Fig. 2: WAKL4 responds to Cd rapidly and specifically.

a WAKL4 responds to Cd treatments at different times. 7-day-old 35Sp:WAKL4-FLAG seedlings were treated with 1/5Hoagland plus 75 μM CdCl₂ for the indicated times. b WAKL4 responds to Cd treatments at different concentrations. 7-day-old 35Sp:WAKL4-FLAG seedlings were treated with the indicated 1/5Hoagland plus concentrations of CdCl₂ for 4 h. c In vivo ubiquitination analyses of WAKL4 (Ubn-WAKL4) in response to Cd. Immunoprecipitation (IP) was performed using anti-FLAG or anti-Ub magnetic beads on solubilized protein extracts from 10-day-old 35Sp:WAKL4-FLAG plants and subjected to Western blot (WB) with anti-Ub (middle) or anti-FLAG (right) antibodies. Cd stress was applied for the indicated times. d WAKL4 protein expression level of 7-d-old 35Sp:WAKL4-FLAG seedlings over 16 h of 75 μM CdCl₂ plus MG132 (50 μM) treatment or 75 μM CdCl₂ plus E-64d (50 μM) before sampling. The total proteins were extracted and detected with anti-FLAG antibodies. The bottom panel shows the protein Coomassie brilliant blue. e 7-d-old 35Sp:WAKL4-GFP seedlings were pre-treated with CHX (50 μM) for 1 h and then treated with CHX (50 μM) plus BFA (50 μM) or CHX (50 μM) plus BFA (50 μM) plus 75 μM CdCl₂ for 2 h. The Mock represented no drug treatment. Images of roots were taken by the Confocal microscope. The white handle arrow indicates the BFA bodies of WAKL4-GFP. BF, bright field. Scale bars, 10 μm. More than 15 images were evaluated for each assay. f–h Fluorescent intensity analysis of plasma membrane (f), BFA bodies (g), and the area of BFA bodies (h) shown in (e). Data are presented as mean values ± SD (n = 20). 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).