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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.

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We present the first cloning and study of glutamine synthetase (GS) genes in wheat (Triticum aestivum L.). Based on sequence analysis, phylogenetic studies and mapping data, ten GS sequences were classified into four sub-families: GS2 (a, b and c), GS1 (a, b and c), GSr (1 and 2) and GSe (1 and 2). Phylogenetic analysis showed that the wheat GS sub-families together with the GS genes from other monocotyledonous species form four distinct clades. Immunolocalisation studies in leaves, stems and rachis in plants at flowering showed GS protein to be present in parenchyma, phloem companion and perifascicular sheath cells. In situ localisation confirmed that GS1 transcripts were present in the perifascicular sheath cells whilst those for GSr were confined to the vascular cells. Studies of the expression and protein profiles showed that all GS sub-families were differentially expressed in the leaves, peduncle, glumes and roots. Expression of GS genes in leaves was developmentally regulated, with both GS2 and GS1 assimilating or recycling ammonia in leaves during the period of grain development and filling. During leaf senescence the cytosolic isozymes, GS1 and GSr, were the predominant forms, suggesting major roles in assimilating ammonia during the critical phases of remobilisation of nitrogen to the grain. A preliminary analysis of three different wheat genotypes showed that the ratio of leaf GS2 protein to GS1 protein was variable. Use of this genetic variation should inform future efforts to modulate this enzyme for pre-breeding efforts to improve nitrogen use in wheat.

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

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

SummaryA novel Arabidopsis thaliana mutant, named hoc, was found to have an high organogenic capacity for shoot regeneration. The HOC locus may be involved in cytokinin metabolism leading to cytokininoverproduction. In vitro, hoc root explants develop many shoots in the absence of exogenous growth regulators. The mutant displays a bushy phenotype with supernumerary rosettes and with normal phyllotaxy, resulting from precocious axillary meristem development. Genetic and molecular analyses show that the high shoot regeneration and the bushy phenotype are controlled by a recessive single gene, located on chromosome I, next to the GAPB CAPS marker. The mapping data and allelism tests reveal that the hoc mutant is not allelic to other reported Arabidopsis growth-regulator mutants. In darkness the hoc mutant is de-etiolated, with a short hypocotyl, opened cotyledons and true leaves. Growth regulator assays reveal that the mutant accumulates cytokinins at about two-and sevenfold the cytokinin level of wild-type plants in its aerial parts and roots, respectively. Consequently, the elevated amounts of endogenous cytokinins in hoc plants are associated with high organogenic capacity and hence bushy phenotype. Thus hoc is the ®rst cytokinin-overproducing Arabidopsis mutant capable of auto-regenerating shoots without exogenous growth regulators.

The mobilization of nitrogen (N) compounds and the roles played by glumes and the flag leaf during grain filling were studied in bread wheat (Triticum aestivum L. cv. Florida) grown under field conditions. Glumes lost twice as much of their total N content as that lost by the flag leaf between the milk and early dough stages. In the flag leaf, glumes and grains, Glu, Asp, Ser and Ala accounted for 85% of all the reductions in the free amino acid pool. Principal component analysis of free amino acid pools separated grains from the glumes and the flag leaf, suggesting grain specific regulations in the use of free amino acids in protein synthesis. In all three organs, no decrease in Gln was detected, probably due to steady glutamine synthetase (GS; EC 6.3.1.2) activities per soluble protein in both the flag leaf and glumes. Compared with the flag leaf, glumes presented relatively smaller amounts of the chloroplast GS associated isoform. This we show is due to a lower relative number of mesophyll cells in glumes as supported by the different anatomy and the cellular pattern of the GS immunolocalization. We argue that cellular distribution plays a key role in supporting metabolism to enable the various functions undertaken by glume tissue.