Characterisation of three functional high affinity ammonium transporters in Lotus japonicus with differential transcriptional regulation and spatial expression

Ammonium is a primary source of nitrogen for plants. In legume plants ammonium can also be obtained by symbiotic nitrogen fixation and NH4+ 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 L. japonicus. Both were able to complement the growth defect of a yeast ammonium transport mutant. Measurement of [14C]methylammonium uptake rates, and competition experiments, revealed that each transporter had a high affinity for NH4+. The Ki 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 where 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 CO2 concentrations, which also suppresses photorespiration. Tissue and cellular localisation of LjAMT1 genes expression, using promoter-GUS and in situ RNA hybridisation approaches, revealed distinct cellular spatial localisation in different organs, including nodules, suggesting differential roles in the N metabolism of these organs.