Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization

Abstract

Ammonium is a preferred source of nitrogen for plants but is toxic at high levels. Plant ammonium transporters (AMTs) play an essential role in NH 4 + uptake, but the mechanism by which AMTs are regulated remains unclear. To study how AMTs are regulated in the presence of ammonium, we used variable-angle total internal reflection fluorescence microscopy and fluorescence crosscorrelation spectroscopy for single-particle fluorescence imaging of EGFP-tagged AMT1;3 on the plasma membrane of Arabidopsis root cells at various ammonium levels. We demonstrated that AMT1;3-EGFP dynamically appeared and disappeared on the plasma membrane as moving fluorescent spots in low oligomeric states under N-deprived and N-sufficient conditions. Under external high-ammonium stress, however, AMT1;3-EGFPs were found to amass into clusters, which were then internalized into the cytoplasm. A similar phenomenon also occurred in the glutamine synthetase mutant gln1;2 background. Single-particle analysis of AMT1;3-EGFPs in the clathrin heavy chain 2 mutant (chc2 mutant) and Flotllin1 artificial microRNA (Flot1 amiRNA) backgrounds, together with chemical inhibitor treatments, demonstrated that the endocytosis of AMT1;3 clusters induced by high-ammonium stress could occur mainly through clathrin-mediated endocytic pathways, but the contribution of microdomain-associated endocytic pathway cannot be excluded in the internalization. Our results revealed that the clustering and endocytosis of AMT1;3 provides an effective mechanism by which plant cells can avoid accumulation of toxic levels of ammonium by eliminating active AMT1;3 from the plasma membrane. VA-TIRFM | FCSA mmonium (NH 4 + ) and nitrate (NO 3 − ) are the primary sources of nitrogen (N) for most plants growing in agricultural soils. Ammonium assimilation requires less energy than nitrate assimilation, and, thus, ammonium is absorbed preferentially when plants are N-deficient. However, high concentrations of ammonium can be toxic (1); therefore, ammonia absorption and metabolism must be strictly controlled. Understanding the mechanisms by which plant cells regulate ammonium uptake and translocation is of critical importance for agricultural improvements in N-use efficiency and avoiding ammonium toxicity.Evidence suggests that membrane ammonium transporters (AMTs) act in NH 4 + uptake into plant cells, serving as the major transporters for high-affinity ammonium uptake (2). In Arabidopsis thaliana, the AMT family comprises six isoforms, of which three (AtAMT1;1, AtAMT1;2, and AtAMT1;3) are responsible for about 90% of the total high-affinity N uptake in roots (3). AMT gene expression in Arabidopsis roots is generally repressed by high N and induced by N deficiency (4). In addition to transcriptional mechanisms, regulation of membrane transporter activity is also involved in the plant's responses to changing nutrient supplies (1). Although posttranscriptional regulation of AMT appears to be N-dependent (5), the question of how ammonium regulates AMT transporter activity, particularly t...

The Kinase CIPK23 Inhibits Ammonium Transport in Arabidopsis thaliana

“…Due to hetero-oligomerization of AMT1;1 and AMT1;3, the phosphorylation of AMT1;3 should almost completely inactivate high-affinity ammonium transport at the epidermis. Furthermore, rhizodermal AMT1s might be internalized upon high external ammonium concentrations, as shown for AtAMT1;3 (Wang et al, 2013). The high background uptake therefore likely consists of an apoplastic flow into the inner root cell layers.…”

Salt-Induced Remodeling of Spatially Restricted Clathrin-Independent Endocytic Pathways in Arabidopsis Root

“…Endocytic uptake helps plants to fine-tune the distribution of transporters and signaling molecules on the PM, which in turn helps plants to cope with situations like high boron or ammonium toxicity (Takano et al, 2005;Wang et al, 2013) or (D) Quantification of VPS9a-GFP intracellular punctae. The numbers of punctae at the midplane of the cells were counted by automatic particle detection (see Methods).…”

The plasma membrane proteome of Medicago truncatula roots as modified by arbuscular mycorrhizal symbiosis

“…Supporting this model, we found evidence at the proteomic level for a depletion in the PM fractions of AM roots of SUT2 and a DRM-associated BAK1 protein, endocytosis of the latter being known to suppress brassinosteroid signaling (Table 1). As plant ammonium transporters (AMT) can dynamically disappear from the PM under N-sufficient conditions though endocytosis (Wang et al 2006), this also may be the case in AM roots for the DRM-associated transporter AMT2;1 (Table 1). In contrast, unlike SUT2, in PMenriched fractions we could not detect any peptide specific to MtPT4, the AM-inducible phosphate transporter that localizes on the branch domain of the periarbuscular membrane even though one might have expected the partitioning of the periarbuscular membrane with the plasmalemma because of the connectivity between the two.…”