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

Yuan, Jun Jie; Zhao, Ya Nan; Yu, Su Hang; Sun, Ying; Li, Gui Xin; Yan, Jing Ying; Xu, Ji Ming; Ding, Wo Na; Benhamed, Moussa; Qiu, Rong Liang; Jin, Chong Wei; Zheng, Shao Jian; Ding, Zhong Jie

doi:10.1038/s41467-024-53898-8

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Published: 04 November 2024

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

Nature Communications volume 15, Article number: 9537 (2024) Cite this article

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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.

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Introduction

Cadmium (Cd) is an extremely hazardous metal to the environment and living organisms and has been inevitably drawing global attention due to its easy accumulation, high mobility, and strong toxicity, ranking first among inorganic pollutants^1,2,3. As the most fundamental part of the food chain, plants are a major source of dietary Cd intake⁴. It is estimated that Cd pollution in crops including cereals, roots, and leafy vegetable consumption, contributes to more than 70% to 90% of Cd exposure in humans^5,6, which can severely threaten human health, such as causing Itai-Itai disease, lung cancer, and kidney dysfunction⁷. Additionally, Cd toxicity limits plant growth and development by inducing oxidative stress and cell death, interfering with chlorophyll biosynthesis and photosynthesis, and hindering the uptake and translocation of other nutrient elements^2,8. Therefore, reducing Cd accumulation in plants is important for improving human health and plants’ tolerance to Cd stress.

The movement of Cd in plants has been revealed during past decades, including Cd uptake from soil roots, root-to-shoot translocation via xylem, and redistribution from the mature parts to young leaves, roots, and seeds through phloem⁹. These transportation processes are mediated by certain transporter proteins responsible for the carriage of nutrient elements structurally similar to Cd. For instance, both the iron Fe (II) transporter IRT1 and the natural resistance-associated macrophage protein 1 (NRAMP1) are involved in Cd uptake in Arabidopsis root epidermal cells¹⁰. AtHMA2/4, expressed mainly in vascular tissues, mediates the translocation of Cd to shoots by loading them into the xylem¹¹. The phloem-specific Fe transporter AtOPT3 plays a role in Cd redistribution in Arabidopsis¹². Thus, blocking these paths of Cd transmission, particularly the uptake process, would reduce Cd accumulation and optimize its distribution in plants. Nevertheless, it remains largely unknown how plants respond to such a non-essential element and actively limits its uptake and translocation.

The NRAMP family, widely found in organisms, has been considered one of the major transporters for Cd uptake and translocation in plants^13,14. For example, OsNRAMP1 and OsNRAMP5 are highly homologous and both implicated in Cd uptake and transport in rice, but their functions in shoot Cd accumulation are likely different^13,14,15. The HvNRAMP5 transporter mediates the uptake of Cd and Mn, but not Fe, in barley¹⁶. Moreover, the roles of NRAMP1 homologs in Cd uptake have also been revealed in tobacco¹⁷, pak choi¹⁸, and Populus⁶. In Arabidopsis, four NRAMP genes (AtNRAMP1/3/4/6) are currently known to be involved in Cd transport. AtNRAMP1 is localized at the plasma membrane (PM), and overexpression of it in yeast leads to increased sensitivity and accumulation of Cd¹⁹. AtNRAMP3 and AtNRAMP4 are vacuolar membrane-localized proteins, and both overexpression of AtNRAMP3 and the nramp3nramp4 double mutant cause Cd hypersensitivity^20,21. The PM- and Golgi/trans-Golgi-localized AtNRAMP6 is highly homologous to AtNRAMP1 and has been demonstrated to affect the distribution of Cd in Arabidopsis²². Recent research revealed that the calcium-dependent protein kinases CPK21/23 enhance the Cd tolerance by phosphorylation and inactivation of AtNRAMP6 without affecting Fe/Mn homeostasis²³, implying a possible active response of plants to Cd. However, how plants sense or respond to Cd and then modulate Cd uptake is obscure.

Receptor-like protein kinases (RLKs) constitute a unique class of transmembrane proteins with large numbers in plants that actively sense and transmit extracellular signals, including peptides, hormones, temperature, pathogens, and salt, regulating various aspects of plant growth and stress responses^24,25,26. We hence wondered if RLK might similarly function in sensing and/or transducing Cd signals and mediating the active response of plants to Cd. Indeed, the cell wall-associated kinases (WAKs) and WAK-like kinases (WAKLs) that form a subfamily of the RLK, were found to be responsive to toxic metals²⁷. The early discovery revealed that overexpression of WAK1 increases the resistance to aluminum (Al) in Arabidopsis²⁸. The rice homolog gene OsWAK11 was shown to be responsive to Al, sodium (Na), and copper (Cu)²⁹. Besides, the lack of the Arabidopsis WAKL4 gene leads to higher sensitivity to potassium (K), Na, Cu, and Zinc (Zn), but increased tolerance to nickel (Ni) toxicity³⁰. A very recent finding showed that the NAC102 − WAKL11 module regulates cell wall pectin metabolism and Cd binding in Arabidopsis³¹. In addition to WAKL proteins, the LRR subfamily member FRONIA (FER) was also demonstrated to be involved in Cd accumulation by affecting Fe-related pathways³². Despite these findings, the molecular mechanism of how RLK responds to Cd and regulates its uptake remains unclear.

In this study, we isolated a Cd-sensitive mutant from an Arabidopsis RLK T-DNA insertion mutant library. The gene encoding WAKL4 was previously known in response to metal ions³⁰. We here show that Cd, but not other metal elements, rapidly induces the accumulation of WAKL4 protein by both inhibiting its proteolysis and promoting its transcription. The accumulated WAKL4 interacts with NRAMP1 and phosphorylates Tyr488 residue to target it to the vacuole for degradation, thereby impeding the uptake of Cd by root. Overall, our findings reveal a presumably Cd-specific mechanism conferred by the WAKL4-NRAMP1 module that enables plants to actively respond to Cd and limit its uptake.

Results

WAKL4 is required for Cd tolerance

To elucidate the probable Cd sensing mechanism in plants, we screened an Arabidopsis RLK T-DNA insertion mutant library and isolated a Cd-sensitive mutant (SALK_002429) with a T-DNA inserted in the promoter of the WAKL4 gene (Supplementary Fig. 1a). The wakl4^T-DNA mutant with a lower transcription level of WAKL4 (Supplementary Fig. 1b), exhibited increased Cd sensitivity versus wild type (WT) (Supplementary Fig. 1c–e). To confirm the role of WAKL4 in Cd response, we next generated two knockout mutants (wakl4-1 and wakl4-2) by using CRISPR/Cas9 (Supplementary Fig. 1f, g) and found that both of them displayed increased sensitivity to Cd (Fig. 1a), showing the significantly decreased length of the roots, biomass of the seedlings and chlorophyll content of the shoots under Cd treatment (Fig. 1b–d). We further generated the WAKL4-complementation lines driven by its native promoter (2413 bp) in the genetic background of wakl4-1 (Com1) and wakl4-2 (Com2), finding that the WAKL4 gene could fully restore the Cd sensitivity of these mutants to that of WT (Fig. 1a–d). The Cd sensitivity of these genotypes was next confirmed with different Cd concentrations (Supplementary Fig. 2a, b). Moreover, we verified the Cd response of wakl4 mutants in the hydroponic condition (Supplementary Fig. 2c, d). These results indicate that WAKL4 is required for Cd tolerance in Arabidopsis. To further investigate how WAKL4 affects Cd tolerance, we measured the Cd content and found that it was remarkably increased in plants lacking WAKL4 (versus WT) (Fig. 1e). These results collectively suggest that WAKL4 regulates Cd tolerance probably via limiting Cd accumulation.

**Fig. 1: Lack of *WAKL4* enhances Cd sensitivity.**

A previous study revealed that a Wassilewskija (Ws)-background wakl4 T-DNA insertion mutant (T-DNA inserted in 5’UTR) showed different responses to various metal elements, e.g. being hypersensitive to Zn but less sensitive to Ni toxicity³⁰. We surprisingly found that the Col-background wakl4-1/−2 mutants showed WT-like responses to Mn, Zn, Ni, cobalt (Co) and Fe (Supplementary Fig. 3). These contradictory results might be attributed to the different genetic backgrounds of wakl4 mutants. Our data suggest that WAKL4 is likely required for metal-specific tolerance to excess Cd in the Columbia (Col-0) background.

Cd induces WAKL4 protein accumulation rapidly and specifically

To evaluate the role of WAKL4, we did a time-course analysis on both the translational and transcriptional levels of WAKL4 under Cd treatment. Using a 35Sp:WAKL4-FLAG line, we found that the WAKL4 protein abundance rapidly accumulated within 1 h after the onset of Cd treatment, peaked at 2-4 h, and began to fall back after 4 h gradually (Fig. 2a). Long-term Cd exposure even inhibited WAKL4 accumulation (Fig. 2a). Furthermore, Cd concentration gradient experiments showed that the WAKL4 protein abundance reached the highest value at 75 μM after 4 h Cd treatment (Fig. 2b). Similarly, the mRNA levels of WAKL4 were also induced by Cd application, with a peak at 4 h of exposure followed by a subsequent drop (Supplementary Fig. 4b). We next addressed whether such an expression pattern of WAKL4 was exclusively conferred by Cd stimulus. Expression analysis under different metal treatments (Mn, Zn, Ni, Co, and Fe) was therefore investigated, showing that the WAKL4 expression levels were only slightly induced by a high concentration of Mn exposure at 4 h, but not by other metal treatments (Supplementary Fig. 4a, b). These results suggest that the response of WAKL4 to Cd treatment is largely specific and rapid.

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

The Cd-dependent changes in WAKL4 protein abundance let us spontaneously associate to dissect the influence of Cd on the degradation fate of WAKL4. As ubiquitination is the predominant pathway of proteolysis, we detected the polyubiquitinated WAKL4-FLAG proteins and found that the ubiquitination levels of WAKL4 were repressed within 4 h of Cd treatment (Fig. 2c), which is in line with the rapid accumulation of WAKL4 proteins (Fig. 2a). This indicates that Cd stimulus may induces WAKL4 protein abundance via inhibition of its ubiquitination levels. Since the turnover of membrane proteins generally relies on vacuolar proteolysis- or 26S proteasome-mediated degradation pathways^33,34, to further investigate which degradation pathway WAKL4 is involved in, we used an endocytosis inhibitor E-64d and a proteasome inhibitor MG132, finding that the E-64d, but not MG132, notably suppressed the degradation of WAKL4 upon CHX and Cd treatment (Fig. 2d). These data suggest that WAKL4 is degraded via the endocytosis pathway.

We next examined the dynamics of WAKL4 under Cd treatment, by visualizing the WAKL4-GFP fusion protein (driven by 35S promoter) in roots. We found that the WAKL4-GFP was primarily localized to the plasma membrane (PM), but was also present at intracellular punctate structures in the root apical meristem zone (Fig. 2e) as previously reported³⁰. We then checked WAKL4 trafficking using the fungal toxin brefeldin A (BFA), which is an inhibitor of ARF-GEF-type vesicle exocytosis regulators^35,36. Following BFA treatment, WAKL4-GFP mainly accumulated in BFA compartments. This signal results from endocytosed proteins because de novo protein synthesis is blocked by CHX addition and the PM proteins dynamically recycle to PM or enter the protein degradation process. Interestingly, we found that Cd treatment enhanced such BFA-induced foci in size and fluorescence intensity (Fig. 2e, g, h). In addition, the PM signals of WAKL4-GFP were also stronger in Cd-supplied plants than in control ones (Fig. 2f). This could be due to the fall-off of WAKL4 in endocytosis caused by Cd-dependent repression of WAKL4 ubiquitination. Altogether, these results indicate that Cd rapidly and specifically induces WAKL4 protein accumulation via repression of WAKL4 degradation and promoting its transcription.

WAKL4 is involved in Cd tolerance through interaction with NRAMP1

Because WAKL4 is required for Cd tolerance by reducing Cd accumulation, we next asked if WAKL4 interacts with and affects the certain PM-localized transporters that are known to be implicated in Cd uptake or movement, such as NRAMP1, NRAMP6, IRT1, MRP7, HMA4, and ZIP6^{18,22,37,38,39,40,41}. To this end, we first employed a bimolecular fluorescence complementation (BiFC) assay in the leaves of Nicotiana benthamiana and found that WAKL4 markedly interacted with NRAMP1 and NRAMP6, but not with other transporters (CPK21 was used as a positive control; Fig. 3a; Supplementary Fig. 5a).

NRAMP1 and NRAMP6 are phylogenetically close but have distinct tissue expression patterns. NRAMP1 is predominantly expressed in roots, which displays a similar expression pattern with WAKL4, whereas NRAMP6 is mainly expressed in shoots of Arabidopsis seedlings^22,42,43. Moreover, while NRAMP1 is responsible for Cd uptake and increasing cellular Cd content, NRAMP6 plays a role in Cd distribution/availability within the cell and does not affect total Cd accumulation in plants^18,22,44. Since WAKL4 affects Cd accumulation, therefore, we focused mainly on NRAMP1, and in turn, confirmed the WAKL4-NRAMP1 interaction in split-LUC and Co-immunoprecipitation (Co-IP) assays (Fig. 3b, c).

To further determine if NRAMP1 is involved in WAKL4-dependent Cd tolerance, we first analyzed the nramp1 loss-of-function mutant and found that lack of NRAMP1 increased Cd tolerance (Fig. 3d), which is in accord with the role of NRAMP1 in Cd uptake. We next generated the wakl4-1 nramp1 and wakl4-2 nramp1 double mutants and found that these mutants exhibited similar Cd responses with the nramp1 single mutant (Fig. 3e–g), suggesting that NRAMP1 is epistatic to WAKL4 in Cd tolerance. Taken together, these results indicate that WAKL4 is involved in Cd tolerance probably via interaction with NRAMP1.

WAKL4 phosphorylates NRAMP1 at Tyr488

Given that the NRAMP1 protein harbors a predictive topology with 5 motifs partially located in the transmembrane domains which are mostly buried in the resolved NRAMP1 structure⁴⁵, to determine which part interacts with WAKL4, we accordingly separated NRAMP1 into 5 relevant sections for interaction analysis (Supplementary Fig. 5c). Following the BiFC assay, we found that WAKL4 interacted with the C-terminus of NRAMP1 in planta (Supplementary Fig. 5b). We next asked if WAKL4 could directly phosphorylate NRAMP1. To test this hypothesis, we conducted an in vitro kinase assay, using a Strep-tagged WAKL4 (Strep-WAKL4) expressed from mammalian cells and a His-tagged C-terminus of NRAMP1 (His-NRAMP1⁽³⁾) purified from E. coli. We demonstrated that WAKL4 was capable of phosphorylating NRAMP1⁽³⁾ in vitro (Fig. 4b).

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

Since there are 9 Ser, Thr and Tyr amino acid (AA) residues in the C-terminus of NRAMP1, to identify potential phosphorylation sites of NRAMP1 by WAKL4, we used an online tool (ESPript 3.0) for multiple sequence alignment, revealing that AtNRAMP1 shares a highly conserved Tyr488 residue at the C-terminal ___domain with the intra or cross-specific homologs (Fig. 4a). In plants, protein tyrosine kinases (PTKs) catalyze the phosphorylation of Tyr residue of target proteins by transferring the γ-phosphate from ATP. Structurally, it has been shown that PTKs share a conserved catalytic ___domain of ~250 to 300 AAs with 11 conserved sub-domains (I to XI)⁴⁶. Sequence comparison revealed that WAKL4 has this catalytic ___domain like dual-specificity kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1) and other classical PTKs in Arabidopsis, suggesting that WAKL4 is likely a PTK (Supplementary Fig. 6). To further verify whether WAKL4 phosphorylates NRAMP1 at Tyr488, we obtained the mutant NRAMP1⁽³⁾ variants either with Tyr488 substituted to Phe (NRAMP1^(3)-Y488F) or with all Ser/Thr substituted to Ala (NRAMP1^(3)-8A). Our data showed that mutation of Tyr488 (NRAMP1^(3)-Y488F), but not of Ser/Thr (NRAMP1^(3)-8A), resulting in a decrease of phosphorylation conferred by WAKL4 (Fig. 4b), compared to the WT NRAMP1⁽³⁾, suggesting that WAKL4 phosphorylates the C-terminus of NRAMP1 mainly at Tyr488.

To further confirm the in vivo phosphorylation, we generated transgenic plants WT/ProNRAMP1:NRAMP1^Y488F-GFP (WT/NRAMP1^Y488F-GFP) and crossed the wakl4-1/-2 mutants with ProNRAMP1:NRAMP1-GFP (wakl4-1/NRAMP1-GFP or wakl4-2/NRAMP1-GFP). Using a P-Tyr antibody, we detected obvious phosphorylation of NRAMP1 in WT/NRAMP1-GFP plants, particularly under 4 h Cd treatment (Fig. 4c), which is in line with the expression pattern of WAKL4 in response to Cd stimulus (Fig. 2a). As expected, this Cd-induced phosphorylation was notably reduced in WT/NRAMP1^Y488F-GFP (Fig. 4c) or wakl4-1/-2/NRAMP1-GFP plants (Fig. 4d). Besides, although treatment with non-cadmium metals such as Mn, Co, Zn, Ni, and Cu could also obviously induce the abundance of NRAMP1 Tyr phosphorylation (Fig. 4d), it still maintained a much higher level than that under Cd treatment in the WT/NRAMP1^Y488F-GFP variant (Fig. 4e). This implied that the Tyr488 phosphorylation is specifically responsive to Cd and that other metals (Mn, Co, Zn, Ni, and Cu) likely induce NRAMP1 phosphorylation in a Tyr488-independent manner. Collectively, these results demonstrate that Cd-induced phosphorylation of Tyr488 in NRAMP1 is predominantly dependent on WAKL4.

NRAMP1^Tyr488 phosphorylation is essential for NRAMP1 function under Cd stress in plants

To determine the function of NRAMP1^Tyr488 phosphorylation in planta, we transformed the nramp1 mutant background with NRAMP1, the phospho-dead variant NRAMP1^Y488F, or the phospho-mimetic variant NRAMP1^Y488E driven by a native NRAMP1 promoter. We found that the expression of NRAMP1 could restore the Cd sensitivity of nramp1 to that of WT even though the NRAMP1 expression level was not completely rescued to that in WT (Supplementary Fig. 7b). Moreover, the nramp1/NRAMP1^Y488F lines showed reduced Cd tolerance relative to the WT and nramp1/NRAMP1 plants, while the nramp1/NRAMP1^Y488E lines displayed comparable Cd tolerance to the nramp1 mutant (Fig. 5a, c–e). These results were next confirmed in the hydroponic condition (Supplementary Fig. 7a, c). Furthermore, we observed that the Cd content was significantly increased in the nramp1/NRAMP1^Y488F lines versus WT and nramp1/NRAMP1 plants, but was markedly reduced in nramp1 and nramp1/NRAMP1^Y488E plants (Fig. 5b). These data demonstrate that the phosphorylation status of Tyr488 is essential for the function of NRAMP1 in Cd tolerance.

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

To further elucidate whether the function of NRAMP1^Tyr488 phosphorylation is specific for Cd tolerance, we first detected the metal-absorbing ability of NRAMP1 by comparing the sensitivities of NRAMP1-GFP overexpression plants to Mn, Cd, Co, Zn, Ni, Cu, Pb and Fe processing (Supplementary Fig. 8a, b). It was noteworthy that NRAMP1 has a relatively high absorption capacity for Mn, Cd, and Co during seed germination and seedling growth (Supplementary Fig. 8c). We next found that, unlike that under Cd treatment, the nramp1/NRAMP1, nramp1/NRAMP1^Y488F, and nramp1/NRAMP1^Y488E seedlings all showed comparable sensitivities under Mn-/Co-treatment (Supplementary Fig. 8d–g), suggesting that the NRAMP1^Tyr488 phosphorylation is likely specific for Cd response. Collectively, these results indicate that phosphorylation of Tyr488 is critical and specific for NRAMP1-mediated Cd tolerance in plants.

Phosphorylation of Tyr488 is indispensable for the Cd-induced reduction of NRAMP1 localization to the PM

To characterize how Tyr488 residue affects NRAMP1 function, we expressed the WT (NRAMP1) and mutant variants of NRAMP1 (NRAMP1^Y488F and NRAMP1^Y488E) in the Cd-hypersensitive yeast strain Δycf1¹⁷. We found that expression of NRAMP1 significantly reduced yeast growth under Cd treatment (Supplementary Fig. 9a), as shown in the previous study¹⁹. By comparison, we did not observe any discernable growth differences among the Δycf1 strains expressing NRAMP1, NRAMP1^Y488F, and NRAMP1^Y488E (Supplementary Fig. 9b, c), indicating that the phosphorylation status of Tyr488 may not affect the NRAMP1 Cd transport activity in yeast.

Considering that Tyr phosphorylation can substantially influence protein function, interaction, subcellular localization, or degradation in plants^47,48,49,50, we next intend to find out whether Tyr488 phosphorylation affects the localization and/or stability of NRAMP1 under Cd-supply conditions. Transient expression analysis in tobacco leaves showed that the mimic phosphorylation on Tyr488 notably compromised the localization of NRAMP1 to the PM (Supplementary Fig. 10a). These results suggest that the phosphorylation status of Tyr488 may affect the localization of NRAMP1 to the PM. To confirm the effect of NRAMP1^Tyr488 phosphorylation in vivo, we overexpressed NRAMP1-YFP with the UBQ promoter (Supplementary Fig. 10b) and found that NRAMP1 and NRAMP1^Y488F mainly resided at the PM of root cells under normal conditions (Fig. 6a). Upon Cd exposure, the NRAMP1-YFP fluorescence descended remarkably in the PM and formed evident foci in the cytosol (Fig. 6a). Conversely, the NRAMP1^Y488F protein was conspicuously retained at the PM, with no obvious fluorescence signals detected in the cytosol (Fig. 6a), yet this phenomenon did not occur under Mn or Co-treatment (Supplementary Fig. 10c). Intriguingly, the mimic phosphorylation of Tyr488 resulted in sharply reduced fluorescence intensity of NRAMP1-YFP protein under either Cd-free or Cd-treated conditions (Fig. 6a). These results collectively demonstrate that the phosphorylation of Tyr488 is a prerequisite for NRAMP1 removal from the PM in response to Cd stress in Arabidopsis.

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

WAKL4-mediated phosphorylation of Tyr488 is required for the vacuole-targeted degradation of NRAMP1 under Cd stress

To further confirm whether the WAKL4-regulated Tyr488 phosphorylation promotes NRAMP1 degradation under Cd stress, we detected the abundance of NRAMP1-GFP protein in a time course after the onset of Cd treatment and found that the endocytosis of NRAMP1-GFP began at 2 h after exposure to Cd and became more pronounced over time in WT roots, with most NRAMP1-GFP being degraded from the PM after 12 h (Fig. 6b). In contrast, such endocytosis was less observed in roots lacking WAKL4 (Fig. 6b; Supplementary Fig. 10d, e), and the NRAMP1-GFP proteolysis was substantially repressed or delayed in the wakl4-1 mutant under Cd stress (Fig. 6c), which was further confirmed by immunoblot assay (Fig. 6d). Accordingly, we found that the ubiquitination levels of NRAMP1 were reduced in the wakl4-1 mutant versus WT under Cd treatment (Fig. 6e). To next determine if the ubiquitinated NRAMP1 was targeted to the vacuole for degradation, we transferred the ProNRAMP1:NRAMP1-GFP transgenic line to the dark condition for Cd treatment, as dark can impair the vacuolar lytic activity of root cells and allow visualization of the pH-resistant GFP fusion proteins targeted to the vacuole⁵¹. We detected obvious vacuolar accumulation of NRAMP1-GFP after 5 h of 50 μM Cd treatment in the continuous dark condition (Fig. 6f, g). To confirm the vacuole-targeted NRAMP1 degradation, we treated the plants with concanamycin A (ConcA), a V-ATPase inhibitor, which leads to vacuole deacidification, thus preventing vacuolar degradation of autophagic bodies^52,53. We found that pharmacological inhibition of V-ATPase activity by ConcA disrupted the Golgi morphology and numerous autophagic bodies accumulated within the vacuole (Supplementary Fig. 10f, g), implicating that NRAMP1-GFP was indeed sorted to the vacuole. Altogether, these results demonstrate that the phosphorylation of NRAMP1^Tyr488 conferred by WAKL4 accelerates NRAMP1 endocytosis and degradation under Cd toxicity.

The Cd-specific response of WAKL4 is likely ecotype dependent

Since the metal response of WAKL4 in Col-0 shown in this study is different from that in Ws ecotype as described previously³⁰, to determine if the role of WAKL4 in Cd tolerance is similar in these two ecotypes, we generated the wakl4 knockout mutants (wakl4-34/-38) in the Ws background by CRISPR/Cas9 (Supplementary Fig. 11). We found that wakl4-34/-38 seedlings were more sensitive to Cd than WT (Ws), but this phenotype was weaker than that of wakl4-1/-2 in Col-0 background (Fig. 1; Supplementary Fig. 11). These results suggest that while WAKL4 plays a role in Cd tolerance in Ws ecotype, its contribution to Cd tolerance is different in Col-0 and Ws ecotypes. Furthermore, we showed that the responses of both WAKL4 transcriptional and protein levels to excess Cd were much weaker in Ws than in Col-0 ecotype (Supplementary Fig. 4a, b; Supplementary Fig. 12a, b), but that the interaction of WAKL4 and NRAMP1 was comparable in these two ecotypes (Supplementary Fig. 12c). These results indicate that the WAKL4-NRAMP1 module is presumably conserved in Col-0 and Ws, but the differential expression of WAKL4 in response to Cd may lead to the different contribution of this module to Cd tolerance in these ecotypes. In addition, we surprisingly found that the response of the wakl4-34/-38 (Ws) mutants and WAKL4 expression to other metal stresses were different from that in the previous study³⁰ (Supplementary Fig. 11; Supplementary Fig. 12), which might be attributed to the different plant growth conditions or distinct wakl4 mutants used in the two studies. Overall, these data indicate that the Cd-specific response of WAKL4 is likely ecotype dependent.

Discussion

Cd amassing in vegetables, fruits, and food crops poses health risks to animals and humans. Understanding how plants resist Cd and reduce Cd accumulation has been fundamentally important to agricultural production and environmental protection. Although achievements have been made in understanding how plants take up and translocate Cd, it remains largely unknown how plants actively respond to Cd and in turn limit its uptake, particularly at the post-translational level. Here, we identified an RLK mutant wakl4 (in Col-0 background) that is more sensitive to Cd stress than WT. We show that Cd rapidly induces WAKL4 protein abundance in a relatively Cd-specific manner (Fig. 7). The accumulated WAKL4 phosphorylates NRAMP1 at Tyr488 residue and promotes NRAMP1 degradation towards the vacuole, in turn reducing Cd uptake and thus enhancing Cd tolerance (Fig. 7). Hence, our findings provide insights into a previously uncharacterized WAKL4-NRAMP1 module that enables plants to actively respond to Cd and limit Cd accumulation.

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

Protein phosphorylation is a representative hallmark of RLK activation, which plays an essential role in transmitting various signals in response to environmental stimuli. In eukaryotes, phosphorylation events primarily occur on Ser, Thr, and Tyr residues, controlling the biochemical and physiological functions^49,54. Tyr phosphorylation is common and has been well-studied in animals^55,56. Although accumulating evidence has shown that Tyr phosphorylation also occurs in plants and acts in more and more molecular functions, such as protein interaction and activation^49,50, the role of Tyr phosphorylation signaling has yet to be determined because of the low Tyr phosphoproteome abundances (~5%) in plants^57,58. In this study, we found that WAKL4 is likely a tyrosine kinase that shares a conserved catalytic ___domain (~250 to 300 AAs) containing 11 sub-domains (I to XI) with the classical PTKs (Supplementary Fig. 6). Consistently, the Tyr488 residue of NRAMP1 is the main phosphorylation site targeted by WAKL4 (Fig. 4). This Tyr phosphorylation does not affect NRAMP1 transport activity (Supplementary Fig. 9), but enables it to be degraded through a vacuole-dependent pathway (Fig. 6f), hence reducing Cd uptake, and thereby enhancing Cd tolerance. Our work therefore provides an additional function of Tyr phosphorylation in plants and establishes the key role of WAKL4 in Cd stress signaling.

NRAMP1 is an essential transporter for Mn, and phosphorylation modification has been revealed recently to affect its function. For example, phosphorylation of Ser20/22/24 at the N-terminus of NRAMP1 is important for the alteration of its membrane distribution⁴⁵. Further study reveals that the Ca-dependent kinases CPK21/23 phosphorylate NRAMP1 at Ser20 and Thr498 residues, with the phosphorylation status of Thr498 being a more crucial determinant of NRAMP1 in Mn-deficiency response⁴². Additionally, CIPK23 activated by CBL1/9 is another kinase for the phosphorylation of NRAMP1^Ser20, which is necessary for NRAMP1 internalization into endosomes⁵⁹. Yet it’s still unclear whether the phosphorylation status of these Ser/Thr residues affects NRAMP1 protein stability under Mn toxicity. Our findings reveal that Tyr488 is a potentially Cd-specific phosphorylation site for NRAMP1 vacuole-targeted degradation, uncovering a previously uncharacterized Cd-induced specific mechanism that restricts the uptake of Cd itself in plants.

It has been demonstrated that excess divalent metal ions can induce the CIPK23-mediated phosphorylation of IRT1 and in turn the recruitment of IDF1 E3 ligase, which is required for IRT1 polyubiquitination, joint endosomal sorting, and vacuolar degradation, thus preventing the uptake of excess amounts of non-iron metals¹⁰. We propose that WAKL4-mediated Tyr488 phosphorylation may similarly promote the recruitment of unknown E3 ligase for ubiquitination of NRAMP1 and targeting it to the vacuole for degradation. Consistent with this, the ubiquitination levels of NRAMP1 are reduced in the wakl4-1 mutant versus WT, leading to increased protein levels of NRAMP1 in the mutants (Fig. 6d, e). Nevertheless, how the Tyr488 phosphorylation induces the ubiquitination of NRAMP1 and which E3 ligase mediates this process needs to be investigated in the future.

WAKL4 may confer an ecotype-dependent specific response to Cd stress. As is known, there are huge differences in genetic genes, morphological development, and physiological responses among different ecotypes of plants^57,60,61,62, and the same gene may function dissimilarly in different ecotypes or genetic backgrounds. For instance, data from recent research revealed that a chilling tolerance QTL, dubbed HAN1, confers chilling tolerance to temperate japonica rice, which is distinct from other ecotypes (e.g. indica rice cultivar) and wild rice populations⁶³. Moreover, the Arabidopsis receptor-like kinase CRK4 displays differences between Col-0 and Landsberg erecta (Ler-0) in stomatal movement and drought resistance⁶⁴. Similar instances are also found in Arabidopsis Ws and Col-0 ecotypes, which mainly differ in photosynthesis, cell wall-related proteins, plant stress responses, ROS homeostasis, and DNA/RNA transcription/translation⁵⁷. Take for example the BON1 gene, the lack of which leads to a dwarf phenotype in Col-0 while a normal phenotype in Ws, indicating the presence of a bon1 suppressor in Ws background⁶⁵. Here, we showed that the responses of both WAKL4 transcriptional and protein levels to excess Cd were much weaker in Ws than in Col-0 ecotype (Fig. 2; Supplementary Fig. 12), leading to compromised Cd-sensitive phenotype of wakl4 loss-of-function mutants in Ws (versus Col-0; Fig. 1; Supplementary Fig. 11). Such differential responses of WAKL4 expression to Cd in Ws and Col-0 might be at least partially resulted from the multiple variations in the promoter and coding regions of WAKL4 gene (Supplementary Fig. 13). For example, the SNPs and/or Indels in WAKL4 promoter may confer Col-0 ecotype specific cis-elements that can be recognized by certain Cd-responsive transcription factors, because it is possible that there are mineral-responsive cis-acting elements located in WAKL4 promoter region, and these elements may have overlapping or opposite roles in responding to various mineral nutrients and in maintaining cellular mineral homeostasis²⁷. The ones in WAKL4 coding region, on the other side, might affect WAKL4 protein stability.

The reason why Col-0 ecotype acquires such a Cd-specific response of WAKL4 (including these genomic variations mentioned above) is presumably attributed to the different genetic backgrounds of Col-0 and Ws that confer altered Cd adaptation strategies. For instance, the vacuolar membrane-localized Cd/Zn transporter HMA3 plays a pivotal role in Arabidopsis Cd tolerance through Cd vacuolar sequestration⁶⁶. However, compared with that in Ws, HMA3 is nonfunctional in Col-0 ecotype, as there is an SNP in HMA3 coding region that causes premature termination of translation⁶⁷. This may explain why Col-0 ecotype is more sensitive to Cd than Ws ecotype^67,68,69,70. As a consequence, it is possible that the Col-0 ecotype may increase the contribution of the WAKL4-NRAMP1 module to Cd tolerance by promoting Cd-dependent WAKL4 protein accumulation, therefore to prevent the excessive accumulation of intracellular free Cd and thus for better adaptation to soils containing Cd.

In summary, our findings establish a specific mechanism conferred by the WAKL4-NRAMP1 module in response to Cd stress. This mechanism can prevent plants from absorbing excess Cd and thus enhance Cd tolerance. Our discoveries will therefore deepen the understanding of how plants restrict Cd accumulation and shed light on the future molecular breeding of low Cd-accumulated crops or vegetables.

Methods

Plant materials and growth conditions

Seeds of Arabidopsis thaliana wild type (ecotype Columbia, Col-0; ecotype Wassilewskija, Ws) and the T-DNA insertion line wakl4 (AT1G16150, SALK_002429) were obtained from the Nottingham Arabidopsis Stock Centre (NASC). The nramp1 (AT1G80830, SALK_053236) mutant was kindly provided by Prof. Chao Feng Huang (CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai). Homozygous knockout mutant lines in WAKL4 (wakl4-1 and wakl4-2 in the Col-0; wakl4-34 and wakl4-38 in the Ws) were obtained by CRISPR/Cas9 technology. WAKL4 complementary lines Com1 and Com2, WAKL4 overexpression lines 35Sp:WAKL4-FLAG and 35Sp:WAKL4-GFP in the Col-0, 35Sp:WAKL4-GFP overexpression plants in the Ws; nramp1/ProNRAMP1:NRAMP1 and nramp1/ProNRAMP1:NRAMP1^Y488F/E, nramp1/ProNRAMP1:NRAMP1-GFP from Prof. Daiyin Chao (CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai), NRAMP1 overexpression plants 35Sp:NRAMP1-GFP, 35Sp:NRAMP1^Y488F/E-GFP, ProUBQ:NRAMP1-YFP, ProUBQ:NRAMP1^Y488F/E-YFP, and were obtained by transgenesis. Furthermore, FLAG/NRAMP-GFP, WAKL4-FLAG/NRAMP-GFP, wakl4-1 nramp1, wakl4-2 nramp1, wakl4-1/NRAMP1-GFP, and wakl4-2/NRAMP1-GFP were generated by crossing and homozygous F3 plants were selected. All the lines were identified by PCR with primers listed in Supplementary Data 1 and confirmed by Sanger sequencing.

For agar plate experiments, seeds were surface-sterilized with 75% (v/v) ethanol, washed with deionized water 3 times, and then stratified at 4 °C for two days in the dark to synchronize germination. Seeds were sown on 1/2MS medium (pH 5.8) as previously described⁷¹ containing 0.5% (w/v) sucrose, 1% (w/v) Difco agar (Sangon Biotech) for 5 d, then transferred to 1/2MS medium and 1/2MS medium supplemented with different metals for another 7 d. After that, agar plates containing seeds were placed vertically in a growth chamber or room programmed for a 16-h-light/8-h-dark cycle with a daytime temperature of 23 °C and a night temperature of 21 °C.

For hydroponic experiments, 5-d-old seedlings grown in 1/2MS medium were transferred to 1/5Hoagland liquid medium (1.012 mg/L KNO3, 1.89 mg/L Ca(NO₃)₂, 0.16 mg/L NH₄NO₃, 0.272 mg/L KH₂PO₄, 0.482 mg/L MgSO4, 73.4 μg/L FeNaEDTA, 1.66 μg/L KI, 12.4 μg/L H₃BO₃, 44.6 μg/L MnSO₄, 17.2 μg/L ZnSO₄, 0.5 μg/L Na₂M_OO₄, 0.05 μg/L CuSO₄, 0.05 μg/L CoCl₂, and pH 5.8) for 20 d and then transferred to 1/5Hoagland liquid nutrient with or without 15 μM CdCl₂ for 7 d.

Chemical drugs treatments

CHX (200 mM; Sigma-Aldrich), BFA (50 mM; Sigma-Aldrich), MG132 (50 mM; Calbiochem), E-64d (50 mM; Cayman), and Concanamycin A (20 mM; Aladdin) stock solutions were prepared in DMSO. The final working concentration used in 1/5Hoagland liquid plus Cd medium was specified according to the experiment.

Plasmid construction and plant transformation

For generating the wakl4-1/-2 mutants, we first designed a specific target (Supplementary Data 1) through a quick guide-RNA designer for CRISPR/Cas9/Cpf1 genome editing, then the AtU6 promoter, single guide RNA (sgRNA) and sgRNA target of WAKL4 were both inserted in CRISPR/Cas9 binary vector to generate WAKL4-Cas9 construct⁷². To complement the wakl4-1/-2 mutants, the coding sequences of full-length WAKL4 (2280 bp) driven by WAKL4 native promoter (2413 bp) were cloned into pCAMBIA 1301 binary vector to generate Com1 and Com2. For generating 35Sp:WAKL4-FLAG and 35Sp:WAKL4-GFP, full-length WAKL4 coding sequence was cloned into pCAMBIA2300-35S and pCAMBIA1300-35S, respectively. These constructs were transformed into the WT by Agrobacteria strain GV3101.

For the NRAMP1 transgenic complementation test, a full-length NRAMP1 coding sequence (1596 bp) driven by its native promoter (2178 bp) was cloned into pCAMBIA1301 to generate ProNRAMP1:NRAMP1⁴³. The ProNRAMP1:NRAMP1^Y488F/E constructs were generated by site-directed mutagenesis (TOYOBO, SMK-101, Life Science Department, OSAKA JAPAN) in ProNRAMP1:NRAMP1. These constructs were then transformed into nramp1 background by Agrobacteria strain GV3101. To generate transgenic lines overexpressing NRAMP1 (ProUBQ:NRAMP1-YFP and 35Sp:NRAMP1-GFP), the full-length coding sequence of NRAMP1 was amplified and cloned into the pCAMBIA1301-ProUBQ or pCAMBIA1300-35S vectors. The ProUBQ:NRAMP1^Y488F/E-YFP and 35Sp:NRAMP1^Y488F/E-GFP constructs were generated by site-directed mutagenesis in ProUBQ:NRAMP1-YFP and 35Sp:NRAMP1-GFP. These constructs were then transformed into the WT background by Agrobacteria strain GV3101. The primers used for plasmid construction are listed in Supplementary Data 1. All positive transformants were selected on agar plates supplemented with 40 mg/L hygromycin.

Determination of total chlorophyll content

Seedlings were grown on 1/2MS or 1/5Hoagland culture solution with or without Cd application and then plant leaves (per 6 seedlings FW) were collected, grounded into fine powder, and extracted with 1 mL of 80% (v/v) acetone until complete bleaching was achieved. Total Chlorophyll was quantified by reading the absorption at A663 and A645, and the content of chlorophyll was calculated by the method described previousl⁷³.

Analysis of cadmium content

The plant shoots were separately harvested and washed with 5 mM CaCl₂ solution three times and rinsed with deionized water before being dried at 70 °C for 5 days. Tissues were digested completely in 1 mL of 70% (v/v) nitric acid at 80 °C for 1 h, 100 °C for 1 h, and 120 °C for 2 h. After dilution to 20 mL with ultrapure water, the total Cd content from samples was quantified using inductively coupled plasma-atomic emission spectrometry¹⁰ (ICP-MS) (NexIon 300Q, PerkinElmer Instruments, USA).

Bimolecular fluorescence complementation (BiFC) assay

For the BiFC assays⁷⁴, the full-length WAKL4 was fused with N-terminal Yellow Fluorescent Protein (YFP), and the full-length NRAMP1 or other candidates were fused with C-terminal YFP. These vectors were cloned into the pUC35S-YFP^N or pUC35S-YFP^C (BIOGLE GeneTech). For transient expression in Nicotiana benthamiana leaves, the indicated constructs were transformed into the Agrobacterium strain GV3101. Overnight cultures of Agrobacterium were collected by centrifugation at 5,000× g for 5 min. The pellets were washed twice with 1 mL buffer (10 mM MES, pH 5.6, 10 mM MgCl₂, and 100 μM acetosyringone) and resuspended to OD₆₀₀ = 1. Equal volumes of bacterial suspensions were mixed and then injected into the Nicotiana benthamiana leaves. Plants grew in the dark for 1 day and then were transferred to long-day conditions for 2 days.

For transient interaction in Arabidopsis protoplasts, the full-length WAKL4 sequence with the N-terminal YFP fusion and the truncated NRAMP1 sequences with the C-terminal YFP fusion were cloned into pUC35S-YFP^N or pUC35S-YFP^C vectors and then co-transformed into the Arabidopsis protoplast cells as previously reported⁷⁵.

Split-LUC and Co-immunoprecipitation (Co-IP) assays

The coding sequences of WAKL4 and NRAMP1 were cloned into the pCAMBIA-NLUC and pCAMBIA-CLUC vectors, respectively. All these constructs were introduced into Agrobacterium strain GV3101, and then infiltrated into Nicotiana benthamiana leaves as described above. Three days after infiltration, leaves were incubated with 1 mM luciferin, and luminescence was recorded with a low-light cooled charge-coupled device camera⁷⁶.

For the Co-immunoprecipitation assay, stable transgenic plants were used to detect the interaction between WAKL4 proteins and NRAMP1 in planta. Total proteins were extracted from Arabidopsis using IP buffer (10 mM Tris, pH 7.5, 2 mM EDTA, 150 mM NaCl, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail; ThermoFisher SCIENTIFIC)⁷⁷. After removing cellular debris, the supernatant was incubated with anti-GFP magnetic beads (MCE) at room temperature for 2-3 h, and then the beads were washed three times with IP buffer. The associated proteins were analyzed by Western blot and detected with anti-FLAG/GFP antibodies (1:5,000; ABclonal).

Yeast functional analysis

Yeast vectors expressing NRAMP1 and its variants (pYES2; invitrogen^TM) were transformed into the Δycf1 yeast strain. SD/-Ura liquid medium was used to culture the yeast grown to OD₆₀₀ = 1. Four 10-fold dilution series were established under sterile conditions. The OD₆₀₀ values of the transgenic yeast were recorded every 4 h from the start of OD₆₀₀ = 0.4 of growth to prepare the growth curve.

Subcellular localization

For the observation of subcellular localization in the Nicotiana benthamiana leaves, the 35Sp:NRAMP1-GFP construct and its variants were transformed into the Agrobacterium strain GV3101. The transform method is similar to the BiFC mentioned above. 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. Transgenic lines expressing the ProUBQ:NRAMP1-YFP and ProUBQ:NRAMP1^Y488F/E-YFP, 35Sp:NRAMP1-GFP, and 35Sp:NRAMP1^Y488F/E-GFP were grown for 7 d, and then treated with 1/5Hoagland plus different metals for the indicated times. Subsequently, the fluorescence signals in root tip cells were observed using Zeiss confocal microscopy (LSM710).

Recombinant protein expression

For recombinant His-NRAMP1⁽³⁾ and its mutant variants (His-NRAMP1^(3)-Y488F, His-NRAMP1^(3)-8A), the plasmid pET28a was used by fusing with a SUMO tag (11.2 kDa). These plasmids were transformed into E. coli BL21 CondonPlus (DE3) strains. Cultures were grown at 37 °C until OD₆₀₀ = 0.5, and protein expression was induced by 1 mM IPTG at 16 °C for 16 h. After induction, the bacteria fluid was collected by centrifugation at 5,000 x g for 10 min, ultrasonic lysis using PBS buffer, and purified using a Ni-NTA superflow column (QIAGEN, 30622). The purified proteins were finally eluted with 3 mL elution buffer (50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, 2 mM DTT, 1x protease inhibitor cocktail). The protein after dialysis was aliquoted and stored at −80 °C until use. For recombinant pEZT-Strep-WAKL4 purification, a mammalian cell protein expression system was used as previously described⁷⁸.

In vitro and in vivo kinase assays

An in vitro kinase assay was performed using ATP-gamma-S (Abcam) as previously described⁷⁹. His-NRAMP1⁽³⁾, His-NRAMP1^(3)-Y488F, or His-NRAMP1^(3)-8A was incubated with Strep-WAKL4 in 25 μL kinase buffer (50 mM HEPES (pH 7.5), 5 mM MgCl₂, 1 mM dithiothreitol (DTT) and 1 mM ATP-gamma-S). The mixture was incubated at 37 °C for 30 min before the proteins were mixed with 2.5 mM p-Nitrobenzyl mesylate (Abcam) and incubated at 37 °C for 2 h. After adding 5x SDS loading buffer, the phosphorylated proteins were detected by Western blot using an anti-thiophosphate ester antibody (Abcam, ab92570), and total protein inputs were visualized by Coomassie brilliant blue (CBB) staining.

For in vivo phosphorylation assay, 10-day-old WT/NRAMP1-GFP, WT/NRAMP1-GFP^Y488F, and wakl4-1/NRAMP1-GFP transgenic lines were treated with 1/5Hoagland plus 75 μM for 4/12 h. The seedlings were harvested and lysed in 500 μL of IP buffer. The NRAMP1-GFP proteins were immunoprecipitated by anti-GFP magnetic beads (MCE), and the phosphorylation was detected by Western blot with anti-phosphotyrosine (Anti-P-Tyr) antibodies (1:500; ABclonal). The total proteins were detected with anti-GFP antibody (1:5,000; ABclonal).

In vivo ubiquitination assays

10-day-old 35Sp:WAKL4-FLAG, WT/35Sp:NRAMP1-GFP, and wakl4-1/35Sp:NRAMP1-GFP plants grown on medium were treated with or without Cd for the indicated times and ground to powder in liquid nitrogen. Total proteins were extracted with IP buffer. The supernatants were centrifuged twice at 12,000 × g for 10 min and passed through a 0.45 μm filter. The resulting supernatants were incubated with prewashed anti-FLAG/GFP magnetic beads (MCE) at room temperature for 2-3 h. The magnetic beads were washed 3 times with the IP buffer and boiled with 1× SDS loading buffer at 95 °C for 5 min. The proteins were separated on SDS-PAGE and detected with anti-FLAG/GFP (1:5,000; ABclonal) and anti-Ub (1:1,000; ABclonal) antibodies.

Sequence alignment and conservation analysis

To obtain different NRAMPs of different species, we used Arabidopsis NRAMPs to obtain possible NRAMPs of 5 different species by BLAST in NCBI. WAKL4 catalytic ___domain with other protein tyrosine kinases was shown⁴⁶. Multiple sequence alignment analysis was performed using the ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) online tool.

RNA extraction and real-time quantitative PCR

Total RNA was extracted using QIAzol reagent following the manufacturer’s instructions (QIAGEN). The integrity of RNA was verified by agarose gel electrophoresis, and an equal amount of total RNA was used for reverse transcription as previously described⁸⁰. RT-qPCR analysis was carried out using the SYBR Green Realtime PCR Master Mix (TOYOBO) on a Roche LightCycler480 real-time qPCR system. Transcript levels of each mRNA were determined and normalized with the level of ACTIN2 mRNAs using the ΔCt method.

Statistics and reproducibility

The Arabidopsis plants used in this study were grown in a greenhouse or incubator at 16-h-light/8-h-dark cycle with a daytime temperature of 23 °C and a night temperature of 21 °C. To avoid bias, the growth conditions were ensured to be consistent before the start of the metal-stress experiments, and the hydroponic solution was renewed every 4 days during the experiments. For microscopic, physiological and biochemistry experiments, at least three completely independent experiments were performed.

Statistical tests in this study were performed in Prism 9 for macOS (Version 9.5.0 (525), November 8, 2022, CA, USA). An unpaired two-tailed t-test or a one-way analysis of variance (ANOVA) with Tukey’s test was used in most experiments. No data were excluded from the study. Source data underlying the figures are provided as a Source Data file.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data generated and analyzed in this study are available in the article and its supplementary information file. Source data are provided in this paper. Source data are provided with this paper.

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Acknowledgements

We thank Prof. Chao Feng Huang and Prof. Dai Yin Chao (CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai) for kindly providing the nramp1 mutant, nramp1/ProNRAMP1:NRAMP1-GFP line, and ProNRAMP1:NRAMP1-GFP expression vector, respectively. This work was supported by the Guangdong Laboratory for Lingnan Modern Agriculture (NT2021010), the National Key Research and Development Program of China (2022YFA1303402), the Natural Science Foundation of China (31970272), the Ministry of Education and Bureau of Foreign Experts of China (B14027), the ZJU Tang Scholar Foundation, and the Fundamental Research Funds for the Central Universities (226-2024-00102).

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State Key Laboratory of Plant Environmental Resilience, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China
Jun Jie Yuan, Ya Nan Zhao, Su Hang Yu, Ji Ming Xu, Chong Wei Jin, Shao Jian Zheng & Zhong Jie Ding
Guangdong Laboratory for Lingnan Modern Agriculture, College of Natural Resources and Environment, South China Agricultural University, Guangzhou, 5100642, China
Jun Jie Yuan, Rong Liang Qiu & Shao Jian Zheng
School of Agriculture, Ningxia University, Yinchuan, 750021, China
Ying Sun
State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou, 310058, China
Gui Xin Li
Agricultural Experimental Station, Zhejiang University, Hangzhou, 310058, China
Jing Ying Yan
Ningbo Key Laboratory of Agricultural Germplasm Resources Mining and Environmental Regulation, College of Science and Technology, Ningbo University, Ningbo, 315300, China
Wo Na Ding
Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), 10 91405, Orsay, France
Moussa Benhamed

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Jun Jie Yuan
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Ya Nan Zhao
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Su Hang Yu
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Ying Sun
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Gui Xin Li
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Rong Liang Qiu
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Contributions

J.J.Y., S.J.Z., and Z.J.D. designed the research. J.J.Y., Y.N.Z., S.H.Y., G.X.L., and J.M.X. performed experiments, J.J.Y., Y.S., and J.Y.Y. were involved in plant material generation, J.J.Y., Z.J.D., and W.N.D. analysed the data, M.B., R.L.Q. and C.W.J. discussed part of the experimental design, and Z.J.D., S.J.Z., and J.J.Y. contributed to writing the article.

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Correspondence to Zhong Jie Ding.

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Yuan, J.J., Zhao, Y.N., Yu, S.H. et al. The Arabidopsis receptor-like kinase WAKL4 limits cadmium uptake via phosphorylation and degradation of NRAMP1 transporter. Nat Commun 15, 9537 (2024). https://doi.org/10.1038/s41467-024-53898-8

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Received: 25 September 2023
Accepted: 22 October 2024
Published: 04 November 2024
DOI: https://doi.org/10.1038/s41467-024-53898-8

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