Fig. 3: WAKL4 is involved in Cd tolerance through interaction with NRAMP1. | Nature Communications

Fig. 3: WAKL4 is involved in Cd tolerance through interaction with NRAMP1.

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

Fig. 3

a Partial yellow fluorescent protein (YFP) constructs were fused with WAKL4 or NRAMP1, and the fusions were co-expressed transiently in N. benthamiana leaves. CPK21 was used as a positive control. The YFP signal was visualized under confocal microscopy. Scale bars, 50 μm. More than 20 images were evaluated for each assay. b Split luciferase complementation assays show the interaction of WAKL4 with NRAMP1. Fluorescence was detected at 72 h after infiltration of the indicated constructs. c Co-immunoprecipitation was performed to test the interaction of WAKL4 with NRAMP1 in vivo. A pNRAMP1:NRAMP1-GFP construct in Col-0 (WT/NRAMP1-GFP) was crossed with 35Sp:FLAG plants (FLAG/NRAMP1-GFP) or 35Sp:WAKL4-FLAG plants (WAKL4-FLAG/NRAMP1-GFP). Total proteins were extracted from 7-day-old seedlings. NRAMP1-GFP proteins were immunoprecipitated (IP) using anti-GFP magnetic beads. Western blot was performed using anti-GFP and anti-FLAG antibodies. d Phenotypes of nramp1/wakl4-1, nramp1/wakl4-2, wakl4-1, wakl4-2 and nramp1 mutant plants in response to Cd treatment. The 5-day-old seedlings were transferred onto 1/2MS medium (-Cd) or 1/2MS medium supplemented with 75 μM CdCl2 (+Cd). Pictures were taken 7 d after the transfer. Scale bar, 1 cm. Split luciferase complementation assays show the interaction of WAKL4 with NRAMP1. Fluorescence was detected at 72 h after infiltration of the indicated constructs. e–g Statistical analysis of primary root length (e) (n = 24 plants), fresh weight (f) (n = 24 independent shoot pools, 6 plants/pool), and content of chlorophyll A + B (g) (n = 20 independent shoot pools) shown in (d). In e, f, centerlines in the boxplots show the medians, and box limits indicate the 25th and 75th percentile. The whiskers plots represent minimum to maximum values. In g, data are presented as mean values ± SD. All experiments were repeated at least three times with similar results. All data were analyzed by ordinary one-way ANOVA (**P < 0.01, ***P < 0.001, ****P < 0.0001).

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