A revised view on the evolution of glutamine synthetase isoenzymes in plants.

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 genes 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) as a result of 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 new view of GS evolution in plants, including a new cytosolic GS group (GS1a), has important functional implications in the context of plant metabolism adaptation to global changes.

Dissecting the metabolic reprogramming of maize root under nitrogen-deficient stress conditions.

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.

Does Potassium (K+) Contribute to High-Nitrate (NO3-) Weakening of a 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 Arabidopsis thaliana jasmonic acid, a (JA)-mediated defense system against necrotrophic fungi is weakened when NO3- supply is high. Such a 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, which are 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 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 a 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 the JA regulatory pathway, resulting in a weakened JA-mediated plant's defense system against necrotrophic fungi.

Beneficial soil-borne bacteria and fungi: a promising way to improve plant nitrogen acquisition.

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.

Exploiting the Genetic Diversity of Maize Using a Combined Metabolomic, Enzyme Activity Profiling, and Metabolic Modeling Approach to Link Leaf Physiology to Kernel Yield.

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.

Resolving the Role of Plant NAD-Glutamate Dehydrogenase: III. Overexpressing Individually or Simultaneously the Two Enzyme Subunits Under Salt Stress Induces Changes in the Leaf Metabolic Profile and Increases Plant Biomass Production.

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.

Assessing the metabolic impact of nitrogen availability using a compartmentalized maize leaf genome-scale model.

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.

Characterization of a NADH-dependent glutamate dehydrogenase mutant of Arabidopsis demonstrates the key role of this enzyme in root carbon and nitrogen metabolism.

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, γ-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.

An integrated "omics" approach to the characterization of maize (Zea mays L.) mutants deficient in the expression of two genes encoding cytosolic glutamine synthetase.

BACKGROUND: To identify the key elements controlling grain production in maize, it is essential to have an integrated view of the responses to alterations in the main steps of nitrogen assimilation by modification of gene expression. Two maize mutant lines (gln1.3 and gln1.4), deficient in two genes encoding cytosolic glutamine synthetase, a key enzyme involved in nitrogen assimilation, were previously characterized by a reduction of kernel size in the gln1.4 mutant and by a reduction of kernel number in the gln1.3 mutant. In this work, the differences in leaf gene transcripts, proteins and metabolite accumulation in gln1.3 and gln1.4 mutants were studied at two key stages of plant development, in order to identify putative candidate genes, proteins and metabolic pathways contributing on one hand to the control of plant development and on the other to grain production. RESULTS: The most interesting finding in this study is that a number of key plant processes were altered in the gln1.3 and gln1.4 mutants, including a number of major biological processes such as carbon metabolism and transport, cell wall metabolism, and several metabolic pathways and stress responsive and regulatory elements. We also found that the two mutants share common or specific characteristics across at least two or even three of the "omics" considered at the vegetative stage of plant development, or during the grain filling period. CONCLUSIONS: This is the first comprehensive molecular and physiological characterization of two cytosolic glutamine synthetase maize mutants using a combined transcriptomic, proteomic and metabolomic approach. We find that the integration of the three "omics" procedures is not straight forward, since developmental and mutant-specific levels of regulation seem to occur from gene expression to metabolite accumulation. However, their potential use is discussed with a view to improving our understanding of nitrogen assimilation and partitioning and its impact on grain production.

Nitrogen-use efficiency in maize (Zea mays L.): from 'omics' studies to metabolic modelling.

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

Resolving the role of plant glutamate dehydrogenase: II. Physiological characterization of plants overexpressing the two enzyme subunits individually or simultaneously.

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 ß-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.

15N-labelling of Leaves Combined with GC-MS Analysis as a Tool for Monitoring the Dynamics of Nitrogen Incorporation into Amino Acids.

Labeling plant material such as detached leaves with 15NH4+ is a very instrumental method for the characterization of metabolic pathways of mineral nitrogen assimilation and incorporation into amino acids. A procedure of labeling, followed by amino acid extraction, purification, and derivatization for gas chromatography coupled to mass spectrometry (GC/MS) analysis, is presented. The rationale of heavy isotope abundance calculations and amino acid quantification is detailed. This method is adaptable to various plant species and various kinds of investigations, such as elucidating physiological changes occurring as a result of gene mutations (overexpression or inhibition) in natural variants or genetically modified crops, or characterization of metabolic fluxes in genotypes exhibiting contrasted physiological or developmental adaptive responses to biotic and/or abiotic environmental stresses. Furthermore, the benefit of working on detached organs or pieces of organs is to investigate finely the metabolism of species that are not amenable to laboratory work, such as plants growing in natural environments or under agricultural conditions in the field.

Sphingomonas sediminicola Is an Endosymbiotic Bacterium Able to Induce the Formation of Root Nodules in Pea (Pisum sativum L.) and to Enhance Plant Biomass Production.

The application of bacterial bio-inputs is a very attractive alternative to the use of mineral fertilisers. In ploughed soils including a crop rotation pea, we observed an enrichment of bacterial communities with Sphingomonas (S.) sediminicola. Inoculation experiments, cytological studies, and de novo sequencing were used to investigate the beneficial role of S. sediminicola in pea. S. sediminicola is able to colonise pea plants and establish a symbiotic association that promotes plant biomass production. Sequencing of the S. sediminicola genome revealed the existence of genes involved in secretion systems, Nod factor synthesis, and nitrogenase activity. Light and electron microscopic observations allowed us to refine the different steps involved in the establishment of the symbiotic association, including the formation of infection threads, the entry of the bacteria into the root cells, and the development of differentiated bacteroids in root nodules. These results, together with phylogenetic analysis, demonstrated that S. sediminicola is a non-rhizobia that has the potential to develop a beneficial symbiotic association with a legume. Such a symbiotic association could be a promising alternative for the development of more sustainable agricultural practices, especially under reduced N fertilisation conditions.

The Key Role of Glutamate Dehydrogenase 2 (GDH2) in the Control of Kernel Production in Maize (Zea mays L.).

The agronomic potential of glutamate dehydrogenase 2 (GDH2) in maize kernel production was investigated by examining the impact of a mutation on the corresponding gene. Mu-insertion homozygous and heterozygous mutant lines lacking GDH2 activity were isolated and characterized at the biochemical, physiological and agronomic levels. In comparison to the wild type and to the homozygous ghd2 mutants, the heterozygous gdh2 mutant plants were characterized by a decrease in the root amino acid content, whereas in the leaves an increase of a number of phenolic compounds was observed. On average, a 30 to 40% increase in kernel yield was obtained only in the heterozygous gdh2 mutant lines when plants were grown in the field over two years. The importance of GDH2 in the control of plant productivity is discussed in relation to the physiological impact of the mutation on amino acid content, with primary carbon metabolism mostly occurring in the roots and secondary metabolism occurring in the leaves.

A multi-organ maize metabolic model connects temperature stress with energy production and reducing power generation.

Climate change has adversely affected maize productivity. Thereby, a holistic understanding of metabolic crosstalk among its organs is important to address this issue. Thus, we reconstructed the first multi-organ maize metabolic model, iZMA6517, and contextualized it with heat and cold stress transcriptomics data using expression distributed reaction flux measurement (EXTREAM) algorithm. Furthermore, implementing metabolic bottleneck analysis on contextualized models revealed differences between these stresses. While both stresses had reducing power bottlenecks, heat stress had additional energy generation bottlenecks. We also performed thermodynamic driving force analysis, revealing thermodynamics-reducing power-energy generation axis dictating the nature of temperature stress responses. Thus, a temperature-tolerant maize ideotype can be engineered by leveraging the proposed thermodynamics-reducing power-energy generation axis. We experimentally inoculated maize root with a beneficial mycorrhizal fungus, Rhizophagus irregularis, and as a proof-of-concept demonstrated its efficacy in alleviating temperature stress. Overall, this study will guide the engineering effort of temperature stress-tolerant maize ideotypes.

Can genetic variability for nitrogen metabolism in the developing ear of maize be exploited to improve yield?

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

The use of metabolomics integrated with transcriptomic and proteomic studies for identifying key steps involved in the control of nitrogen metabolism in crops such as maize.

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.

An integrated statistical analysis of the genetic variability of nitrogen metabolism in the ear of three maize inbred lines (Zea mays L.).

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 Δ1-pyrroline-5-carboxylate synthetase (P5CS).

Impacts of environmental conditions, and allelic variation of cytosolic glutamine synthetase on maize hybrid kernel production.

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.

Analysis of amino acid metabolism in the ear of maize mutants deficient in two cytosolic glutamine synthetase isoenzymes highlights the importance of asparagine for nitrogen translocation within sink organs.

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.