The roles of two cytosolic maize glutamine synthetase isoenzymes (GS1), products of the Gln1-3 and Gln1-4 genes, were investigated by examining the impact of knockout mutations on kernel yield. In the gln1-3 and gln1-4 single mutants and the gln1-3 gln1-4 double mutant, GS mRNA expression was impaired, resulting in reduced GS1 protein and activity. The gln1-4 phenotype displayed reduced kernel size and gln1-3 reduced kernel number, with both phenotypes displayed in gln1-3 gln1-4. However, at maturity, shoot biomass production was not modified in either the single mutants or double mutants, suggesting a specific impact on grain production in both mutants. Asn increased in the leaves of the mutants during grain filling, indicating that it probably accumulates to circumvent ammonium buildup resulting from lower GS1 activity. Phloem sap analysis revealed that unlike Gln, Asn is not efficiently transported to developing kernels, apparently causing reduced kernel production. When Gln1-3 was overexpressed constitutively in leaves, kernel number increased by 30%, providing further evidence that GS1-3 plays a major role in kernel yield. Cytoimmunochemistry and in situ hybridization revealed that GS1-3 is present in mesophyll cells, whereas GS1-4 is specifically localized in the bundle sheath cells. The two GS1 isoenzymes play nonredundant roles with respect to their tissue-specific localization.

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.

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.

SummaryIn this article, we discuss the ways in which our understanding of the controls of nitrogen remobilisation in model species and crop plants have been increased through classical physiological studies and the use of transgenic plants or mutants with modified capacities for nitrogen or carbon assimilation and recycling. An improved understanding of the transition between nitrogen assimilation and nitrogen recycling will be vital, if improvements in crop nitrogen use efficiency are to reduce the need for excessive input of fertilisers and improve or stabilise yield. In this review, we present an overall view of past work and more recent studies on this topic, using different plants systems and models depicting the biochemical and molecular events occurring during the transition between sink leaves and source leaves. These models may provide a way to identify the nature of the metabolic or developmental signals triggering in a coordinate manner nitrogen and carbon recycling during leaf senescence.Another way of developing crop varieties with improved nitrogen use efficiency, and identifying key elements controlling the process of nitrogen remobilisation, is the use of quantitative genetics. We present and discuss recent findings on the genetic variability and basis of nitrogen use efficiency in crops in general and in maize in particular. A genetic approach using maize recombinant inbred lines was undertaken allowing the detection of Quantitative Trait Loci (QTLs) for morphological traits, grain yield and its components under high nitrogen or low nitrogen input. Co‐mapping was observed between genes encoding enzymes involved in nitrogen assimilation (nitrate reductase, glutamine synthetase) and these Quantitative Trait Loci. All coincidences were consistent with the expected physiological function of the corresponding enzyme activities. This work strongly suggests that in maize, nitrogen use efficiency can be improved both by marker‐assisted selection and genetic engineering.

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