Receptor-Like Cytoplasmic Kinase STK Confers Salt Tolerance in Rice
Rice volume 16, Article number: 21 (2023)
Soil salinization is a major abiotic environmental stress factor threatening crop production throughout 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 overexpression 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.
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.
These findings suggest that STK plays a positive regulatory role in salt stress tolerance by inducing antioxidant defense and associated with the ABA signaling pathway in rice.
Salinity is one of the major abiotic stresses which influences plant growth and can lead to severe crop production losses (Chen et al. 2021). Approximately 46 million hectares of farmland in the world is subject to salt conditions, and this proportion is increasing every year due to inappropriate crop irrigation, over fertilization and excessive plowing, as well as natural reasons, such as salt intrusion into coastal zones caused by rising sea levels (Liu et al. 2021). Understanding the mechanisms of salt tolerance in rice will allow improving their yield and quality in areas subject to salt stress.
Reactive oxygen species (ROS) in plants include superoxide anion (O2), singlet oxygen (1O2), hydroxyl radical (OH−), and hydrogen peroxide (H2O2), which serve as key signaling molecules in many biological processes, involving biotic and abiotic stress tolerance (Mittler et al. 2011; Schippers et al. 2012; Schmidt et al. 2013). ROS production is enhanced in plants suffering from various abiotic stresses, such as salt, drought, and temperature (Mittler et al. 2004). Low ROS concentrations can function as a signal to activate salt stress responses, but excess ROS can result in oxidative damage to cellular membranes and other cellular components, which ultimately leads to cell death (Apel and Hirt 2004). To balance ROS production and destruction, plants have evolved an antioxidant system that involves both antioxidant enzymes, such as ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR), and glutathione peroxidase (GPX), and antioxidant compounds, such as ascorbate (ASC) and glutathione (GSH), to scavenge ROS (Mittler 2002; Apel and Hirt 2004; Foyer and Noctor 2011). Identifying genes modulating the activities of antioxidant enzymes and antioxidant compounds content will be essential for improving plant stress tolerance. Overexpression of a receptor-like kinase gene OsSIK1 in rice enhanced plant tolerance to drought and salt stress through activating the antioxidant enzymes to scavenging and detoxification of ROS (Ouyang et al. 2010). The genes encoding ROS scavenging enzymes were down-regulated in the ospp18 mutant, and the mutant showed reduced activities of ROS scavenging enzymes and increased sensitivity to oxidative stresses (You et al. 2014). A loss of function of CSN5B enhances tolerance to salt by increasing ascorbic acid (AsA) synthesis (Wang et al. 2013).
Receptor-like kinases (RLKs) are transmembrane proteins with an extracellular domain, a transmembrane domain, and an intracellular kinase domain (Torii 2000). RLKs contain a large plant protein family with over 610 members in Arabidopsis and over 1131 in rice (Shiu et al. 2004). However, there are plant-specific RLKs without an extracellular domain and these RLKs contain only the transmembrane domain and intracellular kinase domain or only an intracellular kinase domain (Vij et al. 2008). This group are referred to as receptor-like cytoplasmic kinases (RLCKs). There are 200 genes encoding RLCKs in Arabidopsis and 379 in rice (Jurca et al. 2008; Vij et al. 2008). Eighty-two OsRLCKs are predicted to participate in abiotic stresses in rice (Vij et al. 2008). However, only a few RLCKs have been functionally characterized to be involved in abiotic stress response. A rice receptor-like cytoplasmic kinase, GUDK, was reported to phosphorylate OsAP37 and activate stress-induced gene expression, which improve drought tolerance and grain yield (Ramegowda et al. 2014). Cold-responsive protein kinase 1 (CRPK1) was reported to negatively regulate cold stress by phosphorylating 14-3-3 proteins, which interact with C-repeat binding factor (CBF) proteins and promote their degradation in the nucleus (Liu et al. 2017). A novel rice RLCK, STRK1, improves salt and oxidative tolerance by phosphorylating and activating CatC and thereby regulating H2O2 homeostasis (Zhou et al. 2018). Overexpression of OsRLCK241 improved tolerance of rice plants to salt and drought stresses with improved ROS detoxification and altered expression of stress-responsive genes (Zhang et al. 2021).
Previously, we have identified RLCK genes from rice and examined their expression patterns in response to salt stress (Zhou et al. 2018). One of the salt-responsive RLCK genes, SALT TOLERANCE KINASE (STK), which encodes a putative receptor-like cytoplasmic kinase was selected for further analysis. This gene was induced by salt stress. To explore the potential function of STK in the salt stress response, transgenic plants were created with the overexpression of STK and knockout of STK in kitaake, respectively. Overexpression of STK improved tolerance to salt and oxidative stress through the enhanced activity of ROS scavenging enzyme GST and regulating the expression of multiple stress-related genes, whereas the STK knockout mutants increased salt sensitivity.
The Expression Profiles and Subcellular Localization of STK
To elucidate important factors for mediating salt stress signaling, we identified RLCK genes whose expression was induced by salt stress from our previous study (Zhou et al. 2018). Among these genes, we selected a salt stress-responsive RLCK gene named SALT TOLERANCE KINASE (STK), which belongs to the RLCK-VIIa sub-family (Gao and Xue 2012). To examine the physiological function of STK under abiotic stress conditions, the expression pattern of STK was examined in leaves of 3-week-old plants (Oryza. sativa cv Kitaake) in response to various abiotic stresses and treatments by reverse transcription quantitative PCR (RT-qPCR). Expression of STK was apparently induced under six treatments, including NaCl (150 mM), PEG (20%), H2O2 (1%), Cold (4 °C), ABA (100 μM) and GA (100 μM). ABA treatment led to earlier expression peak of STK than other treatments (Fig. 1A). These results suggested that STK is involved in responses to multiple abiotic stresses and treatments.
To investigate the tissue-specific expression of STK in the rice cultivar kitaake, the expression of STK was measured by RT-qPCR in various rice organs during the seedling and heading stages. The expression level of STK was higher in the shoot than in the root at the seedling stage (Fig. 1B). In addition, STK was expressed in all the tissues at the heading stage, with the highest level of expression in the stem, and the lowest level of expression in the leaf (Fig. 1B).
To determine the subcellular localization of STK, the full-length STK coding region was fused in frame to the yellow fluorescent protein (YFP) marker gene under the control of cauliflower mosaic virus 35S promoter. Rice protoplasts prepared from an etiolated shoot were cotransformed with 35S::STK-YFP and 35::PIP2A-mCherry by polyethylene glycol treatment. PIP2A was used as a marker since it has been reported as a plasma membrane aquaporin (Nelson et al. 2007). As shown in Fig. 2, the STK-YFP fusion protein was located in the plasma membrane; the plasma membrane localization was confirmed by its colocalization with the red fluorescence protein (mCherry)-fused plasma membrane protein PIP2A, indicating that STK is a plasma membrane protein.
STK Positively Regulates Salt Tolerance in Rice
To evaluate the biological role of STK, transgenic rice plants with STK overexpressed under the control of the 35S promoter (STK-OE) and knockout by CRISPR/Cas9 system (STK-KO) were generated. One target site within the second exon was designed and an expression vector was constructed according to previously described CRSPR/Cas9 vector (Additional file 1: Fig. S1A, B) (Zhou et al. 2022). Two homozygous mutants (KO1 and KO3) were identified by sequencing (Additional file 1: Fig. S1C). KO1 contains an A insertion and KO3 contains a G deletion in the CDS, which caused a frameshift mutation (Additional file 1: Fig. S1D). Two independent STK-OE transgenic lines (OE3 and OE5; Additional file 1: Fig. S2) and two STK-KO lines (KO1 and KO3; Additional file 1: Fig. S1) of STK were selected for further analysis.
Under salt stress using 140 mM NaCl for 12 d and recovery 4 d, STK-OE lines showed greater resistance than wild-type plants (Fig. 3A). Almost 70% to 73% of the STK-OE plants survived, while only 43% of the wild-type plants survived under this treatment (Fig. 3B). As shown in Fig. 3C–E, compared with wild-type plants, STK-OE plants accumulated more chlorophyll and less malondialdehyde (MDA) and relative ion leakage. In contrast, when wild-type and STK-KO plants were subjected to 140 mM NaCl for 12 d, 41% of the wild-type plants recovered 4 d after watering was restored, but only 14% to 18% of the STK-KO plants recovered (Fig. 4A, B). Further physiological analyses revealed that loss of function of STK reduced the content of chlorophyll and increased levels of MDA and relative ion leakage in leaves (Fig. 4C–E). Previous studies showed that the ability to avoid Na+ excessive accumulation and keep a low Na+/K+ ratio contributes to salt tolerance in plants (Zhu 2003). We further measured Na+ and K+ contents in the shoot of wild-type, STK-OE and STK-KO plants. Less Na+ but higher K+ content was detected in STK-OE plants under salt stress, resulting in a lower Na+/K+ ratio in the shoot of STK-OE plants than wild-type (Fig. 3F–H). In contrast, compared with the wild-type plants, the STK-KO plants accumulated more Na+ and less K+, leading to a higher Na+/K+ ratio under salt stress (Fig. 4F–H). These findings indicated that STK confers tolerance to salt stress via relief of the membrane damage caused by salt stress and avoidance of Na+ excessive accumulation in plants.
Overexpression of STK Enhances ABA Sensitivity
Recent study revealed that STK was up-regulated under ABA treatment (Gao and Xue 2012). As shown previously, the expression of STK was also induced by ABA (Fig. 1A). Consequently, we determined whether STK-OE plants and STK-KO plants showed differences in performance under ABA treatment compared with wild-type plants. The ABA sensitivity of rice plants was investigated at the germination stage. Seeds of STK-OE plants, STK-KO plants, and the wild-type were germinated in one-half-strength Murashige and Skoog (1/2MS) medium containing ABA with a gradient of concentrations (0, 1, 3 and 6 μM). The germination rate of the STK-OE plants and STK-KO plants was identical to the wild-type at 0 μM ABA treatment (Fig. 5A, B, D, E). However, compared with the wild-type, the germination rate was reduced in STK-OE plants and increased in STK-KO plants under 1, 3 and 6 μM ABA treatment, respectively (Fig. 5B, E). We also investigated the ABA sensitivity of rice plants at the post-germination stage. Compared with the wild-type, the shoot length was significantly shorter in STK-OE plants but markedly longer in STK-KO plants under 1, 3 and 6 μM ABA treatment, respectively (Fig. 5C, F). No significant difference in shoot length was observed among STK-OE, STK-KO and wild-type under without ABA treatment (Fig. 5C, F). These results suggested that STK is a positive regulator of ABA signaling in rice.
STK Positively Regulates Oxidative Tolerance in Rice
Salt stress usually cause damage in plants via oxidative stress involving the generation of reactive oxygen species (ROS), such as H2O2 (Chen 2021). As shown previously, the expression of STK was induced by H2O2 (Fig. 1A) and STK-OE plants improved the salt tolerance of transgenic rice plants, we examined whether STK functions in salt tolerance through detoxification of ROS. Three-leaf stage seedlings of STK-OE plants, STK-KO plants and wild-type were treated with 100 mM H2O2. After 2 d H2O2 treatment, severe necrosis and leaf rolling were observed in STK-KO plants (Fig. 6A). Moreover, wild-type plants exhibited stronger leaf rolling and more chlorosis than STK-OE plants after H2O2 treatment. Then, the chlorophyll content in these seedlings were determined. Under oxidative stress, STK-OE plants showed higher chlorophyll content compared with wild-type, whereas the STK-KO plants exhibited the opposite results (Fig. 6B). Nevertheless, the chlorophyll content was not significantly different among STK-OE plants, STK-KO plants and wild-type under normal growth conditions. The accumulation of H2O2 was further determined by 3,3′-diaminobenzidine (DAB) staining. After 1d H2O2 treatment, the intensity of DAB staining was less in leaves of the STK-OE plants than in those of wild-type plants (Fig. 6C). The intensity of DAB staining was greater in the leaves of STK-KO plants than in those of wild-type plants. Thus, the STK-OE plants and the STK-KO plants showed opposite trends in ROS accumulation. Under normal growth conditions, no obvious staining was observed in STK-OE, STK-KO and wild-type. These results suggest that STK is involved in the elimination of H2O2 produced under oxidative stress.
Identification of Stress-Related Genes Controlled by STK
To understand the mechanisms and downstream components of the STK-mediated salt-stress signaling pathways, transcriptome deep sequencing (RNA-seq) analysis was performed using leaves from wild-type and STK-OE (OE5) rice plants under salt stress conditions. The threshold for significantly differentially expressed genes (DEGs) was set at a (log2 scale)-fold change (FC) value of > 1 or < − 1 and adjusted p-value < 0.05. Using these criteria, we identified 1076 DEGs (677 upregulated and 399 downregulated) in OE5 compared to wild-type. These DEGs altered by salt stress are shown in the volcano plots, which illustrate the asymmetry between upregulated (red) and downregulated (green) DEGs (Fig. 7A). Gene ontology (GO) enrichment analysis showed that the DEGs affected by the overexpression of STK were enriched mainly in glutathione transferase activity and glutathione metabolic process (Fig. 7B). The GST (Glutathione S-transferase) genes (LOC_Os03g17480, LOC_Os10g38140 and LOC_Os10g38710) detected in the RNA-seq were further investigated for their expression changes in the STK-OE, STK-KO and wild-type under normal and salt stress conditions by RT-qPCR. Under normal conditions, no significant differences in expression levels were observed among the STK-OE, STK-KO and wild-type. On the contrary, compared with the wild-type plants, the expression levels of the GST genes were significantly increased in the STK-OE plants but markedly lower in STK-KO plants under salt stress conditions (Fig. 7C–E). GST is the ubiquitous enzymes that play a key role in cellular detoxification (You et al. 2014). Thus, STK maybe involved in the detoxification and ROS scavenging mechanisms of rice by GST under salt stress. To check this hypothesis, GST activities and ROS levels in the salt-stressed and unstressed leaves of STK-OE, STK-KO and wild-type were measured. Compared with the wild-type, GST activity was significantly higher in the STK-OE plants but markedly lower in STK-KO plants under salt stress conditions (Fig. 7F). Similarly, the H2O2 content was also significantly lower in STK-OE plants but markedly higher in STK-KO plants under salt stress conditions (Fig. 7G). Under normal growth conditions, no significant difference of GST activity and H2O2 content were detected among the STK-OE, STK-KO and wild-type plants, respectively. These results indicated that STK was involved in the detoxification and ROS scavenging mechanisms of rice under salt stress conditions, which may have contributed to their improved tolerance to salt stress.
Previously identified several stress-responsive genes involved in various pathways, including OsABAR1, Os3BGlu6, OSBZ8 and OsSIK1 (Mukherjee et al. 2006; Ouyang et al. 2010; Wang et al. 2020a, b; Zheng et al. 2020) were up-regulated at 48 h of salt stress treatment (Additional file 2: Table S2). Expression levels of these genes under normal or salt conditions was further verified by RT-qPCR in STK-OE, STK-KO and wild-type plants. In agreement with the analysis of DEGs, the expression of these genes was up-regulated and downregulated in STK-OE plants and STK-KO plants under salt stress condition, respectively (Additional file 1: Fig. S3). These results suggested that STK may promote salt tolerance through activated stress-related genes.
Excessive accumulation of ROS is a frequent event in plants suffering from diverse abiotic stresses, including drought, high salinity, and extreme temperatures, and can cause damage to plants (Mittler 2002; Miller et al. 2010). Overaccumulation of ROS can damage DNA, proteins and carbohydrates, resulting in cell death (Mittler et al. 2004). ROS also cause MDA production, cell membrane damage and lipid peroxidation. Previous studies showed that the plant response to abiotic stresses occurs through the regulation of ROS metabolism (Schmidt et al. 2013; Li et al. 2014; Fang et al. 2015; Zhou et al. 2018; Zhao et al. 2019). For example, Salt Intolerance 1 (SIT1), a rice L-type LecRLK, could be activated by salt and mediate salt sensitivity by activation of MPK3/MPK6 leading to higher ethylene production and downstream ROS accumulation (Li et al. 2014; Zhao et al. 2019). Overexpression of a NAC protein, SNAC3, improves heat and drought tolerance by modulating ROS homeostasis through regulation of the expression of genes encoding ROS production and ROS-scavenging enzymes (Fang et al. 2015). STRK1 (Salt Tolerance Receptor-like Cytoplasmic Kinase 1), which activates CatC activity through phosphorylation at Tyr-210 of CatC to regulate H2O2 homeostasis and improve salt tolerance (Zhou et al. 2018). In this study, induction of STK upon H2O2 treatment indicated that it functions in oxidative stress adaptation (Fig. 1A). Reduced MDA levels in the STK-OE plants under salt stress treatment (Fig. 3D) implied that less oxidative damage occurred in the cells of the STK-OE plants during the salt stress conditions. Relevant to this finding, STK-OE plants showed better growth under oxidative stress caused by H2O2 (Fig. 6A, B). In STK-OE plants, one ROS, H2O2, was present in low levels under salt stress, as revealed by DAB staining (Fig. 6C). To the contrary, STK-KO plants showed reduced tolerance to oxidative stress. These results suggested that the function of STK in salt tolerance may be associated with the regulation of antioxidation ability.
To maintain cellular redox homeostasis and scavenge excess stress-induced ROS, plants have evolved antioxidation systems comprising nonenzymatic and enzymatic antioxidants (Miller et al. 2010). The maintenance of high activity of various antioxidant enzymes, such as SOD, CAT, GST, GR, and glutathione peroxidase, to scavenge the toxic ROS has been linked to improved tolerance of plants to abiotic stresses (Apel and Hirt 2004; Mittler 2002; Miller et al. 2010). GST plays an important role in the reduction of organic hydroperoxides formed during oxidative stress using the tripeptide glutathione (GSH) as a cosubstrate or coenzyme (Dixon et al. 2002). Overexpression of a GST gene from Escherichia coli in transgenic tobacco enhanced salt and cold tolerance (Le Martret et al. 2011). The ospp18 mutant showed increased drought and oxidative sensitivity through reduced the expression of GST genes and the activity of GST which significantly contribute to excessive H2O2 accumulation (You et al. 2014). Overexpression of a GST gene (ThGSTZ1) from Tamarix hispida improves drought and salinity tolerance by enhancing the ability to scavenge ROS (Yang et al. 2014). In the present study, the RNA-seq assay revealed that under salt conditions, STK increased the expression of GST genes (LOC_Os03g17480, LOC_Os10g38140 and LOC_Os10g38710) and further confirmed by RT-qPCR. Under salt stress, the expression of GST genes and the activity of GST were found to be higher in the STK-OE plants than in wild-type, and STK-KO plants showed the reverse results (Fig. 7C–F). The low level of H2O2 in STK-OE plants was most likely a result of high levels of the enzyme GST (Fig. 7G). These results suggested that the enhanced activity of ROS scavenging enzymes may significantly contribute to scavenge H2O2 accumulation and reduce oxidative damage in STK-OE plants, which is associated with the increased tolerance of the plants to salt stress.
High concentrations of salt in plant cells can induce ionic stress. Ionic stress is caused by sodium (Na+) and chlorine (Cl−) accumulation in plant cells, eventually resulting in premature leaf senescence and even plant death (Liu et al. 2021). The toxicity of Na+ mainly manifested in its inhibitory effect on enzyme activities, negatively affecting metabolism, including the Calvin cycle and other pathways (Cheeseman 2013; Wu et al. 2018). Potassium (K+), a vital ion for plant growth, plays a critical role in salt tolerance regulation as the cytosolic Na+/K+ ratio appears to determine plant salt tolerance (Shabala and Cuin 2008; Wu et al. 2018). Na+ and K+ are imported into the plant cell using the same set of transporters, and the two cations compete with each other (Greenway and Munns 1980). In glycophytes, excessive Na+ commonly results in K+ deficiency under salt stress (Yang and Guo 2018). Therefore, keeping a low Na+/K+ ratio is an important mechanism used by plants to adapt to salt stress. OsSOS1, a rice plasma membrane Na+/H+ antiporter, excludes Na+ from the shoot, promoting a lower cellular Na+/K+ ratio and improving salt tolerance (Martínez-Atienza et al. 2007; El Mahi et al. 2019). The K+ channel OsAKT1 and OsKAT1 are involved in salt tolerance via maintaining a low Na+/K+ ratio owing to the increase of K+ uptake (Fuchs et al. 2005; Obata et al. 2007). Salt Intolerance 2 (SIT2), a rice LecRLK, was identified for its role in salinity stress tolerance potentially via its function in Na+ extrusion by regulating SOS pathway (Sun et al. 2020). Our results demonstrate that less Na+ but higher K+ content was detected in STK-OE plants under salt stress, resulting in a lower Na+/K+ ratio in the shoot of STK-OE plants than wild-type (Figs. 3H, 4H). These results indicated that STK may be involved in the transport of Na+ and K+, regulating rice tolerance to salt stress by avoiding Na+ accumulation and promoting K+ uptake.
A better understanding of the mechanisms underlying salt stress sensing and signal transduction will help researchers design novel approaches to increase plant tolerance to salt stress. In response to high salinity, the initial signal activates the production of compounds that trigger activity in many metabolic and developmental pathways (Li et al. 2019). One of the most important compounds is abscisic acid (ABA; Finkelstein and Gibson 2002). ABA plays an important role in regulating the response of plants to environmental stress (Sah et al. 2016; Vishwakarma et al. 2017). Salt stress can trigger the ABA-dependent signaling pathway in plants (Zhu 2002). GmWRKY16 as a WRKY transcription factor may promote tolerance to drought and salt stresses through an ABA-mediated pathway (Ma et al. 2019). Overexpression of OsSRK1 enhances salt tolerance probably by regulating stress-related genes and ABA pathway (Zhou et al. 2020). OsNAC45 positively regulates ABA signal pathway and is required for salt tolerance in rice (Zhang et al. 2020). Overexpression of PpSnRK1α in tomato is beneficial for enhancing salt tolerance by regulating ABA signaling pathway and reactive oxygen metabolism (Wang et al. 2020a, b). ABA plays important roles in many aspects of plant development and physiological processes, such as seed maturation, germination, seedling growth and stomatal movement, is the central regulator of abiotic stress resistance in plants (Finkelstein et al. 2008; Kim et al. 2010). In the present study, an expression pattern analysis showed that STK expression was strongly induced by ABA (Fig. 1A), indicating that STK is involved in ABA signaling pathway. In the absence of ABA, the germination rates of the WT, STK-OE and STK-KO plants were similar, and no significant difference in shoot length was observed among wild-type, STK-OE and STK-KO plants after germinating (Fig. 5). In the presence of ABA, the germination and growth of the STK-OE plants were severely inhibited compared with the wild-type. By contrast, the STK-KO plants germinated more quickly and grew faster than the WT. This phenomenon may be in part explained by the RNA-seq data. OsSAPK10 is a positive regulator of the ABA signal pathway in seed germination and early seedling growth (Wang et al. 2020a, b). OsSAPK10 overexpression lines displayed more severe ABA-mediated repression on seed germination and seedling growth (Wang et al. 2020a, b). The RNA-seq showed that the expression of OsSAPK10 in STK-OE plants is significantly higher than wild-type plants under salt stress conditions (Additional file 2: Table S2), which is further confirmed by RT-qPCR (Additional file 1: Fig. S4). These results showed that STK confers tolerance to salt stress through an ABA-dependent signaling pathway.
To illustrate the molecular mechanism underlying the enhanced salt tolerance of STK-OE plants, RNA-seq was performed. Under salt conditions, 677 genes were upregulated and 399 genes were downregulated (Fig. 7A). GO annotation analysis showed that these genes mainly belong to following categories: glutathione transferase activity and glutathione metabolic process (Fig. 7B). Many abiotic stress-related genes were regulated in STK-OE transgenic plants compared with WT (Additional file 2: Table S2). After salt treatment, the relative expression levels of OsABAR1, Os3BGlu6, OSBZ8 and OsSIK1 increased more obviously in STK-OE plants compared with wild-type. The OsABAR1-overexpressing plants showed increased tolerance to drought and salinity, whereas the OsABAR1 knockout plants had the opposite phenotypes (Zheng et al. 2020). Overexpression of OsSIK1 increased tolerance of rice plants to salt and drought stresses, and the knockout mutants sik1-1 and sik1-2, as well as RNA interference (RNAi) plants, are sensitive to drought and salt stresses (Ouyang et al. 2010). Os3BGlu6, a chloroplast localized β-glucosidase, significantly affected cellular ABA pools, and playing an important role in drought stress and photosynthesis under normal growth and drought conditions (Wang et al. 2020a, b). OSBZ8, a bZIP class of ABRE-binding transcription factor, is shown to be highly expressive in response to tolerance against salt stress cultivars as compared to those that are sensitive to salt stress (Mukherjee et al. 2006). It is worth noting that previous reports have shown that OSBZ8 is a marker for ABA-responsive genes (Miyoshi et al. 1999; Nakagawa et al. 1996). Therefore, the higher expression levels of these stress-related genes in STK-OE transgenic plants could contributed to increased tolerance of the transgenic plants to salt stress.
A novel rice RLCK gene, STK, which was rapidly induced by ABA as a prominent regulator of the response to salt stress while causing rice to be sensitive to exogenous ABA. Overexpression of STK improved tolerance to salt and oxidative stresses than wild-type. On the contrary, the knockout mutants STK-KO were sensitive to salt and oxidative stresses. The activity of GST was enhanced significantly in STK-OE plants. Also, the accumulation of H2O2 in leaves of STK-OE plants was much less than that of the STK-KO plants and wild-type. STK improved salt tolerance by inducing antioxidant defense and associated with the ABA signaling pathway in rice. In summary, STK enhanced ROS scavenging capacity by regulating the expression of GST genes, thereby increasing the salt tolerance of rice.
Plant Materials and Growth Conditions
Rice cultivars Kitaake (Oryza sativa ssp. japonica) were used for RT-qPCR analysis under various stresses and phytohormone treatments, and Kitaake was used for all transgenic experiments. For gene expression analysis, rice seeds were first sterilized with 15% NaClO, germinated at 30 °C for 3 days, and then grown in hydroponic culture solution (Ren et al. 2005) with a 14-h light/10-h dark photoperiod, a 28 °C (light)/25 ℃ (dark) temperature range, 350 μmol m−2 s−2 light intensity, and 85% relative humidity. Rice seedlings at the three-leaf stage were immersed with their roots in NaCl (150 mM), PEG (20%), H2O2 (1%), cold (4 °C), ABA (100 μM), GA (100 μM) for the indicated times. Their leaves were harvested at 0, 1, 3, 6, 12, and 24 h after the beginning of treatments. All harvested leaf samples were rapidly frozen in liquid nitrogen and stored at − 80 °C for RNA extraction.
RNA Extraction and RT-qPCR Analysis
Total RNA was extracted from rice leaves using TRIzol reagent (Sangon Biotech, Shanghai, China) and treated with gDNA Eraser to eliminate any DNA contamination. The quality of the total RNA was evaluated using a NanoDrop 2000 (Thermo Fisher Scientific, Shanghai, China). First-stand cDNAs were synthesized using PrimeScript RT reagent kit (Takara, Beijing, China) following the manufacturer’s instruction. RT-qPCR was performed with ABI 7500 Real Time PCR System (Applied Biosystems, Foster City, USA) using TB Green Premix Ex Taq II (Takara, Beijing, China) to monitor dsDNA synthesis, according to the manufacturer’ s instruction. The primers used for RT-qPCR are listed in Additional file 2: Table S1. The expression of rice Actin gene (LOC_Os03g50885) was used as an internal control. The relative expression levels were measured as previously described (Zhou et al. 2022).
Plasmid Constructions and Generation of STK-OE and STK-KO Plants
To generate the STK-OE plants, the coding region of the STK was cloned in the BamHI and EcoRI sites of the pCAMBIA1300-YFP vector using a specific primer (Additional file 2: Table S1). To generate the STK-KO plants, the target sites of STK were selected by the CRISPR-Plant Web server (Xie et al. 2014) and were constructed into the CRISPR/Cas9 vector as described previously (Zhou et al. 2022). The plasmids were introduced into Agrobacterium tumefaciens strain EHA105. Agrobacterium-mediated transformation of embryogenic calli generated from the japonica rice cultivar Kitaake was performed as previously described (Zhou et al. 2015). To determine whether genome editing of STK was successful, genomic DNA extracted from regenerated transgenic plants was amplified by PCR with the primers listed in Additional file 2: Table S1, followed by sequencing of the PCR products.
For salt stress tolerance assays, uniformly germinated seeds of WT, STK-OE, and STK-KO lines were grown in hydroponic culture solution. 15-d-old plants (40 plants each genotype) were transferred to a hydroponic culture solution containing 140 mM NaCl, and 12 d NaCl treatments were applied for STK-KO and STK-OE plants as well as their corresponding wild-type, respectively. The 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 d, and the color of the leaves was observed. If the leaves were green, they were counted as surviving plants, if the leaves were withered brown, they were counted as dead plants, and finally the survival rate was counted.
For testing the ABA sensitivity of transgenic plants at the germination stage, seeds of WT, STK-OE, and STK-KO lines (30 seeds each, three repeats) were sterilized with 15% NaClO and cultured on hydroponic culture solution containing a gradient concentration of ABA (0, 1, 3, and 6 μM). After 5 d treatment, the number of germinated seeds with emerged coleoptiles and germination rate were calculated.
For the H2O2 treatment, three-week-old plants (30 plants each genotype) were irrigated with hydroponic culture solution containing 100 mM H2O2 solution. After 1 d, the leaves were stained with DAB following the previously described method (You et al. 2014). After 2 d, the leaves were photographed.
The total chlorophyll content was determined according to the method described previously (Zhou et al. 2018). About 100 mg of leaf blades were ground in liquid nitrogen and then immersed in the extract solution (80% acetone) under darkness overnight at 4 °C. The absorbance of the extracts was read at 663 and 645 nm with a spectrophotometer (BioTek). The total chlorophyll content was calculated and expressed as mg g−1 FW. The content of MDA was measured as previously described (Ouyang et al. 2010) with slight modification. About 100 mg of leaf blades were homogenized in 1 mL of 10% trichloroacetic acid (TCA). The homogenate was centrifuged at 5000 g for 10 min at 4 °C. To 800 μl of the supernatant was added 800 μl of thiobarbituric acid (TBA) (made in 10% TCA). The mixture was boiled for 15 min and centrifuged at 12,000g at 4 °C for 5 min. The absorbance of the supernatant was read at 450, 532, and 600 nm. The MDA content was determined using the extinction coefficient of 155 (nmol/L/cm) and expressed as mmol g−1 FW. The relative ion leakage was assayed according to the method described previously (Cao et al. 2007). The activity of glutathione S-transferase (GST) and the concentration of H2O2 were measured as described by using rice leaf blades using commercial kits (D799774-0100, Sangon Biotech, Shanghai, China; D799612-0100, Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. One unit of GST activity was defined as the amount of enzyme depleting 1 μM GSH in 1 min. H2O2 was detected by DAB staining as described previously (Zhou et al. 2018).
Subcellular Localization of STK
For detection of the subcellular localization of STK, the coding sequences of the STK were fused to yellow fluorescent protein (YFP) reporter coding sequences and subcloned into the pCAMBIA1300-YFP vector, in which the YFP-coding sequence was fused in frame to the 3′ end of the STK gene sequence. The plasmid was transformed into isolated rice protoplasts using polyethylene glycol (PEG)-mediated transformation methods (Zhou et al. 2018).
Protoplasts were then transferred into multi-well plates and cultured in the dark at room temperature for 6–16 h. After incubation, green fluorescence signals from transfected protoplasts were observed using a confocal laser scanning microscope (Olympus FV1000) with 488 nm excitation and 505–530 nm emission wavelengths.
RNA Sequencing (RNA-seq) Analysis
For RNA-seq, three-week-old seedlings of WT and STK-OE plants were treated with 150 mM NaCl for 48 h. Total RNA was extracted from leaves of three-week-old WT and STK-OE plants with the TRIzol reagent (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. Library preparation and Illumina sequencing was performed at Novogene from three biological repeats of samples. Raw reads were trimmed by cutadapt to get clean reads. The trimmed reads were then mapped onto the reference rice genome from RAP-DB (https://rapdb.dna.affrc.go.jp). DEGs responding to salt stress were defined by a ≥ twofold expression change and p-value < 0.05, and genes which were up regulated between WT and OE plants were subjected to further gene ontology (GO) enrichment analysis using the ClusterProfiler (Yu 2018). The expression levels of selected DEGs were verified by RT-qPCR.
Availability of Data and Materials
All data supporting the findings of this study are available from the corresponding author on reasonable request.
Receptor-like cytoplasmic kinase
Yellow fluorescent protein
Reactive oxygen species
Reverse transcription quantitative PCR
Differentially expressed gene
False discovery rate
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We are very grateful to Prof. Yaoguang Liu from South China Agricultural University for kindly providing us with the pYLCRISPR/Cas9-MH vector.
This work was supported by the National Natural Science Foundation of China (No. 31901516) and the special funds for the construction of innovative provinces in Hunan (No. 2020RC3085).
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Identification of CRISPR/Cas9-STK rice mutant plants. Fig. S2: Characterization of STK overexpressionplants. Fig. S3: Relative expression levels of four previously known stress-related genes up-regulated in STK-OE pants and down-regulated in STK-KO plants at 48 h under salt stress condition in rice seedlings. Fig. S4: Relative expression levels of OsSAPK10 in leaves of wild-type, STK-OE and STK-KO plants under salt stress condition.
List of primers used in this study. Table S2: Differential expression data of stress-related in the STK-OE plants.
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Zhou, Y., Zhang, Z., Zhao, X. et al. Receptor-Like Cytoplasmic Kinase STK Confers Salt Tolerance in Rice. Rice 16, 21 (2023). https://doi.org/10.1186/s12284-023-00637-0