Fig. 6: WAKL4-mediated phosphorylation of Tyr488 is required for the vacuole-dependent degradation of NRAMP1 under Cd stress. | Nature Communications

Fig. 6: WAKL4-mediated phosphorylation of Tyr488 is required for the vacuole-dependent degradation of NRAMP1 under Cd stress.

From: The Arabidopsis receptor-like kinase WAKL4 limits cadmium uptake via phosphorylation and degradation of NRAMP1 transporter

Fig. 6

a Confocal imaging of roots of transgenic lines expressing YFP-fused NRAMP1, NRAMP1Y488F, and NRAMP1Y488E isoforms. The 7-day-old seedlings were treated with 1/5Hoagland (-Cd) or 1/5Hoagland plus 30 μM CdCl2 (+Cd) for 4 h in the presence of 50 μM CHX. Scale bars, 10 μm. Seedlings were stained for 10 s with 10 μM PI (Propidium Iodide). More than 20 images were evaluated for each assay. b Time-course confocal imaging of (Fig. 4c) plant roots. The 7-day-old seedlings were treated with 1/5Hoagland (-Cd) or 1/5Hoagland plus 30 μM CdCl2 (+Cd) for the indicated times in the presence of 50 μM CHX. Scale bars, 10 μm. More than 15 images were evaluated for each assay. c Fluorescence intensity analysis of plasma membrane shown in (b). Data are presented as mean values ± SD (n = 10). d Analysis of NRAMP1 protein abundance in WT/NRAMP1-GFP or wakl4-1/-2/NRAMP1 plants shown in (b) with or without Cd for 8 h. The total proteins were extracted and detected with anti-GFP and anti-ACTIN antibodies. The relative intensity of NRAMP1 by ImageJ was as shown (d). e In vivo ubiquitination of NRAMP1 (Ubn-NRAMP1) in WT/NRAMP1-GFP or wakl4-1/NRAMP1 plants shown in (b) with or without Cd for 8 h. Immunoprecipitation (IP) was performed using anti-GFP or anti-Ub magnetic beads on solubilized protein and subjected to Western blot (WB) with anti-Ub (middle) or anti-GFP (right) antibodies. f Sensitivity of NRAMP1-GFP to dark growth conditions. Light-grown seedlings were treated with 50 μM CHX (-Cd) or 50 μM CHX plus 50 μM CdCl2 (+Cd) in the dark for 5 h before confocal imaging. Scale bars, 10 μm. The arrowheads shown in (f) indicate vacuole lumen structures. More than 15 images were evaluated for each assay. g Quantification of the ratio between the plasma membrane and intracellular fluorescence signal intensities of NRAMP1-GFP shown in (f). Data are presented as mean values ± SD (n = 15). All experiments were repeated at least three times with similar results. All data were analyzed by two-tailed unpaired t-test (**P < 0.01, ***P < 0.001, ****P < 0.0001).

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