Single-Molecule and Vesicle Trafficking Analysis of Ubiquitination Involved in the Activity of Ammonium Transporter AMT1;3 in Arbidopsis under High Ammonium Stress

Abstract

Plants absorb nitrogen from the soil using ammonium transporters (AMTs). Plants can precisely regulate AMT1;3 levels using sophisticated regulatory systems, ensuring adequate nitrogen uptake without hazardous ammonium production. Here, we demonstrated that ubiquitylation can contribute to AMT1;3 degradation under high ammonium stress. Using the ubiquitin site mutant AMT1;3_K75R,K233R-EGFP, we demonstrated that the loss of ubiquitination affects the dynamic characteristics of AMT1;3 proteins on the plasma membrane and markedly inhibits the endocytosis of AMT1;3 proteins under high ammonium stress. AMT1;3_K75R,K233R-EGFP plants also showed inhibition of protein degradation that targets the vesicular pathway after being exposed to high levels of ammonium. Our findings showed that the dynamic properties, endocytosis, and vesicle trafficking pathways of AMT1;3 proteins are altered in AMT1;3_K75R,K233R-EGFP under high ammonium conditions.

Keywords: 3; AMT1; dynamics; endocytosis; ubiquitylation; vesicle trafficking.

Figure 1

Ubiquitylation participates in the degradation of AMT1; 3 proteins under high ammonium stress. (A) Total proteins were isolated from AMT1;3-EGFP seedlings that had been treated with DMSO and MG132, in the presence of CHX, with high ammonium treatment for 0, 15, 30, 60 min. The total proteins were separated by SDS-PAGE and immune detected with GFP antibody. (B) Relative contents of AMT1;3-EGFP (per unit of total protein). Error bars represent standard errors.

Figure 2

Ubiquitylation participates in the degradation of AMT1;3 proteins under high ammonium stress. (A,B) The distribution coefficient statistics of AMT1;3 and AMT1;3_K75R,K233R-EGFP proteins. (C,D) Motion range of AMT1;3 and AMT1;3_K75R,K233R-EGFP in Arabidopsis. (E,F) Distribution of AMT1;3 and AMT1;3_K75R,K233R-EGFP velocity on the cell plasma membrane. * p < 0.05, t test. Error bars represent the mean ± SD.

Figure 3

The ubiquitinated forms of AMT1;3 proteins affect its endocytosis. (A) The partial colocalization of AMT1;3/AMT1;3_K75R,K233R-EGFP with FM4-64. The left photographs represent GFP signal showing subcellular localization of AMT1;3/AMT1;3_K75R,K233R-EGFP. The middle photographs represent red fluorescence signal showing the plasma membrane stained by FM4-64. The right photographs represent the merge of AMT1;3/AMT1;3_K75R,K233R-EGFP and FM4-64 staining. Scale bars = 20 µm. White arrows indicate the colocalization spots of AMT1;3/AMT1;3_K75R,K233R-EGFP and FM4-64. (B) Statistics of the colocalization of AMT1;3/AMT1;3_K75R,K233R-EGFP with FM4-64. ** p < 0.01, t test. Error bars represent the mean ± SD.

Figure 4

Ubiquitylation affects the colocalization of AMT1;3 with vesicle-transport-labeled proteins under high ammonium stress. (A) VHA-a1-mRFP endosomal markers partially colocalized with AMT1;3/AMT1;3_K75R,K233R-EGFP. Colocalizing endosomes are indicated by white arrows. AMT1;3/AMT1;3_K75R,K233R-EGFP is shown in green, VHA-a1-mRFP in red, and the merged image indicates colocalization (yellow). (B) Statistics of the colocalization of AMT1;3/AMT1;3_K75R,K233R-EGFP with VHA-a1 proteins. (C) AMT1;3/AMT1;3_K75R,K233R-EGFP partially colocalizes with late endosomal markers. The AMT1;3/AMT1;3_K75R,K233R-EGFP is shown in green, the AtSNX 1-mRFP is shown in red, and the merged image shows colocalization (yellow). (D) Statistics of the colocalization of AMT1;3/AMT1;3_K75R,K233R-EGFP with AtSNX1 proteins. (E) SYP22-mRFP vacuole markers partially colocalized with AMT1;3/AMT1;3_K75R,K233R-EGFP. Colocalizing endosomes are indicated by white arrows. AMT1;3/AMT1;3_K75R,K233R-EGFP is shown in green, SYP22-mRFP in red, and the merged image indicates colocalization (yellow). (F) Statistics of the colocalization of AMT1;3/AMT1;3_K75R,K233R-EGFP with SYP22 proteins. Scale bars = 10 μm. White arrows indicate endosomes showing colocalization. * p < 0.05, t test. Error bars represent the mean ± SD.