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Background A better understanding of the physiological response of silage maize to a mild reduction in nitrogen(N) fertilization and the identification of predictive biochemical markers of N utilization efficiency could contribute to limit the detrimental effect of the overuse of N inputs. Results To identify predictive biochemical markers of nitrogen (N) utilization and metabolism of silage maize in relation to growth and productivity, a metabolomic and a proteomic approach were combined. These analyses were performed on young leaves of a core panel of 29 European genetically diverse dent hybrids cultivated in the field under non-limiting and reduced N fertilization conditions in order to identify such predictive markers at an early stage of plant development. Metabolome and proteome data were analyzed either individually or in an integrated manner together with eco-physiological, developmental and yield-related traits. These analyses allowed to depict the physiology underlying plant response to the mild N deficit often occurring when maize is grown under agronomic conditions. Moreover, the genetic diversity of the 29 hybrids was exploited to identify common N-responsive metabolites and protein that could be used as predictive markers to monitor N fertilization and to identify silage maize hybrids representing possible ideotypes that exhibit improved agronomic performance when N fertilization is reduced. Conclusion Among the N-responsive metabolites and proteins identified, a cytosolic NADP-dependent malic enzyme and four metabolite signatures stand out as promising markers that could be used for both breeding and agronomic purposes.

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.

In this opinion article, we have analyzed the relevancy of a hypothesis which is based on the idea that in Arabidopsis thaliana jasmonic acid (JA)-mediated defense system against necrotrophic fungi is weakened when NO3- supply is high. Such hypothesis is based on the fact that when NO3- supply is high, it induces an increase in the amount of bioactive ABA which induces the sequestration of the phosphatase ABI2 (PP2C) into the PYR/PYL/RCAR receptor. Consequently, the Ca sensors CBL1/9 - CIPK23 are not dephosphorylated by ABI2, thus remaining able to phosphorylate targets such as AtNPF6.3 and AtKAT1, a NO3- and K+ transporters respectively. Therefore, the impact of phosphorylation on the regulation of these two transporters, could 1) reduce NO3- influx as in its phosphorylated state AtNPF6.3 shifts to low capacity state and 2) increase K+ influx, as in its phosphorylated state KAT1 becomes more active. It is also well known that in the roots K+ loading in the xylem and its transport to the shoot is activated in the presence of NO3-. As such, the enrichment of plant tissues in K+ can impair jasmonic acid (JA) regulatory pathway and the induction of the corresponding biomarkers. The latter are known to be up-regulated under K+ deficiency and inhibited when K+ is resupplied. We therefore suggest that increased K+ uptake and tissue content induced by high NO3- supply modifies JA regulatory pathway, resulting in weakened JA-mediated plant’s defense system against necrotrophic fungi.

In this opinion article, we have analyzed the relevancy of a hypothesis which is based on the idea that in

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.

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.

Cytosolic glutamine synthetase (GS1) is the enzyme mainly responsible of ammonium assimilation and reassimilation in maize leaves. The agronomic potential of GS1 in maize kernel production was investigated by examining the impact of an overexpression of the enzyme in the leaf cells. Transgenic hybrids exhibiting a three-fold increase in leaf GS activity were produced and characterized using plants grown in the field. Several independent hybrids overexpressing Gln1-3, a gene encoding cytosolic (GS1), in the leaf and bundle sheath mesophyll cells were grown over five years in different locations. On average, a 3.8% increase in kernel yield was obtained in the transgenic hybrids compared to controls. However, we observed that such an increase was simultaneously dependent upon both the environmental conditions and the transgenic event for a given field trial. Although variable from one environment to another, significant associations were also found between two GS1 genes (Gln1-3 and Gln1-4) polymorphic regions and kernel yield in different locations. We propose that the GS1 enzyme is a potential lead for producing high yielding maize hybrids using either genetic engineering or marker-assisted selection. However, for these hybrids, yield increases will be largely dependent upon the environmental conditions used to grow the plants., Amiour et al. use a multi-year field trial evaluation and association mapping to determine if increased enzyme activity and native allelic variations at the GS1 loci in maize contribute to differences in grain yield. Overexpression of GS1 and polymorphisms in the corresponding loci were associated with kernel yield, indicating that GS1 expression can directly control kernel production and that GS1 has a potential lead in the production of high yielding maize hybrids depending on environmental conditions.

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.

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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.

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.

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 (NO

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

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; 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.

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.

International audience; Synthetic nitrogen (N) fertilisers are widely used for enhancing agrosystem productivity and are thus thought to increase organic inputs from crop residues. However, many crop rotations have a low amount of organic residue returned to the soil since the whole aboveground crop biomass is harvested and exported. To compensate for such organic outputs and to improve soil quality, the introduction of winter cover crops in rotations has been suggested. A 4-year controlled field experiment was conducted to quantify the respective and combined effects of chemical N fertilisation and winter cover crops on plant productivity, organic carbon (C) and N inputs from crop residues and cover crops, changes in soil C and N concentrations, C:N ratio, soil mineral N, pH, soil moisture and soil bacterial biodiversity. A ploughing tillage system with low organic input was assessed, for which the main crops were spring wheat, green pea, forage maize, along with cover crops of different legume and non-legume species. N fertilisation did not have an impact on the aboveground biomass except following forage maize. Cover crops increased the total amount of C and N inputs, irrespective of N fertilisation which had no significant effect The soil N concentration decreased in all treatments, particularly when N fertilisers were applied under bare-fallow conditions. The latter treatment also caused decreased soil C concentrations (slightly increased in the other treatments) and decreased bacterial biodiversity (no change in the other treatments). Bacteria from the Proteobacteria and Bacterioidetes phyla were highly correlated with soil from fertilised bare fallow conditions. While Verrucomicrobia was characteristic of non-fertilised bare fallow soils, Acidobacteria and Cyanobacteria were associated with the high C and N concentrations present in soils following cover crop treatments. Taken together, these results demonstrate that in ploughing systems, under low organic restitution regimes, intensive N fertilisation decreases the diversity of the bacterial soil community and reduces soil C and N concentrations, but only in bare fallow conditions. There is a protective effect of winter cover crops against the deleterious effect of chemical N fertilisation on soil biodiversity and nutrient cycling, since they can maintain soil C and N concentrations. The use of winter cover crops containing legumes is thus a practice that is able to meet the criteria of a sustainable agriculture.

In this review, we present the recent developments and future prospects of improving nitrogen use efficiency (NUE) in crops using various complementary approaches. These include conventional breeding and molecular genetics, in addition to alternative farming techniques based on no-till continuous cover cropping cultures and/or organic nitrogen (N) nutrition. Whatever the mode of N fertilization, an increased knowledge of the mechanisms controlling plant N economy is essential for improving NUE and for reducing excessive input of fertilizers, while maintaining an acceptable yield and sufficient profit margin for the farmers. Using plants grown under agronomic conditions, with different tillage conditions, in pure or associated cultures, at low and high N mineral fertilizer input, or using organic fertilization, it is now possible to develop further 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 a critical overview as to how our understanding of the agro-ecophysiological, physiological and molecular controls of N assimilation in crops, under varying environmental conditions, has been improved. We have used combined approaches, based on agronomic studies, whole plant physiology, quantitative genetics, forward and reverse genetics and the emerging systems biology. Long-term sustainability may require a gradual transition from synthetic N inputs to legume-based crop rotation, including continuous cover cropping systems, where these may be possible in certain areas of the world, depending on climatic conditions. Current knowledge and prospects for future agronomic development and application for breeding crops adapted to lower mineral fertilizer input and to alternative farming techniques are explored, whilst taking into account the constraints of both the current world economic situation and the environment.

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.

In higher plants, two vascular tissues, phloem and xylem, are responsible for the transport of water, minerals and nutrients between organs. The xylem transports water and minerals absorbed by the roots and the phloem transports from source to sink organs photoassimilates synthesized in the photosynthetic leaves. The phloem is essential for higher plants in many developmental and physiological processes. It not only provides the route for the distribution of assimilates but also redistribute mineral nutrients. Additionally, the phloem is essential for sending information between distant plant organs and steering developmental and defence processes. For example, flowering and tuberization time are controlled by phloem-mobile signals and important defence reactions on the whole plant level, like systemic acquired resistance or systemic gene silencing, are spread through the phloem. In addition, recent results demonstrate that also the allocation of mineral nutrients is coordinated by phloem mobile signaling molecules. However, in many studies the important analysis of phloem sap is neglected, probably because the content of sieve tubes is not easy to access. We used the EDTA-facilitated exudation method to collect the phloem sap from three plant species, Maize, Tomato and Arabidopsis. The metabolites present in the samples were analysed by GC-MS or GC-TOF to determine the range of metabolites present in the phloem sap. We detected more than 50 metabolites, including sugars, amino acids and organic acids. Because of an intrinsic biological variability in the efficiency of exudation depending on the plant samples, we developed a method for the normalisation and the statistical analysis of the phloem sap metabolite profiles. This study showed that for each species, there is a significant conservation of the phloem sap content, revealing the strong physical and physiological constraints on phloem transport. However we observed important differences in the phloem sap metabolic profiles between plant species, showing different strategies for the allocation of sugars and amino acids between organs.