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

Abstract

Cadmium (Cd) is a detrimental heavy metal propagated from soil to the food chain via plants, posing a great risk to human health upon consumption. Despite the understanding of Cd tolerance mechanisms in plants, whether and how plants actively respond to Cd and in turn restrict its uptake and accumulation remain elusive. Here, we identify a cell wall-associated receptor-like kinase 4 (WAKL4) involved in specific tolerance to Cd stress. We show that Cd rapidly and exclusively induces WAKL4 accumulation by promoting WAKL4 transcription and blocking its vacuole-dependent proteolysis in roots. The accumulated WAKL4 next interacts with and phosphorylates the Cd transporter NRAMP1 at Tyr488, leading to the enhanced ubiquitination and vacuole-dependent degradation of NRAMP1, and consequently reducing Cd uptake. Our findings therefore uncover a mechanism conferred by the WAKL4-NRAMP1 module that enables plants to actively respond to Cd and limit its uptake, informing the future molecular breeding of low Cd accumulated crops or vegetables.

Fig. 1. Lack of WAKL4 enhances Cd sensitivity.

a Phenotypes of wakl4 CRISPR/Cas9 mutants in response to Cd treatment. 5-day-old seedlings were transferred onto 1/2MS medium supplemented with (+Cd) or without 75 μM CdCl₂ (-Cd). Pictures were taken 7 d after the transfer. Scale bar, 1 cm. b–c Statistical analysis of primary root length (b) (n = 24 plants) and fresh weight (c) (n = 24 independent shoot pools, 6 plants/pool) of seedlings shown in (a). Centerlines in the boxplots show the medians, and box limits indicate the 25th and 75th percentile. The whiskers go down to the smallest value and up to the largest. d Content of chlorophyll A and B shown in (a) (n = 20 independent shoot pools). e ICP-MS analysis of shoot Cd concentrations shown in hydroponics (n = 8 independent pools). Data are presented as mean values ± SD (d, e). Three independent repeats were done with similar results. Data were analyzed by ordinary one-way ANOVA (b-d) or two-tailed unpaired t-test (e) (**P < 0.01, ***P < 0.001, ****P < 0.0001).

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).

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

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).

Fig. 4. WAKL4 phosphorylates NRAMP1 at the Tyr488 residue.

a Multiple amino acid sequence alignment of AtNRAMP1 with other NRAMPs. Identical and similar residues are boxed in red highlight/red font. b Analysis of the phosphorylation of NRAMP1 C-terminal (NRAMP1⁽³⁾) by WAKL4 using an in vitro kinase assay. Top: phosphorylated proteins were detected by immunoblotting using an Anti-thiophosphate ester antibody. Bottom: recombinant NRAMP1⁽³⁾ and WAKL4 were detected by CBB staining. c–e Analysis of the phosphorylation of NRAMP1 by WAKL4 in vivo. WAKL4 phosphorylates the Tyr488 site of NRAMP1 in a Cd-induced way (c). Cd-induced phosphorylation of NRAMP1 is dependent on WAKL4 (d). NRAMP1 Tyr488 residue phosphorylation responds to Cd treatment (e). Total proteins were extracted from 10-day-old seedlings treated with 1/5Hoagland plus the indicated concentrations of metal stresses (70 μM CdCl₂, 1.5 mM MnCl₂, 400 μM ZnSO₄, 70 μM NiSO₄, 70 μM CoCl₂, 30 μM CuSO₄ or 400 μM FeEDTA) for 4 h. NRAMP1-GFP protein was immunoprecipitated (IP) by incubating with anti-GFP magnetic beads. Western blot (WB) was performed using anti-phosphotyrosine (Anti-P-Tyr) and anti-GFP antibodies. The relative intensity by ImageJ of phosphorylated NRAMP1-GFP bands (p-NRAMP1) was as shown. The experiments were repeated at least three times with similar results.

Fig. 5. WAKL4-mediated NRAMP1^Tyr488 phosphorylation is vital for its function in Cd tolerance.

a Phenotypes of nramp1, nramp1/ProNRAMP1:NRAMP1, nramp1/ProNRAMP1:NRAMP1^Y488F and nramp1/ProNRAMP1:NRAMP1^Y488E transgenic 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). b ICP-MS analysis of shoot Cd concentrations shown in hydroponics (n = 8 independent pools). c–e Statistical analysis of primary root length (c) (n = 24 plants), fresh weight (d) (n = 24 independent shoot pools, 6 plants/pool), and content of chlorophyll A + B (e) (n > 20 independent shoot pools) shown in (a). In b, e, data are presented as mean values ± SD. In c, d, centerlines in the boxplots show the medians, and box limits indicate the 25th and 75th percentile. The whiskers plots represent minimum to maximum values. 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).

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

a Confocal imaging of roots of transgenic lines expressing YFP-fused NRAMP1, NRAMP1^Y488F, and NRAMP1^Y488E 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).

Fig. 7. A proposed working model of the WAKL4-NRAMP1 module in controlling Cd uptake and tolerance.

In WT plants, exposure to Cd toxicity triggers the accumulation of WAKL4 protein rapidly through enhancement of WAKL4 transcription and suppression of WAKL4 degradation. This process may involve an unknown E3 ligase to sense or respond to Cd. Then, WAKL4 confers the phosphorylation of NRAMP1 at Tyr488 residue. The phosphorylated NRAMP1^Tyr488 further undergoes endocytosis and vacuole-targeted degradation, which may likewise require unknown E3 ligase, thereby restricting Cd uptake and accumulation in plants. This Cd switch mechanism is generally blocked in wakl4 mutants. The constitution of the WAKL4-NRAMP1 signaling pathway allows for effectively regulating plant Cd uptake and tolerance under Cd stress.