The Receptor-Like Cytoplasmic Kinase STRK1 Phosphorylates and Activates CatC, Thereby Regulating H2O2 Homeostasis and Improving Salt Tolerance in Rice

Abstract

Salt stress can significantly affect plant growth and agricultural productivity. Receptor-like kinases (RLKs) are believed to play essential roles in plant growth, development, and responses to abiotic stresses. Here, we identify a receptor-like cytoplasmic kinase, salt tolerance receptor-like cytoplasmic kinase 1 (STRK1), from rice (Oryza sativa) that positively regulates salt and oxidative stress tolerance. Our results show that STRK1 anchors and interacts with CatC at the plasma membrane via palmitoylation. CatC is phosphorylated mainly at Tyr-210 and is activated by STRK1. The phosphorylation mimic form CatC^Y210D exhibits higher catalase activity both in vitro and in planta, and salt stress enhances STRK1-mediated tyrosine phosphorylation on CatC. Compared with wild-type plants, STRK1-overexpressing plants exhibited higher catalase activity and lower accumulation of H₂O₂ as well as higher tolerance to salt and oxidative stress. Our findings demonstrate that STRK1 improves salt and oxidative tolerance by phosphorylating and activating CatC and thereby regulating H₂O₂ homeostasis. Moreover, overexpression of STRK1 in rice not only improved growth at the seedling stage but also markedly limited the grain yield loss under salt stress conditions. Together, these results offer an opportunity to improve rice grain yield under salt stress.

Figure 1.

STRK1 Positively Regulates Salt Tolerance in Rice at the Seedling Stage. (A) Phenotypic comparison of seedlings grown under salt stress at the seedling stage. For STRK1-OE transgenic plants, 15-d-old seedlings were treated with 140 mM NaCl for 10 d and then recovered for 6 d. For STRK1-RNAi transgenic plants, 15-d-old seedlings were treated with 140 mM NaCl for 8 d and then recovered for 6 d. (B) and (C) Survival rates of transgenic and wild-type plants in (A) after 6 d of recovery. Forty plants in each line were used for survival rate analysis. (D) Chlorophyll content in leaves of 15-d-old plants treated with 100 mM NaCl for 7 d. (E) MDA concentrations in leaves of 15-d-old plants after 100 mM NaCl treatment for 24 h. (F) Relative ion leakage in leaves of 15-d-old plants after 100 mM NaCl treatment for 24 h. For all panels, OE indicates STRK1-overexpressing plants; Ri indicates STRK1-RNAi plants; numbers indicate different transgenic lines; data in (B) to (F) are presented as mean ± sd (n = 3, *P ≤ 0.05, **P ≤ 0.01, Student’s t test).

Figure 2.

Subcellular Localization and Expression Pattern of STRK1. (A) Plasma membrane localization of STRK1-YFP anchored by palmitoylation. Amino acid sequence of the N-terminal region of STRK1 showed the three cysteine residues (bold) as putative palmitoylation sites. Confocal images showed localization of STRK1-YFP or STRK1-C5,10,14A-YFP in rice protoplasts. Bar = 10 µm. (B) to (H) STRK1 promoter-GUS expression patterns in transgenic rice plants. GUS expression was observed in young root ([B] and [C]), leaf vein (D), 4-d-old seedling (E), stem (F), leaf sheath (G), and young spikelet (H). (I) and (J) Relative mRNA levels of STRK1 by RT-qPCR in the root (I) and shoot (J) of three-leaf stage rice seedlings treated with 150 mM NaCl and 1% (v/v) H₂O₂. Data are presented as mean ± sd (n = 3).

Figure 3.

STRK1 Physically Interacts with CatC. (A) Yeast two-hybrid assay of STRK1 interaction with CatC. STRK1 (bait) and CatC (prey) were introduced into yeast cells as indicated and grown on selection medium minus leucine and tryptophan (-LW) or leucine, tryptophan, histidine, and adenine (-LWHA). Empty vectors pGBKT7 and pGADT7 were used as controls. (B) BiFC assay for interaction between STRK1 and CatC in Arabidopsis protoplasts. Full-length STRK1 protein was fused to C-terminal CFP (STRK1-cCFP), and full-length CatC was fused to N-terminal Venus (nVenus-CatC). The expression of nVenus/STRK1-cCFP was used as control. Before imaging, the protoplasts were treated with FM4-64 (2 μM) for 5 min to show the plasma membrane. Bar = 10 µm. (C) Pull-down assay for interaction between STRK1 and CatC. Purified His-TF-CatC was used to copurify STRK1-FLAG from OE18 seedling protein extracts (Input) following incubation with Ni-NTA agarose (see Methods). The flow-through was collected after centrifugation (Flow) and two washes (Wash1 and Wash2), and the eluted fractions (Elute) were separated by 10% SDS-PAGE and immunoblotted with anti-FLAG antibodies. His-TF was used as control. (D) Co-IP assay for interaction between STRK1 and CatC in N. benthamiana leaves. Protein extracts (Input) were immunoprecipitated with anti-FLAG antibody (IP). Immunoblots were developed with anti-FLAG antibody to detect STRK1 and with anti-GFP to detect CatC.

Figure 4.

STRK1 Phosphorylates CatC Mainly at Tyr-210 and Activates Its Activity. (A) STRK1 phosphorylates CatC in vitro. His-TF or His-TF-CatC was incubated with GST-STRK1 and [γ-³²P]ATP. Upper panel shows autoradiography and bottom panel Coomassie brilliant blue (CBB) staining of the gel. (B) Phosphorylation of CatC is indeed dependent on the kinase activity of STRK1. His-TF-CatC was incubated with GST-STRK1, GST-STRK1^K95E and ³²P-γ-ATP. GST-STRK1^K95E is the kinase dead form of STRK1. Upper panel shows autoradiography and bottom panel Coomassie brilliant blue (CBB) staining of the gel. (C) STRK1 stimulates CatC activity in vitro. Recombinant His-TF-CatC expressed in E. coli was purified, and the CatC activity was measured with GST or GST-STRK1 in the presence of ATP. Data are presented as mean ± sd (n = 3, **P ≤ 0.01, Student’s t test). (D) An equal amount of His-TF fused CatC, CatC^Y210F, CatC^Y360F, and CatC^Y210,360F was incubated with GST-STRK1 and [γ-³²P]ATP. Upper panel shows autoradiography and bottom panel Coomassie brilliant blue staining of the gel. (E) Phosphorylation of CatC at Tyr-210 is essential for CatC activity. CatC^Y210Fand CatC^Y360F, recombinant CatC with Tyr-210 and Tyr-360 mutated to Phe, CatC^Y210D and CatC^Y360D, and recombinant CatC with Tyr-210 and Tyr-360 mutated to Asp. Each data point represents the mean ± se (n = 6). Asterisk indicates significant difference relative to CatC activity in the absence of STRK1 and ATP (Student’s t test, **P < 0.01). (F) and (G) CAT activity (F) and H₂O₂ contents (G) in transgenic seedlings (T0) overexpressing CatC^Y210F and CatC^Y210D in the Ri11 background, a STRK1 knockdown mutant. The Kitaake (WT) and Ri11 seedlings regenerated from the corresponding calli were used as control. Data are presented as mean ± sd (n = 3, *P ≤ 0.05, **P ≤ 0.01, Student’s t test).

Figure 5.

Salt Stress Induces STRK1 Tyrosine Phosphorylation on CatC. (A) NaCl treatment enhances STRK1 tyrosine phosphorylation on CatC. STRK1-FLAG and CatC proteins were immunoprecipitated from OE18 seedling after treatment with NaCl for the indicated periods and then subjected to an immunoblot assay. Anti-phospho-tyrosine (α-pTyr) antibody was used to probe for STRK1 and CatC phosphorylation. Immunoprecipitated endogenous CatC and STRK1-FLAG were analyzed by immunoblotting with anti-CatC and anti-FLAG antibodies, respectively. The relative intensities of tyrosine phosphorylation of CatC and STRK1-FLAG without NaCl treatment (0 min) were set to 1.00. (B) The CatC activity is associated with its tyrosine phosphorylation levels. The phosphorylated CatC proteins in (A) were used to determine the catalase activity. Data are presented as mean ± sd (n = 3, *P ≤ 0.05, **P ≤ 0.01, Student’s t test). (C) Knockdown of STRK1 reduces the tyrosine phosphorylation level of CatC. Endogenous CatC proteins were immunoprecipitated from Ri11 and Ri16, the STRK1 knockdown mutants, and the wild-type seedlings were grown under normal growth conditions and then detected by immunoblotting. Anti-phospho-tyrosine (α-pTyr) antibody was used to probe for CatC phosphorylation, and anti-CatC antibody was used to show the immunoprecipitated CatC. The relative intensity of tyrosine phosphorylation of CatC in wild-type seedlings was set to 1.00. (D) and (E) CAT activity in rice shoots (D) and roots (E) of seedlings grown under normal conditions or after 100 mM NaCl stress for the indicated periods. Data are presented as mean ± sd (n = 3). (F) and (G) H₂O₂ contents in rice shoots (F) and roots (G) of seedlings grown under normal conditions or after 100 mM NaCl stress for the indicated periods. Data are presented as mean ± sd (n = 3).

Figure 6.

STRK1 Positively Regulates Oxidative Tolerance in Rice. (A) Phenotypic comparison of rice plants subjected to MV stress. The germinated seeds were transplanted into either 0.5× MS medium or 0.5× MS medium supplemented with 2 μM MV for 6 d. (B) to (D) Chlorophyll content in leaves (B), seedling height (C), and CAT activity in leaves (D) of STRK1 transgenic and wild-type plants under normal and MV stress conditions. Data are presented as mean ± sd (n = 3, *P ≤ 0.05, **P ≤ 0.01, Student’s t test). (E) Leaf phenotype of STRK1 transgenic and wild-type plants at the three-leaf stage under normal conditions or after 100 mM H₂O₂ stress for 2 d. (F) DAB staining for H₂O₂ in leaves from unstressed and H₂O₂-stressed STRK1 transgenic and wild-type plants for 1 d.

Figure 7.

STRK1 Improves the Grain Yield of Rice under Salt Stress at the Reproductive Stage. (A) and (B) Phenotypic comparison of rice plants under salt stress. Salt stress of plants was initiated at the panicle development stage (top) by exposure to 1% NaCl as indicated (down), and then the plants were recovered with irrigation for 10 d and harvested. (C) to (E) Panicle number (C), spikelet fertility (D), and grain yield (E) of STRK1-overexpressing and wild-type plants in (A) after 10 d of recovery. Data are presented as mean ± sd (n = 20,*P ≤ 0.05, **P ≤ 0.01, Student’s t test). (F) to (H) Panicle number (F), spikelet fertility (G), and grain yield (H) of STRK1-RNAi and wild-type plants in (B) after 10 d of recovery. Data are presented as mean ± sd (n = 20,*P ≤ 0.05, **P ≤ 0.01, Student’s t test).

Figure 8.

A Proposed Model for the Role of STRK1 in Regulating Salt or Oxidative Stress Tolerance. Salt or oxidative stress induces H₂O₂ accumulation, which causes oxidative damage to the cell. In response to salt or oxidative stress, the extracellular signals are transmitted from an unknown RLK to STRK1, a membrane-anchored RLCK by palmitoylation, through phosphorylation. The phosphorylated STRK1 stimulates CatC activity via phosphorylation (P) mainly at Tyr-210, which in turn inhibits H₂O₂ accumulation and, ultimately, increases the salt or oxidative stress tolerance in plants. The question mark indicates an unknown RLK. The wavy lines indicate the palmitoyl groups. The step marked with a black dashed line with an arrow between the unknown RLK and STRK1 is currently unknown.