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The development of nitrogen-fixing nodules in legumes is induced by perception of lipochitin-oligosaccharide signals secreted by a bacterial symbiont. Nitrogen (N) starvation is a prerequisite for the formation, development, and function of root nodules, and high levels of combined N in the form of nitrate or ammonium can completely abolish nodule formation. We distinguished between nitrate and ammonium inhibitory effects by identifying when and where these combined N sources interfere with the Nod-factor-induced pathway. Furthermore, we present a small-scale analysis of the expression profile, under different N conditions, of recently identified genes involved in the Nod-factor-induced pathway. In the presence of high levels of nitrate or ammonium, the NIN gene fails to be induced 24 h after the addition of Nod factor compared with plants grown under N-free conditions. This induction is restored in the hypernodulating nitrate-tolerant har1-3 mutant only in the presence of 10 and 20 mM KNO3. These results were confirmed in Lotus plants inoculated with Mesorhizobium loti. NIN plays a key role in the nodule organogenesis program and its downregulation may represent a crucial event in the nitrate-dependent pathway leading to the inhibition of nodule organogenesis.

Nitrate is an essential element for plant growth, both as a primary nutrient in the nitrogen assimilation pathway and as an important signal for plant development. Low‐ and high‐affinity transport systems are involved in the nitrate uptake from the soil and its distribution between different plant tissues. By an in silico search, we identified putative members of both systems in the model legume Lotus japonicus. We investigated, by a time course analysis, the transcripts abundance in root tissues of nine and four genes encoding putative low‐affinity (NRT1) and high‐affinity (NRT2) nitrate transporters, respectively. The genes were sub‐classified as inducible, repressible and constitutive on the basis of their responses to provision of nitrate, auxin or cytokinin. Furthermore, the analysis of the pattern of expression in root and nodule tissues after Mesorhizobium loti inoculation permitted the identification of sequences significantly regulated during the symbiotic interaction. The interpretation of the global regulative networks obtained allowed to postulate roles for nitrate transporters as possible actors in the cross‐talks between different signalling pathways triggered by biotic and abiotic factors.

(M.B., A.M.) Ammonium is a primary source of nitrogen for plants. In legume plants ammonium can also be obtained by symbiotic nitrogen fixation, and NH 1 4 is also a regulator of early and late symbiotic interaction steps. Ammonium transporters are likely to play important roles in the control of nodule formation as well as in nitrogen assimilation. Two new genes, LjAMT1;2 and LjAMT1;3, were cloned from Lotus japonicus. Both were able to complement the growth defect of a yeast (Saccharomyces cerevisiae) ammonium transport mutant. Measurement of [ 14 C]methylammonium uptake rates and competition experiments revealed that each transporter had a high affinity for NH 1 4 . The K i for ammonium was 1.7, 3, and 15 mM for LjAMT1;1, 1;2, and 1;3, respectively. Real-time PCR revealed higher expression of LjAMT1;1, 1;2, and 1;3 genes in leaves than in roots and nodule, with expression levels decreasing in the order LjAMT1;1 [ 1;2 [ 1;3 except in flowers, in which LjAMT1;3 was expressed at higher level than in leaves, and LjAMT1;1 showed the lowest level of expression. Expression of LjAMT1;1 and 1;2 in roots was induced by nitrogen deprivation. Expression of LjAMT1;1 was repressed in leaves exposed to elevated CO 2 concentrations, which also suppress photorespiration. Tissue and cellular localization of LjAMT1 genes expression, using promoter-b-glucuronidase and in situ RNA hybridization approaches, revealed distinct cellular spatial localization in different organs, including nodules, suggesting differential roles in the nitrogen metabolism of these organs.

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Plants respond to changes of nutrient availability in the soil by modulating their root system developmental plan. This response is mediated by systemic changes of the nutritional status and/or by local perception of specific signals. The effect of nitrate on Arabidopsis (Arabidopsis thaliana) root development represents a paradigm of these responses, and nitrate transporters are involved both in local and systemic control. Ammonium (NH 4 + ) represents an important nitrogen (N) source for plants, although toxicity symptoms are often associated with high NH 4 + concentration when this is present as the only N source. The reason for these effects is still controversial, and mechanisms associating ammonium supply and plant developmental programs are completely unknown. We determined in Lotus japonicus the range of ammonium concentration that significantly inhibits the elongation of primary and lateral roots without affecting the biomass of the shoot. The comparison of the growth phenotypes in different N conditions indicated the specificity of the ammonium effect, suggesting that this was not mediated by assimilatory negative feedback mechanisms. In the range of inhibitory NH 4 + conditions, only the LjAMT1;3 gene, among the members of the LjAMT1 family, showed a strong increased transcription that was reflected by an enlarged topology of expression. Remarkably, the short-root phenotype was phenocopied in transgenic lines by LjAMT1;3 overexpression independently of ammonium supply, and the same phenotype was not induced by another AMT1 member. These data describe a new plant mechanism to cope with environmental changes, giving preliminary information on putative actors involved in this specific ammonium-induced response.

Atmospheric nitrogen (N 2)-fixing nodules are formed on the roots of legume plants as result of the symbiotic interaction with rhizobia. Nodule functioning requires high amounts of carbon and energy, and therefore legumes have developed finely tuned mechanisms to cope with changing external environmental conditions, including nutrient availability and flooding. The investigation of the role of nitrate as regulator of the symbiotic N 2 fixation has been limited to the inhibitory effects exerted by high external concentrations on nodule formation, development and functioning. We describe a nitrate-dependent route acting at low external concentrations that become crucial in hydroponic conditions to ensure an efficient nodule functionality. Combined genetic, biochemical and molecular studies are used to unravel the novel function of the LjNRT2.4 gene. Two independent null mutants are affected by the nitrate content of nodules, consistent with LjNRT2.4 temporal and spatial profiles of expression. The reduced nodular nitrate content is associated to a strong reduction of nitrogenase activity and a severe N-starvation phenotype observed under hydroponic conditions. We also report the effects of the mutations on the nodular nitric oxide (NO) production and content. We discuss the involvement of LjNRT2.4 in a nitrate-NO respiratory chain taking place in the N 2-fixing nodules.