N availability modulates the role of NPF3.1, a gibberellin transporter, in GA-mediated phenotypes in Arabidopsis.

MAIN CONCLUSION: AtNPF3.1 gene expression is promoted by limiting nitrogen nutrition. Atnpf3.1 mutants are affected in hypocotyl elongation and seed germination under conditions of low-nitrate availability. The NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER (NPF) family encodes nitrate or peptides transporters, some of which are also able to transport hormones. AtNPF3.1 has been described as a nitrate/nitrite/gibberellin transporter. Until now only its gibberellins (GAs) transport capacity have been proven in planta. We further analyzed its substrate specificity towards different GA species using a yeast heterologous system which revealed that (1) NPF3.1 transported not only bioactive GAs but also their precursors and metabolites and (2) the GAs' import activity of NPF3.1 was not affected by the presence of exogenous nitrate. Gene expression analysis along with germination assays and hypocotyl length measurements of loss of function mutants was used to understand the in planta role of NPF3.1. GUS staining revealed that this gene is expressed mainly in the endodermis of roots and hypocotyls, in shoots, stamens, and dry seeds. Germination assays in the presence of paclobutrazol, a GA biosynthesis inhibitor, revealed that the germination rate of npf3.1 mutants was lower compared to wild type when GA was added at the same time. Likewise, hypocotyl length measurements showed that the npf3.1 mutants were less sensitive to exogenous GA addition in the presence of paclobutrazol, compared to wild type. Moreover, this phenotype was observed only when plants were grown on low-nitrate supply. In addition, NPF3.1 gene expression was upregulated by low exogenous nitrate concentrations and the npf3.1 mutants exhibited a not yet described GA-related phenotype under these conditions. All together, these results indicated that NPF3.1 is indeed involved in GAs transport in planta under low-nitrate conditions.

Brachypodium: a promising hub between model species and cereals.

Brachypodium distachyon was proposed as a model species for genetics and molecular genomics in cereals less than 10 years ago. It is now established as a standard for research on C3 cereals on a variety of topics, due to its close phylogenetic relationship with Triticeae crops such as wheat and barley, and to its simple genome, its minimal growth requirement, and its short life cycle. In this review, we first highlight the tools and resources for Brachypodium that are currently being developed and made available by the international community. We subsequently describe how this species has been used for comparative genomic studies together with cereal crops, before illustrating major research fields in which Brachypodium has been successfully used as a model: cell wall synthesis, plant-pathogen interactions, root architecture, and seed development. Finally, we discuss the usefulness of research on Brachypodium in order to improve nitrogen use efficiency in cereals, with the aim of reducing the amount of applied fertilizer while increasing the grain yield. Several paths are considered, namely an improvement of either nitrogen remobilization from the vegetative organs, nitrate uptake from the soil, or nitrate assimilation by the plant. Altogether, these examples position the research on Brachypodium as at an intermediate stage between basic research, carried out mainly in Arabidopsis, and applied research carried out on wheat and barley, enabling a complementarity of the studies and reciprocal benefits.

Nitrate transport and signalling in Arabidopsis.

Plants have developed adaptive responses allowing them to cope with nitrogen (N) fluctuation in the soil and maintain growth despite changes in external N availability. Nitrate is the most important N form in temperate soils. Nitrate uptake by roots and its transport at the whole-plant level involves a large panoply of transporters and impacts plant performance. Four families of nitrate-transporting proteins have been identified so far: nitrate transporter 1/peptide transporter family (NPF), nitrate transporter 2 family (NRT2), the chloride channel family (CLC), and slow anion channel-associated homologues (SLAC/SLAH). Nitrate transporters are also involved in the sensing of nitrate. It is now well established that plants are able to sense external nitrate availability, and hence that nitrate also acts as a signal molecule that regulates many aspects of plant intake, metabolism, and gene expression. This review will focus on a global picture of the nitrate transporters so far identified and the recent advances in the molecular knowledge of the so-called primary nitrate response, the rapid regulation of gene expression in response to nitrate. The recent discovery of the NIN-like proteins as master regulators for nitrate signalling has led to a new understanding of the regulation cascade.

Proanthocyanidin oxidation of Arabidopsis seeds is altered in mutant of the high-affinity nitrate transporter NRT2.7.

NRT2.7 is a seed-specific high-affinity nitrate transporter controlling nitrate content in Arabidopsis mature seeds. The objective of this work was to analyse further the consequences of the nrt2.7 mutation for the seed metabolism. This work describes a new phenotype for the nrt2.7-2 mutant allele in the Wassilewskija accession, which exhibited a distinctive pale-brown seed coat that is usually associated with a defect in flavonoid oxidation. Indeed, this phenotype resembled those of tt10 mutant seeds defective in the laccase-like enzyme TT10/LAC15, which is involved in the oxidative polymerization of flavonoids such as the proantocyanidins (PAs) (i.e. epicatechin monomers and PA oligomers) and flavonol glycosides. nrt2.7-2 and tt10-2 mutant seeds displayed the same higher accumulation of PAs, but were partially distinct, since flavonol glycoside accumulation was not affected in the nrt2.7-2 seeds. Moreover, measurement of in situ laccase activity excluded a possibility of the nrt2.7-2 mutation affecting the TT10 enzymic activity at the early stage of seed development. Functional complementation of the nrt2.7-2 mutant by overexpression of a full-length NRT2.7 cDNA clearly demonstrated the link between the nrt2.7 mutation and the PA phenotype. However, the PA-related phenotype of nrt2.7-2 seeds was not strictly correlated to the nitrate content of seeds. No correlation was observed when nitrate was lowered in seeds due to limited nitrate nutrition of plants or to lower nitrate storage capacity in leaves of clca mutants deficient in the vacuolar anionic channel CLCa. All together, the results highlight a hitherto-unknown function of NRT2.7 in PA accumulation/oxidation.

PII is induced by WRINKLED1 and fine-tunes fatty acid composition in seeds of Arabidopsis thaliana.

The PII protein is an integrator of central metabolism and energy levels. In Arabidopsis, allosteric sensing of cellular energy and carbon levels alters the ability of PII to interact with target enzymes such as N-acetyl-l-glutamate kinase and heteromeric acetyl-coenzyme A carboxylase, thereby modulating the biological activity of these plastidial ATP- and carbon-consuming enzymes. A quantitative reverse transcriptase-polymerase chain reaction approach revealed a threefold induction of the AtGLB1 gene (At4g01900) encoding PII during early seed maturation. The activity of the AtGLB1 promoter was consistent with this pattern. A complementary set of molecular and genetic analyses showed that WRINKLED1, a transcription factor known to induce glycolytic and fatty acid biosynthetic genes at the onset of seed maturation, directly controls AtGLB1 expression. Immunoblot analyses and immunolocalization experiments using anti-PII antibodies established that PII protein levels faithfully reflected AtGLB1 mRNA accumulation. At the subcellular level, PII was observed in plastids of maturing embryos. To further investigate the function of PII in seeds, comprehensive functional analyses of two pII mutant alleles were carried out. A transient increase in fatty acid production was observed in mutant seeds at a time when PII protein content was found to be maximal in wild-type seeds. Moreover, minor though statistically significant modifications of the fatty acid composition were measured in pII seeds, which exhibited decreased amounts of modified (elongated, desaturated) fatty acid species. The results obtained outline a role for PII in the fine tuning of fatty acid biosynthesis and partitioning in seeds.

Metabolite regulation of the interaction between Arabidopsis thaliana PII and N-acetyl-l-glutamate kinase.

The metabolic control of the interaction between ArabidopsisN-acetyl-l-glutamate kinase (NAGK) and the PII protein has been studied. Both gel exclusion and affinity chromatography analyses of recombinant, affinity-purified PII (trimeric complex) and NAGK (hexameric complex) showed that NAGK strongly interacted with PII only in the presence of Mg-ATP, and that this process was reversed by 2-oxoglutarate (2-OG). Furthermore, metabolites such as arginine, glutamate, citrate, and oxalacetate also exerted a negative effect on the PII-NAGK complex formation in the presence of Mg-ATP. Using chloroplast protein extracts and PII affinity chromatography, NAGK interacted with PII only in the presence of ATP-Mg(2+), and this process was antagonized by 2-OG. These results reveal a complex metabolic control of the PII interaction with NAGK in the chloroplast stroma of higher plants.

Assimilation of excess ammonium into amino acids and nitrogen translocation in Arabidopsis thaliana--roles of glutamate synthases and carbamoylphosphate synthetase in leaves.

This study was aimed at investigating the physiological role of ferredoxin-glutamate synthases (EC 1.4.1.7), NADH-glutamate synthase (EC 1.4.1.14) and carbamoylphosphate synthetase (EC 6.3.5.5) in Arabidopsis. Phenotypic analysis revealed a high level of photorespiratory ammonium, glutamine/glutamate and asparagine/aspartate in the GLU1 mutant lacking the major ferredoxin-glutamate synthase, indicating that excess photorespiratory ammonium was detoxified into amino acids for transport out of the veins. Consistent with these results, promoter analysis and in situ hybridization demonstrated that GLU1 and GLU2 were expressed in the mesophyll and phloem companion cell-sieve element complex. However, these phenotypic changes were not detected in the GLU2 mutant defective in the second ferredoxin-glutamate synthase gene. The impairment in primary ammonium assimilation in the GLT mutant under nonphotorespiratory high-CO(2) conditions underlined the importance of NADH-glutamate synthase for amino acid trafficking, given that this gene only accounted for 3% of total glutamate synthase activity. The excess ammonium from either endogenous photorespiration or the exogenous medium was shifted to arginine. The promoter analysis and slight effects on overall arginine synthesis in the T-DNA insertion mutant in the single carbamoylphosphate synthetase large subunit gene indicated that carbamoylphosphate synthetase located in the chloroplasts was not limiting for ammonium assimilation into arginine. The data provided evidence that ferredoxin-glutamate synthases, NADH-glutamate synthase and carbamoylphosphate synthetase play specific physiological roles in ammonium assimilation in the mesophyll and phloem for the synthesis and transport of glutamine, glutamate, arginine, and derived amino acids.

Chloroplast nitrite uptake is enhanced in Arabidopsis PII mutants.

In higher plants, the PII protein is a nuclear-encoded plastid protein that regulates the activity of a key enzyme of arginine biosynthesis. We have previously observed that Arabidopsis PII mutants are more sensitive to nitrite toxicity. Using intact chloroplasts isolated from Arabidopsis leaves and (15)N-labelled nitrite we show that a light-dependent nitrite uptake into chloroplasts is increased in PII knock-out mutants when compared to the wild-type. This leads to a higher incorporation of (15)N into ammonium and amino acids in the mutant chloroplasts. However, the uptake differences do not depend on GS/GOGAT activities. Our observations suggest that PII is involved in the regulation of nitrite uptake into higher plant chloroplasts.

The regulatory PII protein controls arginine biosynthesis in Arabidopsis.

In higher plants, PII is a nuclear-encoded plastid protein which is homologous to bacterial PII signalling proteins known to be involved in the regulation of nitrogen metabolism. A reduced ornithine, citrulline and arginine accumulation was observed in two Arabidopsis PII knock-out mutants in response to NH4+ resupply after N starvation. This difference could be explained by the regulation of a key enzyme of the arginine biosynthesis pathway, N-acetyl glutamate kinase (NAGK) by PII. In vitro assays using purified recombinant proteins showed the catalytic activation of Arabidopsis NAGK by PII giving the first evidence of a physiological role of the PII protein in higher plants. Using Arabidopsis transcriptome microarray (CATMA) and RT-PCR analyses, it was found that none of the genes involved in the arginine biosynthetic or catabolic pathways were differentially expressed in a PII knock-out mutant background. In conclusion, the observed changes in metabolite levels can be explained by the reduced activation of NAGK by PII.

Physiological characterisation of Arabidopsis mutants affected in the expression of the putative regulatory protein PII.

The PII signal transducing protein is involved in carbon/nitrogen (C/N) sensing in bacteria and cyanobacteria. In higher plants the function of the PII homolog GLB1 is not known. GLB1 transcripts were found in all plant organs tested, while in Arabidopsis leaves GLB1 expression and PII protein levels were not significantly affected by either the day/night cycle or N-nutrition. Its putative regulatory role in plants has been studied by analysing Arabidopsis thaliana T-DNA insertion lines in the GLB1 gene. These PII mutants showed an 80% (PIIV1 mutant) and 100% (PIIS2 mutant) reduced AtGLB1 transcript level and no detectable PII protein. They did not display an altered growth or developmental phenotype when grown under non-limiting conditions suggesting that the PII protein does not play a crucial role in plants. However, in vitro grown PII mutants did show a higher sensitivity to nitrite (NO (2) (-) ) compared to the wild-type plants. This observation is reminiscent of the role of PII in the regulation of NO (2) (-) metabolism in cyanobacteria. Furthermore, when grown hydroponically, the PII mutants displayed a slight increase in carbohydrate (starch and sugars) levels in response to N starvation and a slight decrease in the levels of ammonium (NH (4) (+) ) and amino acids (mainly Gln) in response to NH (4) (+) resupply. Although the phenotypic changes are rather small in the mutant lines, these data support the hypothesis of a subtle involvement of the PII protein in the regulation of some steps of primary C and N metabolism.

Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.).

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5' flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, (15)NH4(+) was incorporated into [5-(15)N]glutamine and [2-(15)N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2-(15)N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2-(15)N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15-20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO2 contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation.

Glutamate dehydrogenase of tobacco is mainly induced in the cytosol of phloem companion cells when ammonia is provided either externally or released during photorespiration.

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. Terce-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.

Photorespiration-dependent increases in phospho enolpyruvate carboxylase, isocitrate dehydrogenase and glutamate dehydrogenase in transformed tobacco plants deficient in ferredoxin-dependent glutamine-alpha-ketoglutarate aminotransferase.

The metabolic cross-talk associated with re-assimilation of photorespiratory NH4+ was analysed in transformed tobacco (Nicotiana tabacum L.) plants with low activities of ferredoxin-dependent glutamine-alpha-ketoglutarate aminotransferase (Fd-GOGAT; EC 1.4.7.1). Amounts of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco; EC 4.1.1.39) protein and Rubisco transcripts were similar in all lines whether photorespiration rates were low (4,000 microl l(-1) CO2) or high (air). Leaf sucrose, hexose and starch contents were similar in all lines. In contrast, there was evidence that anaplerotic carbon flow was stimulated in the transformed lines with less than 60% Fd-GOGAT, since phospho enolpyruvate carboxylase (PEPc) activity and (PEPc) protein were increased. A strong positive correlation between leaf PEPc activity and glutamine accumulation was observed, suggesting that the increase in PEPc was related to the accumulation of glutamine. A modest stimulation of total NADP-isocitrate dehydrogenase (ICDH; EC 1.1.1.42) activity was also observed in the transformed lines with less than 60% Fd-GOGAT. This was accompanied by increases in both the cytosolic ICDH and mitochondrial NAD-isocitrate dehydrogenases (IDH; EC 1.1.1.41). IDH protein was also increased in the transformed plants with low Fd-GOGAT, suggesting that both IDH and ICDH are involved in the production of carbon skeletons (and ultimately alpha-ketoglutarate) necessary for the re-assimilation of NH4+. In contrast, PEPc, ICDH and IDH transcripts were similar in all lines. The aminating (but not the de-aminating) activity of NAD(H)-glutamate dehydrogenase (NAD(H)-GDH; EC 1.4.1.2) was greatly increased in plants with less than 60% of Fd-GOGAT after transfer to air. The data confirm that NH4+ or glutamine are involved in signalling, leading to modified gene expression and enzyme activity required for enhanced production of the C skeletons, to accommodate increases in the assimilation of photorespiratory NH4+. In addition, we provide the first demonstration of a compensatory role for NAD(H)-GDH in stabilising the leaf glutamic acid pool when Fd-GOGAT becomes limiting.