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Background Soil salinization is a major abiotic environmental stress factor threatening crop production through- out the world. Salt stress drastically affects the growth, development, and grain yield of rice (Oryza sativa L.), and the improvement of rice tolerance to salt stress is a desirable approach for meeting increasing food demand. Receptor- like cytoplasmic kinases (RLCKs) play essential roles in plant growth, development and responses to environmental stresses. However, little is known about their functions in salt stress. Previous reports have demonstrated that overex- pression of an RLCK gene SALT TOLERANCE KINASE (STK) enhances salt tolerance in rice, and that STK may regulate the expression of GST (Glutathione S-transferase) genes. Results The expression of STK was rapidly induced by ABA. STK was highest expressed in the stem at the heading stage. STK was localized at the plasma membrane. Overexpression of STK in rice increased tolerance to salt stress and oxidative stress by increasing ROS scavenging ability and ABA sensitivity. In contrast, CRISPR/Cas9-mediated knockout of STK increased the sensitivity of rice to salt stress and oxidative stress. Transcriptome sequencing analysis suggested that STK increased the expression of GST genes (LOC_Os03g17480, LOC_Os10g38140 and LOC_Os10g38710) under salt stress. Reverse transcription quantitative PCR (RT-qPCR) suggested that four stress-related genes may be regulated by STK including OsABAR1, Os3BGlu6, OSBZ8 and OsSIK1. Conclusions These findings suggest that STK plays a positive regulatory role in salt stress tolerance by inducing anti- oxidant defense and associated with the ABA signaling pathway in rice. Keywords STK, Salt stress, Rice, ROS scavenging, ABA *Correspondence: Hunan Province Key Laboratory of Plant Functional Genomics Yanbiao Zhou and Developmental Regulation, College of Biology, Hunan University, firstname.lastname@example.org Changsha 410082, Hunan, China Yuanzhu Yang email@example.com State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha 410125, Hunan, China Key Laboratory of Southern Rice Innovation and Improvement, Ministry of Agriculture and Rural Affairs, Yuan Longping High-Tech Agriculture Co., Ltd., Changsha 410001, Hunan, China College of Life Sciences, South China Agricultural University, Guangzhou 510642, China College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China College of Agronomy, Hunan Agricultural University, Changsha 410128, Hunan, China College of Life Sciences, Hunan Normal University, Changsha 410081, Hunan, China © The Author(s) 2023. 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Rice (2023) 16:21 Page 2 of 15 transmembrane domain and intracellular kinase domain Background or only an intracellular kinase domain (Vij et al. 2008). Salinity is one of the major abiotic stresses which influ - This group are referred to as receptor-like cytoplasmic ences plant growth and can lead to severe crop produc- kinases (RLCKs). There are 200 genes encoding RLCKs tion losses (Chen et al. 2021). Approximately 46 million in Arabidopsis and 379 in rice (Jurca et al. 2008; Vij et al. hectares of farmland in the world is subject to salt con- 2008). Eighty-two OsRLCKs are predicted to participate ditions, and this proportion is increasing every year due in abiotic stresses in rice (Vij et al. 2008). However, only to inappropriate crop irrigation, over fertilization and a few RLCKs have been functionally characterized to be excessive plowing, as well as natural reasons, such as salt involved in abiotic stress response. A rice receptor-like intrusion into coastal zones caused by rising sea levels cytoplasmic kinase, GUDK, was reported to phospho- (Liu et al. 2021). Understanding the mechanisms of salt rylate OsAP37 and activate stress-induced gene expres- tolerance in rice will allow improving their yield and sion, which improve drought tolerance and grain yield quality in areas subject to salt stress. (Ramegowda et al. 2014). Cold-responsive protein kinase Reactive oxygen species (ROS) in plants include super- 1 (CRPK1) was reported to negatively regulate cold stress oxide anion (O ), singlet oxygen ( O ), hydroxyl radical 2 2 by phosphorylating 14-3-3 proteins, which interact with (OH ), and hydrogen peroxide (H O ), which serve as 2 2 C-repeat binding factor (CBF) proteins and promote key signaling molecules in many biological processes, their degradation in the nucleus (Liu et al. 2017). A novel involving biotic and abiotic stress tolerance (Mittler et al. rice RLCK, STRK1, improves salt and oxidative toler- 2011; Schippers et al. 2012; Schmidt et al. 2013). ROS ance by phosphorylating and activating CatC and thereby production is enhanced in plants suffering from various regulating H O homeostasis (Zhou et al. 2018). Overex- abiotic stresses, such as salt, drought, and temperature 2 2 pression of OsRLCK241 improved tolerance of rice plants (Mittler et al. 2004). Low ROS concentrations can func- to salt and drought stresses with improved ROS detoxifi - tion as a signal to activate salt stress responses, but excess cation and altered expression of stress-responsive genes ROS can result in oxidative damage to cellular mem- (Zhang et al. 2021). branes and other cellular components, which ultimately Previously, we have identified RLCK genes from rice leads to cell death (Apel and Hirt 2004). To balance ROS and examined their expression patterns in response to production and destruction, plants have evolved an anti- salt stress (Zhou et al. 2018). One of the salt-responsive oxidant system that involves both antioxidant enzymes, RLCK genes, SALT TOLERANCE KINASE (STK), which such as ascorbate peroxidase (APX), superoxide dis- encodes a putative receptor-like cytoplasmic kinase mutase (SOD), catalase (CAT), glutathione S-transferase was selected for further analysis. This gene was induced (GST), glutathione reductase (GR), and glutathione per- by salt stress. To explore the potential function of STK oxidase (GPX), and antioxidant compounds, such as in the salt stress response, transgenic plants were cre- ascorbate (ASC) and glutathione (GSH), to scavenge ated with the overexpression of STK and knockout of ROS (Mittler 2002; Apel and Hirt 2004; Foyer and Noc- STK in kitaake, respectively. Overexpression of STK tor 2011). Identifying genes modulating the activities of improved tolerance to salt and oxidative stress through antioxidant enzymes and antioxidant compounds con- the enhanced activity of ROS scavenging enzyme GST tent will be essential for improving plant stress tolerance. and regulating the expression of multiple stress-related Overexpression of a receptor-like kinase gene OsSIK1 in genes, whereas the STK knockout mutants increased salt rice enhanced plant tolerance to drought and salt stress sensitivity. through activating the antioxidant enzymes to scavenging and detoxification of ROS (Ouyang et al. 2010). The genes Results encoding ROS scavenging enzymes were down-regulated The Expression Profiles and Subcellular Localization of STK in the ospp18 mutant, and the mutant showed reduced To elucidate important factors for mediating salt stress activities of ROS scavenging enzymes and increased sen- signaling, we identified RLCK genes whose expres - sitivity to oxidative stresses (You et al. 2014). A loss of sion was induced by salt stress from our previous study function of CSN5B enhances tolerance to salt by increas- (Zhou et al. 2018). Among these genes, we selected a ing ascorbic acid (AsA) synthesis (Wang et al. 2013). salt stress-responsive RLCK gene named SALT TOLER- Receptor-like kinases (RLKs) are transmembrane pro- ANCE KINASE (STK), which belongs to the RLCK-VIIa teins with an extracellular domain, a transmembrane sub-family (Gao and Xue 2012). To examine the physi- domain, and an intracellular kinase domain (Torii 2000). ological function of STK under abiotic stress conditions, RLKs contain a large plant protein family with over 610 the expression pattern of STK was examined in leaves of members in Arabidopsis and over 1131 in rice (Shiu et al. 3-week-old plants (Oryza. sativa cv Kitaake) in response 2004). However, there are plant-specific RLKs without to various abiotic stresses and treatments by reverse an extracellular domain and these RLKs contain only the Zhou et al. Rice (2023) 16:21 Page 3 of 15 transcription quantitative PCR (RT-qPCR). Expression seedling and heading stages. The expression level of STK of STK was apparently induced under six treatments, was higher in the shoot than in the root at the seedling including NaCl (150 mM), PEG (20%), H O (1%), Cold stage (Fig. 1B). In addition, STK was expressed in all the 2 2 (4 °C), ABA (100 μM) and GA (100 μM). ABA treat- tissues at the heading stage, with the highest level of ment led to earlier expression peak of STK than other expression in the stem, and the lowest level of expression treatments (Fig. 1A). These results suggested that STK in the leaf (Fig. 1B). is involved in responses to multiple abiotic stresses and To determine the subcellular localization of STK, the treatments. full-length STK coding region was fused in frame to the To investigate the tissue-specific expression of STK yellow fluorescent protein (YFP) marker gene under in the rice cultivar kitaake, the expression of STK was the control of cauliflower mosaic virus 35S promoter. measured by RT-qPCR in various rice organs during the Rice protoplasts prepared from an etiolated shoot were Fig. 1 Expression patterns of STK. A Expression profiles of STK in Kitaake rice seedlings in response to salt, drought, H O , cold, ABA and GA 2 2 treatments using RT-qPCR. B Expression of STK in various tissues of rice cultivar Kitaake at different stages using RT-qPCR. Data are means ± SD (n = 3) Zhou et al. Rice (2023) 16:21 Page 4 of 15 cotransformed with 35S::STK-YFP and 35::PIP2A- plants survived under this treatment (Fig. 3B). As shown mCherry by polyethylene glycol treatment. PIP2A was in Fig. 3C–E, compared with wild-type plants, STK-OE used as a marker since it has been reported as a plasma plants accumulated more chlorophyll and less malondial- membrane aquaporin (Nelson et al. 2007). As shown in dehyde (MDA) and relative ion leakage. In contrast, when Fig. 2, the STK-YFP fusion protein was located in the wild-type and STK-KO plants were subjected to 140 mM plasma membrane; the plasma membrane localization NaCl for 12 d, 41% of the wild-type plants recovered 4 d was confirmed by its colocalization with the red fluores - after watering was restored, but only 14% to 18% of the cence protein (mCherry)-fused plasma membrane pro- STK-KO plants recovered (Fig. 4A, B). Further physiolog- tein PIP2A, indicating that STK is a plasma membrane ical analyses revealed that loss of function of STK reduced protein. the content of chlorophyll and increased levels of MDA and relative ion leakage in leaves (Fig. 4C–E). Previous STK Positively Regulates Salt Tolerance in Rice studies showed that the ability to avoid N a excessive + + To evaluate the biological role of STK, transgenic rice accumulation and keep a low Na /K ratio contributes to plants with STK overexpressed under the control of the salt tolerance in plants (Zhu 2003). We further measured + + 35S promoter (STK-OE) and knockout by CRISPR/Cas9 Na and K contents in the shoot of wild-type, STK-OE + + system (STK-KO) were generated. One target site within and STK-KO plants. Less Na but higher K content was the second exon was designed and an expression vec- detected in STK-OE plants under salt stress, resulting in + + tor was constructed according to previously described a lower Na /K ratio in the shoot of STK-OE plants than CRSPR/Cas9 vector (Additional file 1: Fig. S1A, B) (Zhou wild-type (Fig. 3F–H). In contrast, compared with the et al. 2022). Two homozygous mutants (KO1 and KO3) wild-type plants, the STK-KO plants accumulated more + + + + were identified by sequencing (Additional file 1: Fig. Na and less K , leading to a higher Na /K ratio under S1C). KO1 contains an A insertion and KO3 contains a G salt stress (Fig. 4F–H). These findings indicated that STK deletion in the CDS, which caused a frameshift mutation confers tolerance to salt stress via relief of the membrane (Additional file 1: Fig. S1D). Two independent STK-OE damage caused by salt stress and avoidance of N a exces- transgenic lines (OE3 and OE5; Additional file 1: Fig. S2) sive accumulation in plants. and two STK-KO lines (KO1 and KO3; Additional file 1: Fig. S1) of STK were selected for further analysis. Overexpression of STK Enhances ABA Sensitivity Under salt stress using 140 mM NaCl for 12 d and Recent study revealed that STK was up-regulated under recovery 4 d, STK-OE lines showed greater resistance ABA treatment (Gao and Xue 2012). As shown previ- than wild-type plants (Fig. 3A). Almost 70% to 73% of the ously, the expression of STK was also induced by ABA STK-OE plants survived, while only 43% of the wild-type (Fig. 1A). Consequently, we determined whether STK-OE Fig. 2 Subcellular localization of STK in rice protoplast. 35S::STK-YFP and 35S::PIP2A-mCherry were cotransformed into rice etiolated shoot protoplasts. 35S::YFP was transformed as control. Bar = 10 μm Zhou et al. Rice (2023) 16:21 Page 5 of 15 Fig. 3 Overexpression of STK increased salt tolerance in rice. A Phenotype of WT, OE3 and OE5 rice plants under salt stress at seedling stage. For STK-OE transgenic plants, 15-d-old seedlings were treated with 140 mM NaCl for 12 d and recovery 4 d. B Survival rates of WT and transgenic plants in (A) after 4 d of recovery. C Chlorophyll content in leaves of 15-d-old plants treated with 100 mM NaCl for 7 d. D MDA concentrations in leaves of 15-d-old plants after 100 mM NaCl treatment for 24 h. E Relative ion leakage in leaves of 15-d-old plants after 100 mM NaCl treatment for 24 h. F–H Ion content in shoots of 4-week-old plants after 6 d of salt stress (100 mM NaCl). Wild type and STK-OE plants were exposed to salt stress + + (100 mM NaCl) for 6 d and the shoots were harvested for analysis of ion content. The Na and K. contents in seedlings were detected as previously described (Schmidt et al. 2013). Data in B–H are presented as mean ± SD (n = 3, **P ≤ 0.01, Tukey ’s test) plants and STK-KO plants showed differences in perfor - rice plants at the post-germination stage. Compared with mance under ABA treatment compared with wild-type the wild-type, the shoot length was significantly shorter plants. The ABA sensitivity of rice plants was investigated in STK-OE plants but markedly longer in STK-KO at the germination stage. Seeds of STK-OE plants, STK- plants under 1, 3 and 6 μM ABA treatment, respectively KO plants, and the wild-type were germinated in one- (Fig. 5C, F). No significant difference in shoot length was half-strength Murashige and Skoog (1/2MS) medium observed among STK-OE, STK-KO and wild-type under containing ABA with a gradient of concentrations (0, 1, without ABA treatment (Fig. 5C, F). These results sug - 3 and 6 μM). The germination rate of the STK -OE plants gested that STK is a positive regulator of ABA signaling and STK-KO plants was identical to the wild-type at in rice. 0 μM ABA treatment (Fig. 5A, B, D, E). However, com- pared with the wild-type, the germination rate was STK Positively Regulates Oxidative Tolerance in Rice reduced in STK-OE plants and increased in STK-KO Salt stress usually cause damage in plants via oxida- plants under 1, 3 and 6 μM ABA treatment, respectively tive stress involving the generation of reactive oxygen (Fig. 5B, E). We also investigated the ABA sensitivity of species (ROS), such as H O (Chen 2021). As shown 2 2 Zhou et al. Rice (2023) 16:21 Page 6 of 15 Fig. 4 Knockout of STK increased salt sensitivity in rice. A Phenotype of WT, KO1 and KO3 rice plants under salt stress at seedling stage. For STK-KO plants, 15-d-old seedlings were treated with 140 mM NaCl for 12 d and recovery 4 d. B Survival rates of WT, KO1 and KO3 in (A) after 4 d of recovery. C Chlorophyll content in leaves of 15-d-old plants treated with 100 mM NaCl for 7 d. D MDA concentrations in leaves of 15-d-old plants after 100 mM NaCl treatment for 24 h. E Relative ion leakage in leaves of 15-d-old plants after 100 mM NaCl treatment for 24 h. F–H Ion content in shoots of 4-week-old plants after 6 d of salt stress (100 mM NaCl). Wild type and STK-KO plants were exposed to salt stress (100 mM NaCl) for 6 d and the + + shoots were harvested for analysis of ion content. The Na and K. contents in seedlings were detected as previously described (Schmidt et al. 2013). Data in (B) to (H) are presented as mean ± SD (n = 3, **P ≤ 0.01, Tukey ’s test) previously, the expression of STK was induced by H O plants exhibited the opposite results (Fig. 6B). Never- 2 2 (Fig. 1A) and STK-OE plants improved the salt toler- theless, the chlorophyll content was not significantly ance of transgenic rice plants, we examined whether different among STK -OE plants, STK-KO plants and STK functions in salt tolerance through detoxifica - wild-type under normal growth conditions. The accu - tion of ROS. Three-leaf stage seedlings of STK -OE mulation of H O was further determined by 3,3′-diam- 2 2 plants, STK-KO plants and wild-type were treated inobenzidine (DAB) staining. After 1d H O treatment, 2 2 with 100 mM H O . After 2 d H O treatment, severe the intensity of DAB staining was less in leaves of 2 2 2 2 necrosis and leaf rolling were observed in STK-KO the STK-OE plants than in those of wild-type plants plants (Fig. 6A). Moreover, wild-type plants exhibited (Fig. 6C). The intensity of DAB staining was greater stronger leaf rolling and more chlorosis than STK-OE in the leaves of STK-KO plants than in those of wild- plants after H O treatment. Then, the chlorophyll con - type plants. Thus, the STK -OE plants and the STK-KO 2 2 tent in these seedlings were determined. Under oxida- plants showed opposite trends in ROS accumulation. tive stress, STK-OE plants showed higher chlorophyll Under normal growth conditions, no obvious stain- content compared with wild-type, whereas the STK-KO ing was observed in STK-OE, STK-KO and wild-type. Zhou et al. Rice (2023) 16:21 Page 7 of 15 Fig. 5 Increased ABA sensitivity of STK overexpression plants at germination stage. A, D Germination performance of STK-OE (A) and STK-KO (D) seeds on 1/2MS medium containing 0, 1, 3, or 6 μM ABA at 5 d after initiation. B, E The germination rates of STK-OE (B) and STK-KO (E) seeds after 0, 1, 3, or 6 μM ABA treatment. C, F Shoot lengths of STK-OE (C) and STK-KO (F) plants after 0, 1, 3, or 6 μM ABA treatment for 5 d. Data in (B, C, E, F) are presented as mean ± SD (n = 3, **P ≤ 0.01, Tukey ’s test) These results suggest that STK is involved in the elimi- changes in the STK-OE, STK-KO and wild-type under nation of H O produced under oxidative stress. normal and salt stress conditions by RT-qPCR. Under 2 2 normal conditions, no significant differences in expres - Identification of Stress‑Related Genes Controlled by STK sion levels were observed among the STK-OE, STK-KO To understand the mechanisms and downstream com- and wild-type. On the contrary, compared with the wild- ponents of the STK-mediated salt-stress signaling path- type plants, the expression levels of the GST genes were ways, transcriptome deep sequencing (RNA-seq) analysis significantly increased in the STK-OE plants but mark- was performed using leaves from wild-type and STK-OE edly lower in STK-KO plants under salt stress conditions (OE5) rice plants under salt stress conditions. The thresh - (Fig. 7C–E). GST is the ubiquitous enzymes that play a old for significantly differentially expressed genes (DEGs) key role in cellular detoxification (You et al. 2014). u Th s, was set at a (log2 scale)-fold change (FC) value of > 1 STK maybe involved in the detoxification and ROS scav - or < − 1 and adjusted p-value < 0.05. Using these crite- enging mechanisms of rice by GST under salt stress. To ria, we identified 1076 DEGs (677 upregulated and 399 check this hypothesis, GST activities and ROS levels in downregulated) in OE5 compared to wild-type. These the salt-stressed and unstressed leaves of STK-OE, STK- DEGs altered by salt stress are shown in the volcano KO and wild-type were measured. Compared with the plots, which illustrate the asymmetry between upregu- wild-type, GST activity was significantly higher in the lated (red) and downregulated (green) DEGs (Fig. 7A). STK-OE plants but markedly lower in STK-KO plants Gene ontology (GO) enrichment analysis showed that under salt stress conditions (Fig. 7F). Similarly, the H O 2 2 the DEGs affected by the overexpression of STK were content was also significantly lower in STK -OE plants but enriched mainly in glutathione transferase activity and markedly higher in STK-KO plants under salt stress con- glutathione metabolic process (Fig. 7B). The GST (Glu - ditions (Fig. 7G). Under normal growth conditions, no tathione S-transferase) genes (LOC_Os03g17480, LOC_ significant difference of GST activity and H O content 2 2 Os10g38140 and LOC_Os10g38710) detected in the were detected among the STK-OE, STK-KO and wild- RNA-seq were further investigated for their expression type plants, respectively. These results indicated that STK Zhou et al. Rice (2023) 16:21 Page 8 of 15 Fig. 6 Increased oxidative tolerance of STK overexpression plants. A Leaf phenotype of WT, STK-OE and STK-KO plants at the three-leaf stage under normal conditions or after 100 mM H O stress for 2 d. B Total chlorophyll contents of the WT, STK-OE and STK-KO plants under normal and H O 2 2 2 2 stress conditions. FW, Fresh weight. Data are presented as mean ± SD (n = 3, **P ≤ 0.01, Tukey ’s test). C DAB staining for H O in leaves from normal 2 2 and H O -stressed WT, STK-OE and STK-KO plants for 1 d 2 2 was involved in the detoxification and ROS scavenging can cause damage to plants (Mittler 2002; Miller et al. mechanisms of rice under salt stress conditions, which 2010). Overaccumulation of ROS can damage DNA, pro- may have contributed to their improved tolerance to salt teins and carbohydrates, resulting in cell death (Mittler stress. et al. 2004). ROS also cause MDA production, cell mem- Previously identified several stress-responsive genes brane damage and lipid peroxidation. Previous studies involved in various pathways, including OsABAR1, Os3B- showed that the plant response to abiotic stresses occurs Glu6, OSBZ8 and OsSIK1 (Mukherjee et al. 2006; Ouy- through the regulation of ROS metabolism (Schmidt ang et al. 2010; Wang et al. 2020a, b; Zheng et al. 2020) et al. 2013; Li et al. 2014; Fang et al. 2015; Zhou et al. were up-regulated at 48 h of salt stress treatment (Addi- 2018; Zhao et al. 2019). For example, Salt Intolerance 1 tional file 2: Table S2). Expression levels of these genes (SIT1), a rice L-type LecRLK, could be activated by salt under normal or salt conditions was further verified by and mediate salt sensitivity by activation of MPK3/MPK6 RT-qPCR in STK-OE, STK-KO and wild-type plants. In leading to higher ethylene production and downstream agreement with the analysis of DEGs, the expression of ROS accumulation (Li et al. 2014; Zhao et al. 2019). these genes was up-regulated and downregulated in STK- Overexpression of a NAC protein, SNAC3, improves heat OE plants and STK-KO plants under salt stress condition, and drought tolerance by modulating ROS homeostasis respectively (Additional file 1: Fig. S3). These results sug - through regulation of the expression of genes encod- gested that STK may promote salt tolerance through acti- ing ROS production and ROS-scavenging enzymes vated stress-related genes. (Fang et al. 2015). STRK1 (Salt Tolerance Receptor-like Cytoplasmic Kinase 1), which activates CatC activity Discussion through phosphorylation at Tyr-210 of CatC to regu- Excessive accumulation of ROS is a frequent event in late H O homeostasis and improve salt tolerance (Zhou 2 2 plants suffering from diverse abiotic stresses, including et al. 2018). In this study, induction of STK upon H O 2 2 drought, high salinity, and extreme temperatures, and treatment indicated that it functions in oxidative stress Zhou et al. Rice (2023) 16:21 Page 9 of 15 Fig. 7 Differentially expressed genes (DEGs) in the STK-OE plants. A Volcano plots comparing the transcriptomes of OE5 plants with the WT. X-axis and Y-axis represent log fold change (FC) and − log (p-value), respectively. The green and red dots represent downregulated DEGs and 2 10 upregulated DEGs, respectively. The blue dots represent no significant difference in transcriptomes. B Gene ontology (GO) enrichment analysis of DEGs. TOP 20 significantly enriched biological process GO terms are show. Three biological replicates were included for each treatment. C–E Relative expression of GST genes Os03g17480 (C) Os10g38140 (D) and Os10g38710 (E) in rice leaves of seedlings grown under normal conditions or after 150 mM NaCl for 48 h. F GST activity in rice leaves of seedlings grown under normal conditions or after 150 mM NaCl for 48 h. G H O contents 2 2 in rice leaves of seedlings grown under normal conditions or after 150 mM NaCl for 48 h. Data in (C–G) are presented as mean ± SD (n = 3, *P ≤ 0.05, **P ≤ 0.01, Tukey ’s test) adaptation (Fig. 1A). Reduced MDA levels in the STK- STK-OE plants during the salt stress conditions. Relevant OE plants under salt stress treatment (Fig. 3D) implied to this finding, STK -OE plants showed better growth that less oxidative damage occurred in the cells of the under oxidative stress caused by H O (Fig. 6A, B). In 2 2 Zhou et al. Rice (2023) 16:21 Page 10 of 15 + + STK-OE plants, one ROS, H O , was present in low levels (Shabala and Cuin 2008; Wu et al. 2018). Na and K are 2 2 under salt stress, as revealed by DAB staining (Fig. 6C). imported into the plant cell using the same set of trans- To the contrary, STK-KO plants showed reduced toler- porters, and the two cations compete with each other ance to oxidative stress. These results suggested that the (Greenway and Munns 1980). In glycophytes, excessive + + function of STK in salt tolerance may be associated with Na commonly results in K deficiency under salt stress + + the regulation of antioxidation ability. (Yang and Guo 2018). Therefore, keeping a low Na /K To maintain cellular redox homeostasis and scavenge ratio is an important mechanism used by plants to adapt + + excess stress-induced ROS, plants have evolved anti- to salt stress. OsSOS1, a rice plasma membrane N a /H oxidation systems comprising nonenzymatic and enzy- antiporter, excludes Na from the shoot, promoting a + + matic antioxidants (Miller et al. 2010). The maintenance lower cellular Na /K ratio and improving salt tolerance of high activity of various antioxidant enzymes, such as (Martínez-Atienza et al. 2007; El Mahi et al. 2019). The SOD, CAT, GST, GR, and glutathione peroxidase, to K channel OsAKT1 and OsKAT1 are involved in salt + + scavenge the toxic ROS has been linked to improved tol- tolerance via maintaining a low Na /K ratio owing to erance of plants to abiotic stresses (Apel and Hirt 2004; the increase of K uptake (Fuchs et al. 2005; Obata et al. Mittler 2002; Miller et al. 2010). GST plays an important 2007). Salt Intolerance 2 (SIT2), a rice LecRLK, was iden- role in the reduction of organic hydroperoxides formed tified for its role in salinity stress tolerance potentially via during oxidative stress using the tripeptide glutathione its function in Na extrusion by regulating SOS pathway (GSH) as a cosubstrate or coenzyme (Dixon et al. 2002). (Sun et al. 2020). Our results demonstrate that less Na Overexpression of a GST gene from Escherichia coli in but higher K content was detected in STK-OE plants + + transgenic tobacco enhanced salt and cold tolerance under salt stress, resulting in a lower N a /K ratio in the (Le Martret et al. 2011). The ospp18 mutant showed shoot of STK-OE plants than wild-type (Figs. 3H, 4H). increased drought and oxidative sensitivity through These results indicated that STK may be involved in the + + reduced the expression of GST genes and the activ- transport of N a and K , regulating rice tolerance to salt + + ity of GST which significantly contribute to excessive stress by avoiding Na accumulation and promoting K H O accumulation (You et al. 2014). Overexpression of uptake. 2 2 a GST gene (ThGSTZ1) from Tamarix hispida improves A better understanding of the mechanisms underly- drought and salinity tolerance by enhancing the abil- ing salt stress sensing and signal transduction will help ity to scavenge ROS (Yang et al. 2014). In the present researchers design novel approaches to increase plant study, the RNA-seq assay revealed that under salt condi- tolerance to salt stress. In response to high salinity, the tions, STK increased the expression of GST genes (LOC_ initial signal activates the production of compounds Os03g17480, LOC_Os10g38140 and LOC_Os10g38710) that trigger activity in many metabolic and developmen- and further confirmed by RT-qPCR. Under salt stress, tal pathways (Li et al. 2019). One of the most important the expression of GST genes and the activity of GST compounds is abscisic acid (ABA; Finkelstein and Gib- were found to be higher in the STK-OE plants than in son 2002). ABA plays an important role in regulating wild-type, and STK-KO plants showed the reverse results the response of plants to environmental stress (Sah et al. (Fig. 7C–F). The low level of H O in STK-OE plants was 2016; Vishwakarma et al. 2017). Salt stress can trigger the 2 2 most likely a result of high levels of the enzyme GST ABA-dependent signaling pathway in plants (Zhu 2002). (Fig. 7G). These results suggested that the enhanced GmWRKY16 as a WRKY transcription factor may pro- activity of ROS scavenging enzymes may significantly mote tolerance to drought and salt stresses through an contribute to scavenge H O accumulation and reduce ABA-mediated pathway (Ma et al. 2019). Overexpression 2 2 oxidative damage in STK-OE plants, which is associated of OsSRK1 enhances salt tolerance probably by regulating with the increased tolerance of the plants to salt stress. stress-related genes and ABA pathway (Zhou et al. 2020). High concentrations of salt in plant cells can induce OsNAC45 positively regulates ABA signal pathway and ionic stress. Ionic stress is caused by sodium (Na ) and is required for salt tolerance in rice (Zhang et al. 2020). chlorine (Cl ) accumulation in plant cells, eventually Overexpression of PpSnRK1α in tomato is beneficial for resulting in premature leaf senescence and even plant enhancing salt tolerance by regulating ABA signaling death (Liu et al. 2021). The toxicity of Na mainly mani- pathway and reactive oxygen metabolism (Wang et al. fested in its inhibitory effect on enzyme activities, nega - 2020a, b). ABA plays important roles in many aspects of tively affecting metabolism, including the Calvin cycle plant development and physiological processes, such as and other pathways (Cheeseman 2013; Wu et al. 2018). seed maturation, germination, seedling growth and sto- Potassium (K ), a vital ion for plant growth, plays a matal movement, is the central regulator of abiotic stress critical role in salt tolerance regulation as the cytosolic resistance in plants (Finkelstein et al. 2008; Kim et al. + + Na /K ratio appears to determine plant salt tolerance 2010). In the present study, an expression pattern analysis Zhou et al. Rice (2023) 16:21 Page 11 of 15 showed that STK expression was strongly induced by genes (Miyoshi et al. 1999; Nakagawa et al. 1996). There - ABA (Fig. 1A), indicating that STK is involved in ABA fore, the higher expression levels of these stress-related signaling pathway. In the absence of ABA, the germina- genes in STK-OE transgenic plants could contributed to tion rates of the WT, STK-OE and STK-KO plants were increased tolerance of the transgenic plants to salt stress. similar, and no significant difference in shoot length was observed among wild-type, STK-OE and STK-KO Conclusions plants after germinating (Fig. 5). In the presence of ABA, A novel rice RLCK gene, STK, which was rapidly induced the germination and growth of the STK-OE plants were by ABA as a prominent regulator of the response to salt severely inhibited compared with the wild-type. By con- stress while causing rice to be sensitive to exogenous trast, the STK-KO plants germinated more quickly and ABA. Overexpression of STK improved tolerance to salt grew faster than the WT. This phenomenon may be in and oxidative stresses than wild-type. On the contrary, part explained by the RNA-seq data. OsSAPK10 is a posi- the knockout mutants STK-KO were sensitive to salt and tive regulator of the ABA signal pathway in seed germi- oxidative stresses. The activity of GST was enhanced sig - nation and early seedling growth (Wang et al. 2020a, b). nificantly in STK -OE plants. Also, the accumulation of OsSAPK10 overexpression lines displayed more severe H O in leaves of STK-OE plants was much less than that 2 2 ABA-mediated repression on seed germination and seed- of the STK-KO plants and wild-type. STK improved salt ling growth (Wang et al. 2020a, b). The RNA-seq showed tolerance by inducing antioxidant defense and associ- that the expression of OsSAPK10 in STK-OE plants is sig- ated with the ABA signaling pathway in rice. In summary, nificantly higher than wild-type plants under salt stress STK enhanced ROS scavenging capacity by regulating the conditions (Additional file 2: Table S2), which is further expression of GST genes, thereby increasing the salt tol- confirmed by RT-qPCR (Additional file 1: Fig. S4). These erance of rice. results showed that STK confers tolerance to salt stress through an ABA-dependent signaling pathway. Methods To illustrate the molecular mechanism underlying Plant Materials and Growth Conditions the enhanced salt tolerance of STK-OE plants, RNA- Rice cultivars Kitaake (Oryza sativa ssp. japonica) were seq was performed. Under salt conditions, 677 genes used for RT-qPCR analysis under various stresses and were upregulated and 399 genes were downregulated phytohormone treatments, and Kitaake was used for all (Fig. 7A). GO annotation analysis showed that these transgenic experiments. For gene expression analysis, genes mainly belong to following categories: glutathione rice seeds were first sterilized with 15% NaClO, germi - transferase activity and glutathione metabolic process nated at 30 °C for 3 days, and then grown in hydroponic (Fig. 7B). Many abiotic stress-related genes were regu- culture solution (Ren et al. 2005) with a 14-h light/10-h lated in STK-OE transgenic plants compared with WT dark photoperiod, a 28 °C (light)/25 ℃ (dark) tempera- −2 −2 (Additional file 2: Table S2). After salt treatment, the rela- ture range, 350 μmol m s light intensity, and 85% rel- tive expression levels of OsABAR1, Os3BGlu6, OSBZ8 ative humidity. Rice seedlings at the three-leaf stage were and OsSIK1 increased more obviously in STK-OE plants immersed with their roots in NaCl (150 mM), PEG (20%), compared with wild-type. The OsABAR1-overexpress - H O (1%), cold (4 °C), ABA (100 μM), GA (100 μM) for 2 2 ing plants showed increased tolerance to drought and the indicated times. Their leaves were harvested at 0, 1, 3, salinity, whereas the OsABAR1 knockout plants had the 6, 12, and 24 h after the beginning of treatments. All har- opposite phenotypes (Zheng et al. 2020). Overexpres- vested leaf samples were rapidly frozen in liquid nitrogen sion of OsSIK1 increased tolerance of rice plants to salt and stored at − 80 °C for RNA extraction. and drought stresses, and the knockout mutants sik1-1 and sik1-2, as well as RNA interference (RNAi) plants, RNA Extraction and RT‑qPCR Analysis are sensitive to drought and salt stresses (Ouyang et al. Total RNA was extracted from rice leaves using TRIzol 2010). Os3BGlu6, a chloroplast localized β-glucosidase, reagent (Sangon Biotech, Shanghai, China) and treated significantly affected cellular ABA pools, and playing with gDNA Eraser to eliminate any DNA contamination. an important role in drought stress and photosynthe- The quality of the total RNA was evaluated using a Nan - sis under normal growth and drought conditions (Wang oDrop 2000 (Thermo Fisher Scientific, Shanghai, China). et al. 2020a, b). OSBZ8, a bZIP class of ABRE-binding First-stand cDNAs were synthesized using PrimeScript transcription factor, is shown to be highly expressive in RT reagent kit (Takara, Beijing, China) following the response to tolerance against salt stress cultivars as com- manufacturer’s instruction. RT-qPCR was performed pared to those that are sensitive to salt stress (Mukher- with ABI 7500 Real Time PCR System (Applied Biosys- jee et al. 2006). It is worth noting that previous reports tems, Foster City, USA) using TB Green Premix Ex Taq have shown that OSBZ8 is a marker for ABA-responsive II (Takara, Beijing, China) to monitor dsDNA synthesis, Zhou et al. Rice (2023) 16:21 Page 12 of 15 according to the manufacturer’ s instruction. The primers 1 d, the leaves were stained with DAB following the pre- used for RT-qPCR are listed in Additional file 2: Table S1. viously described method (You et al. 2014). After 2 d, the The expression of rice Actin gene (LOC_Os03g50885) leaves were photographed. was used as an internal control. The relative expression levels were measured as previously described (Zhou et al. Physiological Measurements 2022). The total chlorophyll content was determined according to the method described previously (Zhou et al. 2018). Plasmid Constructions and Generation of STK‑OE About 100 mg of leaf blades were ground in liquid nitro- and STK‑KO Plants gen and then immersed in the extract solution (80% ace- To generate the STK-OE plants, the coding region of tone) under darkness overnight at 4 °C. The absorbance the STK was cloned in the BamHI and EcoRI sites of of the extracts was read at 663 and 645 nm with a spec- the pCAMBIA1300-YFP vector using a specific primer trophotometer (BioTek). The total chlorophyll content (Additional file 2: Table S1). To generate the STK- −1 was calculated and expressed as mg g FW. The content KO plants, the target sites of STK were selected by the of MDA was measured as previously described (Ouy- CRISPR-Plant Web server (Xie et al. 2014) and were con- ang et al. 2010) with slight modification. About 100 mg structed into the CRISPR/Cas9 vector as described previ- of leaf blades were homogenized in 1 mL of 10% trichlo- ously (Zhou et al. 2022). The plasmids were introduced roacetic acid (TCA). The homogenate was centrifuged at into Agrobacterium tumefaciens strain EHA105. Agro- 5000 g for 10 min at 4 °C. To 800 μl of the supernatant bacterium-mediated transformation of embryogenic calli was added 800 μl of thiobarbituric acid (TBA) (made in generated from the japonica rice cultivar Kitaake was 10% TCA). The mixture was boiled for 15 min and centri - performed as previously described (Zhou et al. 2015). To fuged at 12,000g at 4 °C for 5 min. The absorbance of the determine whether genome editing of STK was success- supernatant was read at 450, 532, and 600 nm. The MDA ful, genomic DNA extracted from regenerated transgenic content was determined using the extinction coefficient plants was amplified by PCR with the primers listed in −1 of 155 (nmol/L/cm) and expressed as mmol g FW. The Additional file 2: Table S1, followed by sequencing of the relative ion leakage was assayed according to the method PCR products. described previously (Cao et al. 2007). The activity of glutathione S-transferase (GST) and the concentration Stress Treatments of H O were measured as described by using rice leaf 2 2 For salt stress tolerance assays, uniformly germinated blades using commercial kits (D799774-0100, Sangon seeds of WT, STK-OE, and STK-KO lines were grown in Biotech, Shanghai, China; D799612-0100, Sangon Bio- hydroponic culture solution. 15-d-old plants (40 plants tech, Shanghai, China) according to the manufacturer’s each genotype) were transferred to a hydroponic culture instructions. One unit of GST activity was defined as the solution containing 140 mM NaCl, and 12 d NaCl treat- amount of enzyme depleting 1 μM GSH in 1 min. H O 2 2 ments were applied for STK-KO and STK-OE plants as was detected by DAB staining as described previously well as their corresponding wild-type, respectively. The (Zhou et al. 2018). hydroponic solution was changed every two days during salt stress treatment. Then, the plants were transferred to a normal hydroponic culture solution to recover for 4 Subcellular Localization of STK d, and the color of the leaves was observed. If the leaves For detection of the subcellular localization of STK, the were green, they were counted as surviving plants, if the coding sequences of the STK were fused to yellow fluo - leaves were withered brown, they were counted as dead rescent protein (YFP) reporter coding sequences and plants, and finally the survival rate was counted. subcloned into the pCAMBIA1300-YFP vector, in which For testing the ABA sensitivity of transgenic plants at the YFP-coding sequence was fused in frame to the 3′ the germination stage, seeds of WT, STK-OE, and STK- end of the STK gene sequence. The plasmid was trans - KO lines (30 seeds each, three repeats) were sterilized formed into isolated rice protoplasts using polyethylene with 15% NaClO and cultured on hydroponic culture glycol (PEG)-mediated transformation methods (Zhou solution containing a gradient concentration of ABA (0, et al. 2018). 1, 3, and 6 μM). After 5 d treatment, the number of ger- Protoplasts were then transferred into multi-well plates minated seeds with emerged coleoptiles and germination and cultured in the dark at room temperature for 6–16 h. rate were calculated. After incubation, green fluorescence signals from trans - For the H O treatment, three-week-old plants (30 fected protoplasts were observed using a confocal laser 2 2 plants each genotype) were irrigated with hydroponic scanning microscope (Olympus FV1000) with 488 nm culture solution containing 100 mM H O solution. After excitation and 505–530 nm emission wavelengths. 2 2 Zhou et al. Rice (2023) 16:21 Page 13 of 15 Funding RNA Sequencing (RNA‑seq) Analysis This work was supported by the National Natural Science Foundation of For RNA-seq, three-week-old seedlings of WT and China (No. 31901516) and the special funds for the construction of innovative STK-OE plants were treated with 150 mM NaCl for provinces in Hunan (No. 2020RC3085). 48 h. Total RNA was extracted from leaves of three- Availability of Data and Materials week-old WT and STK-OE plants with the TRIzol All data supporting the findings of this study are available from the corre - reagent (Sangon Biotech, Shanghai, China) according sponding author on reasonable request. to the manufacturer’s instructions. Library prepara- tion and Illumina sequencing was performed at Novo- Declarations gene from three biological repeats of samples. Raw Ethical Approval and Consent to Participate reads were trimmed by cutadapt to get clean reads. The This study complied with the ethical standards of China, where this research trimmed reads were then mapped onto the reference work was carried out. rice genome from RAP-DB (https:// rapdb. dna. affrc. Consent for Publication go. jp). DEGs responding to salt stress were defined by All authors are consent for publication. a ≥ twofold expression change and p-value < 0.05, and Competing interests genes which were up regulated between WT and OE The authors declare that they have no competing interests. plants were subjected to further gene ontology (GO) enrichment analysis using the ClusterProfiler (Yu Received: 5 September 2022 Accepted: 16 April 2023 2018). The expression levels of selected DEGs were ver - ified by RT-qPCR. Abbreviations References RLK Receptor-like kinase Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, RLCK Receptor-like cytoplasmic kinase and signal transduction. Annu Rev Plant Biol 55:373–399 OE Overexpression Cao WH, Liu J, He XJ, Mu RL, Zhou HL, Chen SY, Zhang JS (2007) Modulation KO Knockout of ethylene responses affects plant salt-stress responses. 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Rice – Springer Journals
Published: Dec 1, 2023
Keywords: STK; Salt stress; Rice; ROS scavenging; ABA
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