbaginifolia and Arabidopis thaliana enzyme is encoded by two distinct genes encoding either an alpha- or a beta-subunit. Using antisense plants and mutants impaired in the expression of either of the two genes, we showed that in leaves and stems both the alpha- and beta-subunits are targeted to the mitochondria of the companion cells. In addition, we found in both species that there is a compensatory mechanism up-regulating the expression of the alpha-subunit in the stems when the expression of the beta-subunit is impaired in the leaves, and of the beta-subunit in the leaves when the expression of the alpha-subunit is impaired in the stems. When one of the two genes encoding glutamate dehydrogenase is ectopically expressed, the corresponding protein is targeted to the mitochondria of both leaf and stem parenchyma cells and its production is increased in the companion cells. These results are discussed in relation to the possible signalling and/or physiological function of the enzyme which appears to be coordinated in leaves and stems.

The meaning of variation in 15 N/ 14 N isotope ratio in plants grown in the field is better known when variation is due to environment than when it is due to plant genotype. To study the physiological and genetic meaning of variation of such a ratio, a set of 99 recombinant inbred lines of maize were evaluated at low and high N-input and organ 15 N abundances were correlated to agronomic and physiological traits. At the level of means, at high N-input there appeared no difference in 15 N partitioning according to plant organs, with the same abundances for blades, stalks + sheaths and kernels. However, at low N-input blades and kernels were 15 N-enriched, whereas stalks were significantly 15 N-depleted with an abundance close to that observed for high N-input. 15 N abundance of whole-plant and organs showed significant genotypic effects and genotype by nitrogen input interaction, varying according to the organ and the stage, silking and grain maturity. Genetic variation for 15 N abundance and correlations involving 15 N abundance were always lower at high N-input than at low N-input. 15 N abundances of blades and stalks + sheaths were negatively related to silking date whatever the stage (silking or maturity) and N-fertilization whereas kernel 15 N abundance was not affected by silking date. At low N-input, whole-plant 15 N abundance at maturity was positively correlated to whole-plant and kernel protein content whereas at high N-input such correlation disappeared. Whole-plant 15 N abundance at silking was negatively related to root fresh weight and to glutamine synthetase activity measured in young plants grown in hydroponics. Twelve QTLs for 15 N abundance were detected, mainly at high N-input; among them, 10 coincided with QTLs involved in nitrogen use efficiency (grain yield, N-uptake and N remobilization) and the root system. Interpretation of all results leads to the conclusion that two mechanisms could explain genetic variation in 15 N discrimination ability: morpho-physiological differences, in particular in the root system, and activities of the first enzymes of nitrogen metabolism, with a positive relationship between enzyme activity and discrimination abilities.

In maize (Zea mays), nitrogen (N) remobilization and postflowering N uptake are two processes that provide amino acids for grain protein synthesis. To study the way in which N is allocated to the grain and to the stover, two different 15N-labelling techniques were developed. 15NO(3-) was provided to the soil either at the beginning of stem elongation or after silking. The distribution of 15N in the stover and in the grain was monitored by calculating relative 15N-specific allocation (RSA). A nearly linear relationship between the RSA of the kernels and the RSA of the stover was found as a result of two simultaneous N fluxes: N remobilization from the stover to the grain, and N allocation to the stover and to the grain originating from N uptake. By modelling the 15N fluxes, it was possible to demonstrate that, as a consequence of protein turnover, a large proportion of the amino acids synthesized from the N taken up after silking were integrated into the proteins of the stover, and these proteins were further hydrolysed to provide N to the grain.

A soybean cytosolic glutamine synthetase gene ( GS15 ) fused to a constitutive promoter (CaMV 35S), a putative nodule-specific promoter ( LBC 3 ), or a putative root-specific promoter ( rolD ) was transformed into Pisum sativum L. cv. Greenfeast. Four lines with single copies (Lines 1, 7, 8 and 9) and four lines with two copies each of GS15 (Lines 2, 4, 6 and 11) were compared to the wild-type (WT) parental line for levels of cytosolic glutamine synthetase (GS1), glutamine synthetase (GS) activity, N accumulation, N derived form the atmosphere (NDFA), and biomass of plants grown on 0.0, 0.1, 1.0 or 10.0 mM NH 4 + . Enhanced levels of GS1 were detected in leaves of one of the two lines transformed with the 35S-GS15 construct, and all three lines containing the rolD-GS15 construct. All three lines containing the LBC 3 -GS15 construct had increased levels of GS1 in nodules. Despite the increased levels of GS1 in many transformants, only the roots of lines containing the rolD-GS15 construct consistently demonstrated enhanced levels of GS activity (up to 12-fold). Positive responses in plant N content, NDFA, and biomass were rare, but increases in plant biomass and N content of up to 17% and 54%, respectively, occurred in some of the rolD-GS15 lines at certain levels of ammonium. In general, GS15 copy number did not seem to differentially affect phenotype of the transformants, and transformants respond to ammonium concentrations in similar patterns to that previously observed with nitrate. Despite the fact that the rolD-GS15 transformants consistently resulted in increased GS activity in roots and resulted in some occurrences of increases in biomass and plant N content, the lack of consistent positive growth effect across all transformants indicates that the generalized overexpression of GS1 in tissues holds little potential for positive growth responses in pea.

Maize (Zea mays L., line F2) plants were grown in the field under high or low fertilization input to monitor the metabolic, biochemical and molecular events occurring in young vegetative leaves and in the different leaf stages along the main axis in plants harvested 15 days after silking. This study shows that in maize which possess large sinks represented by the seeds, nitrogen (N) management is different compared with tobacco in which sink strength is much lower and mostly limited to young developing leaves. Although in young leaves nitrate assimilation predominates in both species, ammonium assimilation exhibits some species-specific differences with respect to inorganic and organic N metabolite accumulation during leaf ageing. These differences are likely to be related to the high sink strength of the ear in maize, which continuously imports carbon and N assimilates during grain filling. Consequently, a number of cytosolic glutamine synthetase isoenzymes are expressed during leaf ageing to maintain a constant flux of reduced N necessary for the synthesis of organic N molecules used either for leaf protein synthesis or directly translocated to the grain. This situation contrasts with that found in tobacco for which leaf ammonium assimilation in the plastids is shifted to the cytosol during the transition from sink leaves to source leaves. These species-specific differences for N assimilation and recycling are discussed in relation to the evolution of leaf photosynthetic activity and leaf senescence, which both seem to be largely dependent on the different sink strength in each species.

To illustrate the development of the source-to-sink transition in maize leaves during the grain-filling period, an integrated physiological-agronomic approach is presented in this study. The evolution of physiological markers such as total leaf nitrogen (N), chlorophyll, soluble protein, amino acid and ammonium contents was monitored from silking to a period close to maturity in different leaf stages of three maize genotypes grown at high and low levels of N fertilization. In addition, the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH), two enzymes known to play a direct or an indirect role during leaf N remobilization, were measured. In the three genotypes examined, we found that a general decrease of most metabolic and enzyme markers occurred during leaf ageing and that this decrease was enhanced when plants were N starved. In contrast, such variations were not observed between different sections of a single leaf even at an advanced stage of leaf senescence. We found that there is a strong correlation between total N, chlorophyll, soluble protein and GS activity, which is not dependent upon the N fertilization level, which indicates the N status of the plant, either in a single leaf or during ageing. In contrast, ammonium, amino acids and GDH activity were not subject to such variations, thus suggesting that they are indicators of the metabolic activity of the whole plant in response to the level of N fertilization. The use of these markers to predict the N status of maize as a function of both plant development and N availability is discussed.

Nitrogen deficiency tolerance is necessary to breed for wheat varieties adapted to low input management systems. The aim of this study was to identify root proteins differentially expressed in response to a nitrogen stress. Two wheat varieties were experimented at low (0.5 mM nitrate) and at high (3.0 mM nitrate) nitrogen levels. After 8 weeks, root samples were taken to assess enzyme activities and to perform two dimensional gel electrophoresis. A total of 860 root protein spots were qualitatively or quantitatively analysed. A significant variety effect existed for 74 spots, while a significant nitrate supply-dependent variation was observed for 56 spots. Moreover 20 spots showed a significant variety × N-supply effect. Correlations were made between physiological parameters (nitrogen content and enzyme activities) and the intensity of variable spots. Positive correlations were observed for nitrate reductase and glutamate dehydrogenase activities and the intensity of a number of root proteins. This approach may be a way to identify new structural or regulatory proteins involved in the response to the level of nitrogen fertilisation in different wheat varieties.

Glutamate (Glu) dehydrogenase (GDH) catalyses the reversible amination of 2-oxoglutarate for the synthesis of Glu using ammonium as a substrate. This enzyme preferentially occurs in the mitochondria of companion cells of a number of plant species grown on nitrate as the sole nitrogen source. For a better understanding of the controversial role of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate (F. Dubois, T. Tercé-Laforgue, M.B. Gonzalez-Moro, M.B. Estavillo, R. Sangwan, A. Gallais, B. Hirel [2003] Plant Physiol Biochem 41: 565–576), we studied the localization of GDH in untransformed tobacco (Nicotiana tabacum) plants grown either on low nitrate or on ammonium and in ferredoxin-dependent Glu synthase antisense plants. Production of GDH and its activity were strongly induced when plants were grown on ammonium as the sole nitrogen source. The induction mainly occurred in highly vascularized organs such as stems and midribs and was likely to be due to accumulation of phloem-translocated ammonium in the sap. GDH induction occurred when ammonia was applied externally to untransformed control plants or resulted from photorespiratory activity in transgenic plants down-regulated for ferredoxin-dependent Glu synthase. GDH was increased in the mitochondria and appeared in the cytosol of companion cells. Taken together, our results suggest that the enzyme plays a dual role in companion cells, either in the mitochondria when mineral nitrogen availability is low or in the cytosol when ammonium concentration increases above a certain threshold.

The metabolic, biochemical and molecular events occurring in the different leaf stages along the main axis of tobacco (Nicotiana tabacum) plants grown either on a nitrogen-rich medium, on a medium containing ammonium as sole nitrogen source or on a nitrogen-depleted medium, are presented. This study shows that the highest induction of cytosolic glutamine synthetase (GS1) protein and transcript occurs when nitrogen remobilization is maximal as the result of nitrogen starvation, whereas both glutamate dehydrogenase (GDH) transcript and activity remain at a very low level. In contrast, GDH is highly induced when plants are grown on ammonium as sole nitrogen source, a physiological situation during which leaf protein nitrogen remobilization is limited. It is therefore concluded that GDH does not play a direct role during the process of nitrogen remobilization but is rather induced following a built up of ammonium provided externally or released as the result of protein hydrolysis during natural leaf senescence.