In plants, nutrient transporters require tight regulation to ensure optimal uptake in complex environments. The activities of many nutrient transporters are post-translationally regulated by reversible phosphorylation, allowing rapid adaptation to variable environmental conditions. Here, we show that the Arabidopsis root epidermis-expressed ammonium transporter AtAMT1;3 was dynamically (de-)phosphorylated at multiple sites in the cytosolic C-terminal region (CTR) responding to ammonium and nitrate signals. Under ammonium resupply rapid phosphorylation of a Thr residue (T464) in the conserved part of the CTR (CTRC) effectively inhibited AtAMT1;3-dependent NH4+ uptake. Moreover, phosphorylation of Thr (T494), one of three phosphorylation sites in the non-conserved part of the CTR (CRTNC), moderately decreased the NH4+ transport activity of AtAMT1;3, as deduced from functional analysis of phospho-mimic mutants in yeast, oocytes, and transgenic Arabidopsis. Double phospho-mutants indicated a role of T494 in fine-tuning the NH4+ transport activity when T464 was non-phosphorylated. Transient dephosphorylation of T494 with nitrate resupply closely paralleled a transient increase in ammonium uptake. These results suggest that T464 phosphorylation at the CTRC acts as a prime switch to prevent excess ammonium influx, while T494 phosphorylation at the CTRNC fine tunes ammonium uptake in response to nitrate. This provides a sophisticated regulatory mechanism for plant ammonium transporters to achieve optimal ammonium uptake in response to various nitrogen forms.

High-affinity ammonium uptake in roots mediate by AMT1-type ammonium transporters, which are tightly controlled at multiple regulatory levels for adapting various nitrogen availability. For Arabidopsis AtAMT1;1 gene, in addition to the transcriptional and post-translational controls, an organ-dependent and N-dependent post-transcriptional regulation was suggested as an additional regulatory step for fine tuning ammonium uptake, but the underlying mechanisms remain to be elucidated. Here, we showed that degradation of AtAMT1;1 transcript in roots of Pro35s:AtAMT1;1-transformed atamt1;1-1 Arabidopsis plants resulted from RDR6-dependent sense transgene-induced post-transcriptional gene silencing (S-PTGS). The siRNAs for S-PTGS may derive from the aberrant RNA, of which the production was co-determined by sequence feature and excessive expression of AtAMT1;1. Switching to the expression of AtAMT1;1 driven by ProAtUBQ10 or of AtAMT1;1 mutated at two siRNA-targeted hotspots reduced AtAMT1;1-specific siRNAs and overcame S-PTGS in roots. In roots of these lines, however, the steady-state transcript levels of AtAMT1;1 still significantly decreased under conditions of N-sufficiency compared with N-deficiency, confirming a N-dependent post-transcriptional regulatory manner. A crucial role of the 207-bp 3 0 -end sequence of AtAMT1;1 was further demonstrated by N-dependent accumulation of chimeric-AtAMT1;1 transcript in T-DNA insertion lines and of GFP-tagged chimeric-AtAMT1;1 transcript in transgenic lines. A novel non-coding RNA (ncRNA), which was highly abundant in N-sufficient roots, may target the above-identified 3 0 -end region for the degrading AtAMT1;1 transcript. This degradation could be prevented by a mutation on the AtAMT1;1 transcript at a potential cleavage site (+1458). These results suggested two distinct mechanisms of regulating AtAMT1;1 mRNA turnover by ncRNA for strictly control of ammonium uptake in roots.