OsWRKY76 positively regulates drought stress via OsbHLH148-mediated jasmonate signaling in rice

doi:10.21203/rs.3.rs-2501309/v1

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OsWRKY76 positively regulates drought stress via OsbHLH148-mediated jasmonate signaling in rice

https://doi.org/10.21203/rs.3.rs-2501309/v1

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Drought stress is a major environmental threat that limits plant growth and crop productivity. Therefore, it is necessary to uncover the molecular mechanisms behind drought tolerance in crops. Here, OsWRKY76 positively regulated drought stress in rice. OsWRKY76 expression was induced by PEG treatment, dehydration stress, and exogenous MeJA rather than by no treatment. Notably, OsWRKY76 knockout weakened drought tolerance at the seedling stage and decreased MeJA sensitivity. OsJAZ12 was significantly induced by drought stress, and its expression was significantly higher in OsWRKY76-knockout mutants than in wild-type ZH11 under drought stress. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that OsWRKY76 interacted with OsJAZ12. OsWRKY76 weakened the interaction between OsbHLH148 and OsJAZ12 in yeast cells. The OsJAZ12 protein repressed the transactivation activity of OsbHLH148, and this repression was partly restored by OsWRKY76 in rice protoplasts. Moreover, OsDREB1E expression was lower in OsWRKY76-knockout mutants than in wild-type ZH11 under drought stress, but it was upregulated under normal growth conditions. Yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays showed that OsWRKY76 and OsbHLH148 bound directly to the OsDREB1E promoter and activated OsDREB1E expression in response to drought stress. These results suggest that OsWRKY76 confers drought tolerance through OsbHLH148-mediated jasmonate signaling in rice, offering a new clue to uncover the mechanisms behind drought tolerance.

drought stress

jasmonate signaling

OsWRKY76

OsbHLH148

rice (Oryza sativa)

Drought is one of the most serious environmental stressors in crops (Sinclair, 2011). Rice (Oryza sativa) is a staple crop that feeds over half of the world’s population (Seck et al., 2012). Since the rice cultivation process relies heavily on fresh water resources, shortages of fresh water availability and adverse climate change threaten rice growth and productivity (Hu and Xiong, 2014). This necessitates the identification of drought-resistance genes to create drought-tolerant rice varieties.

Plants have developed complex strategies against drought stress, such as modifying root growth, regulating osmotic substances to maintain intracellular water content, and scavenging excessive reactive oxygen species (ROS) to prevent ROS toxicity caused by drought stress (Gupta et al., 2020; Rodrigues et al., 2019). Numerous rice genes have been reported to play vital roles in drought stress. HDG11 confers drought tolerance and increases grain yield in rice by improving levels of soluble sugar and proline, and ROS-scavenging enzyme activities (Yu et al., 2013). OsTF1L enhances stomatal closure and lignin biosynthesis and positively regulates drought tolerance in rice (Bang et al., 2019). AtNOG1-1 or AtNOG1-2 overexpression in rice increases drought tolerance without affecting grain yield, whereas OsNOG1-RNAi rice plants are more sensitive to drought stress than wild-type plants (Pant et al., 2022). Moreover, the primary signal caused by drought is a water deficit, leading to osmotic stress. Under osmotic stress created by PEG treatment, the shoots and roots of OsWIH2-overexpressing rice plants are longer than those of wild-type rice plants (Gu et al., 2021). OsWIH2-overexpressing rice plants have a significantly higher survival rate and a lower water loss rate compared with wild-type rice plants (Gu et al., 2021). Glutathione peroxidase 1 (GPX1) acts as a redox transducer and confers osmotic stress tolerance in rice by interacting with bZIP68 and promoting the oxidation of bZIP68 (Zhou et al., 2022).

Phytohormones, such as jasmonates (JAs), play a crucial role in responding to stress (Y. Wang et al., 2021). JAs are a general term for JA and its active derivatives, which comprise its isoleucine conjugate (JA-Ile) and its methyl ester (MeJA). JAs participate in plant growth and development, such as stomatal opening, root formation, and leaf senescence (H. Huang et al., 2017). Furthermore, a number of recent reports have revealed that JAs can increase plant stress tolerance through the JA signaling pathway. The core component of the JA signaling pathway is JA-Ile (Marquis et al., 2020). Under normal growth conditions, intracellular JA-Ile levels are very low, and JA signaling occurs during the inhibition state. JAZ proteins that feature the jasmonate zinc-finger inflorescence meristem domain interact with various transcription factors (TFs) participating in the JA signaling pathway. JAZ proteins can suppress this TF activity by recruiting the co-repressor topless (TPL) and the adaptor protein of JAZ (NINJA) to form a JAZ–NINJA–TPL complex or histone-modifying proteins HDA6 and HDA19 (Ke et al., 2015; Pauwels et al., 2010; Zhu et al., 2011). When plants suffer from stress, JA-Ile levels are elevated in the cytosol, and JA-Ile is transported into the nucleus and promotes the interaction of JAZ proteins with the F-box protein COI1 within a kinetochore protein 1–cullin 1–F-box protein COI1 (SCF^COI1) complex, which leads to JAZ protein degradation via the 26S proteasome and activation of various JA- and stress-responsive genes (Mosblech et al., 2011; Wasternack and Song, 2017; Wasternack and Hause, 2013).

Basic helix–loop–helix (bHLH) TFs play a vital role in the JA signaling pathway. In Arabidopsis thaliana, the expression of the bHLH family MYC2 is rapidly upregulated by JA (Lorenzo et al., 2004). MYC2 and its paralogs MYC3 and MYC4 interact with JAZ proteins, are directly repressed by JAZ proteins, and positively regulate JA-dependent plant defense (Fernández-Calvo et al., 2011; Schweizer et al., 2013). OsbHLH148, whose expression is rapidly increased under dehydration stress and MeJA treatment, confers drought tolerance in rice and regulates the JA signaling pathway (Seo et al., 2011). OsbHLH148 interacts with some OsJAZ proteins, including OsJAZ7, OsJAZ8, OsJAZ11, and OsJAZ12, but OsJAZ12 interacts with OsCOI1 only in the presence of coronatine, causing OsJAZ12 degradation (Seo et al., 2011). In addition, OsJAZ1 attenuates drought tolerance in rice and negatively regulates the JA signaling pathway (Fu et al., 2017).

Studies have identified that some WRKY TFs participate in drought stress in rice. OsWRKY47 positively regulates tolerance to water deficit stress (Raineri et al., 2015). OsWRKY11 activates the transcription of the drought-responsive gene RAB21 and enhances drought stress tolerance (Lee et al., 2018). However, OsWRKY55 positively regulates OsAP2-39 expression and negatively regulates drought stress (K. Huang et al., 2021). OsWRKY5 represses ABA-induced stomatal closure and weakens drought tolerance in rice (Lim et al., 2022). Furthermore, WRKY proteins interact with other proteins, such as kinases and TFs, regulate their downstream target genes, and then form complex regulatory networks. OsMPK3, OsMPK4, and OsMPK7 interact with OsWRKY30 and phosphorylate OsWRKY30, which is essential for OsWRKY30 to confer drought tolerance in rice (Shen et al., 2012). OsWRKY87 interacts with SAPK10 and is phosphorylated by it. OsWRKY87 can activate ABF1 transcription and enhance rice drought tolerance (Yan et al., 2021).

Our previous data showed that OsWRKY76 interacts with OsbHLH148 and coordinately plays a positive role in cold tolerance in rice (Zhang et al., 2022). Moreover, OsbHLH148 confers drought tolerance in rice and regulates the JA signaling pathway (Seo et al., 2011). However, whether and how OsWRKY76 mediates the drought stress response and the JA signaling pathway in rice is unknown. In this study, we proposed that OsWRKY76 plays a crucial role in regulating drought stress and jasmonate signaling in rice. OsWRKY76 knockout weakened drought tolerance at the seedling stage and decreased MeJA sensitivity. OsWRKY76 interacted with OsJAZ proteins and activated OsbHLH148 transcriptional activity by interfering with the association of OsJAZ12 with OsbHLH148. OsWRKY76 and OsbHLH148 directly activated the transcription of OsDREB1E under drought stress.

Expression of the OsWRKY76 gene in response to drought stress and MeJA treatment

To investigate the responses of OsWRKY76 to drought stress and MeJA treatment, we examined the expression of OsWRKY76 under PEG treatment, dehydration stress, and exogenous MeJA in two-week-old seedlings of wild-type rice using reverse transcription quantitative PCR (RT-qPCR) analysis. OsWRKY76 expression was induced rapidly within 30 min of exposure to dehydration stress, showing an 8-fold increase over that in control plants and then showing a gradual descent over a period of 6 h (Fig. 1A). OsWRKY76 was strongly upregulated by PEG treatment and exogenous MeJA (Fig. 1B,C). By contrast, OsWRKY76 expression showed no significant differences under no treatment (Fig. 1D). These results suggest that OsWRKY76 can respond to drought stress and MeJA treatment and may play an important role in the drought tolerance of rice and the JA signaling pathway.

Oswrky76 Confers Drought Tolerance In Rice

To test the effect of OsWRKY76 on the drought tolerance of rice, OsWRKY76-knockout mutants (w76-1 and w76-2) were obtained using a CRISPR/Cas9 genome editing approach in the japonica rice accession ‘ZH11’ background, resulting in frameshift mutations (Zhang et al., 2022). Analysis of drought stress tolerance showed no significant differences in two-week-old rice seedling growth between wild-type ZH11 and OsWRKY76-knockout mutants (w76-1 and w76-2) under normal conditions (Fig. 2A,F). By contrast, w76-1 and w76-2 plants treated with 20% PEG showed more severe wilting, chlorosis, electrolyte leakage, and water loss than wild-type ZH11 (Figs. 2B–D and S1), together with lower survival rates after 10 days of recovery (Fig. 2E). We further investigated drought stress conditions. When watering was stopped for 7 days and then resumed for 7 days, OsWRKY76-knockout mutants (w76-1 and w76-2) showed more severe wilting and lower survival rates than wild-type ZH11 (Fig. 2G,H). These observations suggest that loss of OsWRKY76 function renders rice more sensitive to drought stress than the wild type.

Drought stress triggers ROS accumulation in rice cells, and ROS are biomarkers of rice drought tolerance (Li et al., 2021). Using 3, 3’-diaminobenzidine (DAB) staining, we determined the presence of hydrogen peroxide (H₂O₂) in rice leaves. DAB staining was light in wild-type ZH11 and OsWRKY76-knockout mutants (w76-1 and w76-2) under normal conditions (Fig. 2I). However, DAB staining was stronger in OsWRKY76-knockout mutants (w76-1 and w76-2) than in wild-type ZH11 after 6 h of dehydration treatment (Fig. 2I). These leaf phenotypes demonstrated that more H₂O₂ accumulated in OsWRKY76-knockout mutants (w76-1 and w76-2) than in wild-type ZH11 after drought stress. Taken together, these results suggest that OsWRKY76 positively regulates drought tolerance by repressing ROS accumulation.

Oswrky76 Positively Regulates Ja Signaling

Given that OsWRKY76 expression levels were induced by MeJA treatment, we hypothesized that OsWRKY76 is involved in the JA signaling pathway. To test whether OsWRKY76 knockout affected sensitivity to MeJA, we performed the effects of MeJA on seed germination or seedling development among the wild-type ZH11 plants and the OsWRKY76 mutants. As shown in Fig. 3A and B, OsWRKY76-knockout mutants (w76-1 and w76-2) showed hyposensitivity to exogenous MeJA compared to wild-type ZH11 during seed germination. Furthermore, MeJA treatment significantly inhibited shoot and root growth in wild-type ZH11 compared with OsWRKY76-knockout mutants (w76-1 and w76-2) (Fig. 3C–E). These results suggest that the loss of the OsWRKY76 function decreases MeJA sensitivity.

To further confirm this speculation, we performed RT-qPCR to examine the expression of JA signaling and drought-responsive genes under drought stress in OsWRKY76-knockout mutants. The expression of OsJAZ12 (related to JA signaling) was significantly induced at 3 h of exposure to 20% PEG treatment, showing a 15-fold increase over that in wild-type ZH11 (Fig. 3F). OsJAZ12 expression was notably higher in OsWRKY76-knockout mutants (w76-1 and w76-2) than in wild-type ZH11 under PEG treatment (Fig. 3F). OsDREB1E positively regulates tolerance to drought stress (Chen et al., 2008). OsDREB1E expression was lower in OsWRKY76-knockout mutants (w76-1 and w76-2) than in wild-type ZH11 subjected to drought stress, but it was upregulated under normal growth conditions (Fig. 3G). These results indicate that the JA signaling pathway is attenuated in the OsWRKY76-knockout mutants under drought stress. The expression level of the drought-tolerance gene OsDREB1E was also repressed under drought stress. Taken together, these results suggest that OsWRKY76 positively regulates JA signaling under drought stress.

Oswrky76 Interacts With Osjaz12

OsbHLH148 interacted with some OsJAZ proteins. The interaction between OsbHLH148 and OsJAZ12 was relatively strong, and the interaction with OsJAZ7, 8, and 11 was relatively weak (Seo et al., 2011). Our previous data showed that OsWRKY76 interacts with OsbHLH148 in vitro and in vivo (Zhang et al., 2022). To investigate whether OsWRKY76 interacted with these OsJAZ proteins, we performed a yeast two-hybrid assay. In yeast cells, OsWRKY76 interacted with OsJAZ8, 11, and 12 but not with OsJAZ7 (Fig. 4A). The interaction between OsWRKY76 and OsJAZ12 was further confirmed using a bimolecular fluorescence complementation (BiFC) assay in rice protoplasts (Fig. 4B). These results indicate that OsWRKY76, OsbHLH148, and OsJAZ12 form a heterotrimer.

Oswrky76 Competes With Osjaz12 To Bind Osbhlh148

To test the effect of OsWRKY76 on the interaction between OsbHLH148 and OsJAZ12, we performed a yeast three-hybrid assay. When co-transformed with pGADT7 (AD) and pbridge-OsbHLH148 (BD-b148), β-galactosidase activity was significantly higher than that of the empty vector, indicating that OsbHLH148 has transcriptional activation activity in yeast cells (Fig. 5A,B). When co-transformed with pGADT7-OsJAZ12 (AD-JAZ12) and BD-b148, β-galactosidase activity was significantly higher than when co-transformed with AD and BD-b148, and it was also significantly higher than when co-transformed with AD-JAZ12 and pbridge-OsbHLH148-OsWRKY76 (BD-b148-W76), indicating that OsWRKY76 weakens the interaction between OsbHLH148 and OsJAZ12 in yeast cells (Fig. 5A,B).

To analyze the effect of OsWRKY76 and OsJAZ12 proteins on OsbHLH148 transcriptional activity, we performed a Gal4-dependent chimeric transactivation assay. The full-length coding sequence (CDS) of OsbHLH148 was amplified and fused into the Gal4 DNA-binding domain (GD), creating the GD-b148 effector vector (Fig. 5C). The Gal4-dependent chimeric transactivation assay showed that the firefly luciferase (FLUC) activity of the GD-b148 vector was 6-fold higher than that of the GD vector, indicating that OsbHLH148 is a transcriptional activator in rice protoplasts (Fig. 5D). The OsWRKY76 protein activated the transactivation activity of OsbHLH148. However, the OsJAZ12 protein repressed the transactivation activity of OsbHLH148, and this repression was partly restored by OsWRKY76 (Fig. 5D). Taken together, these results indicate that OsWRKY76 activates OsbHLH148 transcriptional activity by interfering with the association of OsJAZ12 with OsbHLH148.

OsWRKY76 directly activates OsDREB1E expression in response to drought stress

OsDREB1E was activated in OsWRKY76-knockout mutants (w76-1 and w76-2) under normal conditions, but the activity showed repression under chilling stress (Fig. 4B). There were several W-box elements (TTGACT/C, WRKY recognition sites) and E-box elements (CANNTG, bHLH recognition sites) in the OsDREB1E promoter region using the PlantCARE database (Fig. 6A). Therefore, we investigated whether OsWRKY76 and OsbHLH148 bound to the OsDREB1E promoter to confer drought tolerance in rice. We performed a yeast one-hybrid assay, which revealed direct binding of OsWRKY76 and OsbHLH148 to the OsDREB1E promoter (Fig. 6B). To further verify that OsWRKY76 bound to the OsDREB1E promoter, we performed an electrophoretic mobility shift assay (EMSA). Strong mobility shift bands in the OsDREB1E probe containing the W-box element (Fig. 6C) were detected for the glutathione S-transferase (GST)-OsWRKY76 protein (lane 3) but not for the GST protein (lane 2) or the OsDREB1E probe alone (lane 1) (Fig. 6C). Strong mobility shift bands were reduced when increasing amounts (10× and 50×) of the unlabeled OsDREB1E competitor probe were added (lanes 3–5) (Fig. 6C). Thus, EMSA showed that OsWRKY76 bound directly to the OsDREB1E promoter containing the W-box element in vitro.

To investigate whether OsWRKY76 and OsbHLH148 regulated the transcription of OsDREB1E, we conducted dual-luciferase assays in rice protoplasts. The OsDREB1E promoter was fused with the FLUC reporter gene, and OsWRKY76 or OsbHLH148, driven by 35S, was the effector (Fig. 6D). There was no significant difference in the relative FLUC/RLUC activity (Renilla luciferase, RLUC) of co-transfection with the 35S:OsWRKY76 vector and with the empty vector under normal conditions (Fig. 6E). When the transformed protoplasts were treated with PEG treatment for 20 min, the transcription of OsDREB1E was strongly activated by OsWRKY76 (Fig. 6E). Under normal conditions and PEG treatment, the relative FLUC/RLUC activity co-transfected with 35S:OsbHLH148 was significantly higher than that of the empty vector, indicating that OsbHLH148 elevated OsDREB1E promoter activity in rice protoplasts (Fig. 6E). These results revealed that OsWRKY76 and OsbHLH148 directly activated the transcription of OsDREB1E under drought stress.

As global climate change continues to cause water scarcity, drought is emerging as a prevalent and important stress factor for crop production worldwide. The latest FAO data have shown that drought has cost the world $29 billion in crop losses over the past decade ( https://www.fao.org/ , accessed 24 May 2022). Rice serves as a staple crop for more than half of the world’s population (Seck et al., 2012). The identification of drought-tolerance genes can speed up the development of drought-tolerant rice varieties. A few WRKY TFs conferring drought tolerance in rice, including OsWRKY11, OsWRKY30, OsWRKY47, and OsWRKY87, have been identified (Lee et al., 2018; Shen et al., 2012; Raineri et al., 2015; Yan et al., 2021). In this study, OsWRKY76 was regulated by drought stress, and its knockout weakened drought tolerance at the seedling stage (Figs. 1 and 2).

Under drought stress, plants regulate adaptive and physiological responses through the metabolism of plant hormones and their signaling pathways. JA is a key phytohormone that regulates plant drought stress adaptation. In pepper (Capsicum annuum L.), CaCIPK3 increases drought and MeJA tolerance by enhancing the expression of JA-related genes (Ma et al., 2021). In Arabidopsis, JASMONIC ACID OXIDASES (JAO1–4) catalyze the specific oxidation of JA to 12OH-JA for attenuating JA-Ile formation and the JA signaling pathway (Smirnova et al., 2017). JAO2 mutants possess higher JA-Ile signaling and drought survival compared with wild-type plants, which is further enhanced by JAO3 and JAO4 deficiency (Marquis et al., 2022). AtNOG1-1 or AtNOG1-2 overexpression in rice positively regulates the expression of genes related to the JA signaling pathway and stomata regulation to prevent water loss (Pant et al., 2022). In this study, OsWRKY76 knockout decreased sensitivity to exogenous MeJA in seed germination and seedling development (Fig. 3A–E). JAZ proteins suppress various TF activities and negatively regulate the JA signaling pathway (Ke et al., 2015). OsJAZ12 was significantly induced by drought stress, and its expression was significantly higher in OsWRKY76-knockout mutants than in wild-type ZH11 under drought stress (Fig. 3F). Based on these findings, we suggest that OsWRKY76 positively regulates drought stress in a JA-dependent signaling pathway.

Under normal conditions, JAZ proteins interacted with bHLH148 and suppressed its activity, causing an inhibition state of JA signaling in rice (Seo et al., 2011; Ke et al., 2015). When rice plants suffer from drought stress, JA levels are elevated, and JA promotes the interaction of JAZ proteins with COI1 of the SCF^COI1 complex and leads to JAZ protein degradation, which releases bHLH148 and activates bHLH148 activity (Mosblech et al., 2011; Seo et al., 2011; Wasternack and Song, 2017). Our data showed that OsWRKY76 interacted with OsJAZ proteins and weakened the interaction between OsbHLH148 and OsJAZ12 in yeast cells (Figs. 4 and 5B). The OsJAZ12 protein repressed the transactivation activity of OsbHLH148, and this repression was partly restored by OsWRKY76 (Fig. 5D). JA OsJAZ12 expression was notably higher in OsWRKY76-knockout mutants than in wild-type ZH11 under drought treatment (Fig. 3F). Based on the results, we propose that OsWRKY76 plays an essential role in OsbHLH148-mediated JA signaling.

Induced stress responses allow plants to survive under a wider range of environmental conditions (VanWallendael et al., 2019). The extreme environment triggers overall and rapid reprogramming events in cells (Wu et al., 2021). Transcription of most DREB genes in various plant species exhibits relatively low basal expression levels but is rapidly induced by different environmental stressors (Agarwal et al., 2017). In rice, OsDREB1E could be significantly induced and positively regulate tolerance to drought stress (Chen et al., 2008). Here, OsDREB1E expression was lower in OsWRKY76-knockout mutants than in wild-type ZH11 under drought stress, but it was upregulated under normal growth conditions (Fig. 4G). OsWRKY76 bound directly to the OsDREB1E promoter and activated OsDREB1E expression in response to drought stress (Fig. 6). Therefore, OsWRKY76 activated OsDREB1E transcription under drought stress instead of under normal growth conditions, which avoids incurring costly fitness trade-offs and may act as a molecular link between drought tolerance and plant growth.

In the present study, our findings support a model in which OsWRKY76 acts as a key regulator in drought stress and jasmonate signaling in rice (Fig. 7). Under normal growth conditions, OsWRKY76 interacted with OsJAZ proteins and weakened the interaction between OsbHLH148 and OsJAZ12. Furthermore, the OsJAZ12 protein repressed the transactivation activity of OsbHLH148, and this repression was partly restored by OsWRKY76. Under drought stress, JA-Ile levels were elevated, leading to the degradation of JAZ proteins and the activation of OsWRKY76 and OsbHLH148 expressions. OsWRKY76 and OsbHLH148 directly activated the transcription of OsDREB1E and positively regulated drought tolerance.

Plant materials and growth conditions

The OsWRKY76-knockout mutants and OsbHLH148-knockout mutants in the background of rice variety ZH11 (Oryza sativa L. ssp. japonica) were obtained from a mutant library (BIOGLE, Changzhou, Jiangsu, China) using a CRISPR/Cas9 genome editing approach. All homozygous T₃ seedlings (w76-1, w76-2, b148-1, and b148-2) were used for this study. All rice seedlings were planted in a growth room with a cycle of 14 h light at 30°C and 10 h dark at 26°C.

Drought-tolerance Assays And Treatments

For analysis of drought stress tolerance, 2-week-old seedlings of wild-type ZH11 rice and OsWRKY76-knockout mutants (w76-1 and w76-2) were transferred to the hydroponic culture medium with or without 20% PEG 6000 for 10 days, and their survival rates were counted after recovery for 10 days. To further test the effect of OsWRKY76 on drought tolerance, 10-day-old seedlings of wild-type ZH11 rice and OsWRKY76-knockout mutants (w76-1 and w76-2) grown in soil suffered from drought stress for one week by stopping irrigation until the leaves of wild-type ZH11 were completely wilted. After recovery with rewatering for one week, the survival rates of the plants were counted visually.

For drought stress and MeJA treatments, two-week-old ZH11 seedlings were treated under dehydration stress (air-dried), 20% PEG 6000 treatment, and MeJA (100 µM) treatment. The leaves of the ZH11 seedlings were harvested at different time points after treatment.

For the analysis of MeJA on seed germination, seeds of wild-type ZH11 rice and OsWRKY76-knockout mutants (w76-1 and w76-2) were germinated on 1/2 MS medium containing 0, 5, or 50 µM MeJA for 3 days, and then germination was scored. To determine the effect of MeJA on seedling development, 1-d-old seedlings of wild-type ZH11 rice and OsWRKY76-knockout mutants (w76-1 and w76-2) were transferred to 1/2 MS medium containing 0, 5, or 50 µM MeJA for 5 days, and then shoot length and root length were measured.

Rt-qpcr Analysis

Total RNA was extracted from rice leaves using TRIzol reagent (Tiangen, Beijing, China), and then reverse transcription was performed using a StarScript II RT Kit with gDNA Remover (Genstar, Beijing, China). RT-qPCR was performed with the SYBR Green-based Fast Mixture (Genstar, Beijing, China) using an Mx3005P instrument (Stratagene, La Jolla, CA, USA). Relative expression levels of genes were calculated by normalization to OsActin and Ubiquitin5 genes (Mao et al., 2019).

Dab Staining

To determine the presence of H₂O₂ in the rice leaves, DAB staining was performed using two-week-old rice seedlings of wild-type ZH11 rice and OsWRKY76-knockout mutants (w76-1 and w76-2) with or without dehydration treatment. The rice leaves were cut into 2-cm pieces, immersed in 1 mg ml^–1 DAB in 50 mM Tris–acetate buffer (pH 5.0), and then vacuum infiltrated for 40 min. After incubation at 30°C for 3 h in the dark, the rice leaves were immersed in 95% ethanol until the chlorophyll was completely removed.

Yeast Two- And Three-hybrid Assays

The full-length CDSs of OsbHLH148, OsJAZ7, OsJAZ8, OsJAZ11, and OsJAZ12 were cloned and fused into the vector pGADT7 (AD) to generate AD-OsbHLH148, AD-OsJAZ7, AD-OsJAZ8, AD-OsJAZ11, and AD-OsJAZ12, respectively, as prey. The full-length CDSs of OsWRKY76 and OsbHLH148 were inserted into the vector pbridge (BD) to form BD-OsWRKY76 and BD-OsbHLH148, respectively, as bait. In the yeast three-hybrid assay, the full-length CDSs of OsWRKY76 were inserted into the BD-OsbHLH148 vector to form BD-OsbHLH148-OsWRKY76 in which OsWRKY76 driven by the Met25 promoter was expressed in the absence of methionine. These prey and bait vectors were transformed into Y2HGold cells grown on SD/-Trp-Leu dropout plates for the yeast two-hybrid assay and into Y190 cells grown on SD/-Trp-Leu-Met dropout plates for the yeast three-hybrid assay. The positive transformants were grown on SD/-Trp-Leu and SD/-Trp-Leu-His dropout plates for the yeast two-hybrid assay, and β-galactosidase activity levels were detected for the yeast three-hybrid assay.

Bifc

To confirm the in vivo interaction between OsWRKY76 and OsJAZ12, a BiFC assay was performed. The CDSs of OsWRKY76 and OsJAZ12 were amplified and fused into the pxy103 (nYFP) and apXY105 (cYFP) vectors, respectively. Thus, OsWRKY76-nYFP and cYFP-OsJAZ12 fusion constructs were obtained. cYFP or cYFP-OsJAZ12 and nYFP or OsWRKY76-nYFP were transformed into Arabidopsis protoplasts (S. C. Wang et al., 2005). Fluorescence signals were analyzed using a Zeiss Axio Observer A1 (Carl Zeiss, Jena, Germany).

Gal4-dependent Chimeric Transactivation Assay

To determine OsbHLH148 transcriptional activity, we created a recombinant effector vector encoding the GD-OsbHLH148 fusion protein. Moreover, the full-length CDSs of OsWRKY76 and OsJAZ12 were cloned into the effector vector without GD. Combinations of these effector vectors, reporter vector (35S-Gal4:FLUC), and internal control (35S:RLUC) were transformed into rice protoplasts followed by incubation at 30°C for 15 h in the dark to allow transient expression. FLUC and RLUC activities were measured using a Dual-Luciferase reporter assay system (Promega, Madison, Wisconsin, USA).

Yeast One-hybrid Assay

The yeast one-hybrid assay was performed as previously described (Lin et al., 2007). An upstream 2000-bp sequence of OsDREB1E’s start codon as a promoter sequence of OsDREB1E was cloned and fused into the pLacZi2µ vector to generate OsDREB1Epro:LacZ. The full-length CDSs of OsWRKY76 and OsbHLH148 were ligated into the pJG4-5 (GAD) vector to form GAD-OsWRKY76 (GAD-W76) and GAD-OsbHLH148 (GAD-b148), respectively. The GAD, GAD-W76, and GAD-b148 constructs were separately co-transformed with the OsDREB1Epro:LacZ vector into the yeast strain EGY48. The positive transformants were identified using SD/-Trp-Ura dropout plates containing X-Gluc for blue color development.

EMSA

EMSA was conducted as previously described (Gao et al., 2019). The full-length CDS of OsWRKY76 was inserted into the pGEX-4T-3 vector to fuse it to the GST coding region and to create a recombinant vector encoding a GST-WRKY76 fusion protein. The recombinant vector and empty GST vector were then transformed into the E. coli strain BL21 (DE3), which was purified using GST Resin. The 6-FAM 5’ end-labeled and unlabeled OsDREB1E probe was synthesized by a Bio-Tech Company (Sangon, Shanghai, China). Fluorescence signals in the gel were detected using a Tanon 5200 Multi imaging system (Tanon, Shanghai, China).

Dual-luciferase Assay

The OsDREB1E promoter was cloned into the double vector pGreenII 0800-LUC, in which the OsDREB1E promoter was fused with the FLUC reporter gene, and the Renilla luciferase gene was driven by the 35S promoter as an internal control. The full-length CDSs of OsWRKY76 and OsbHLH148 were inserted into pCAMBIA1300 as the effector vector. The effector vectors were co-transformed with the double vector into rice protoplasts, followed by incubation at 30°C for 15 h in the dark to allow transient expression. The transformed protoplasts were treated with and without 10% PEG 6000 treatment for 20 min. The FLUC and RLUC activities were measured with a Dual-Luciferase reporter assay system (Promega, Madison, Wisconsin, USA).

Primers And Accession Numbers

The sequences of all primers used for this study are listed in Table S1. The sequence data from this study can be found in the Rice Genome Annotation Project Database (http://rice.uga.edu./), and the accession numbers of genes are listed in Table S2.

ROS

reactive oxygen species

GPX1

Glutathione peroxidase 1

JAs

jasmonates

JA-Ile

JA isoleucine conjugate

MeJA

JA methyl ester

TFs

transcription factors

TPL

co-repressor topless

NINJA

adaptor protein of JAZ

SCF^COI1

kinetochore protein 1–cullin 1–F-box protein COI1

bHLH

basic helix–loop–helix

RT-qPCR

reverse transcription quantitative PCR

DAB

3, 3’-diaminobenzidine

H₂O₂

hydrogen peroxide

BiFC

bimolecular fluorescence complementation

CDS

coding sequence

Gal4 DNA-binding domain

FLUC

firefly luciferase

EMSA

electrophoretic mobility shift assay

GST

glutathione S-transferase

RLUC

Renilla luciferase.

Acknowledgements

We thank LetPub (Accdon LLC) for his help in polishing the English text.

Author contributions

T.W., X.D., and M.Z. designed the study; M.Z. performed experiments, data analysis, and wrote the manuscript; R.Z., K.H., Z.W., and B.G. assisted in performing part of the experiments; K.H., S.H., Z.L., and W.J. assisted in analyzing the data; X.D. and T.W. supervised the project and modified the manuscript.

Funding

This research was supported by Science and Technology Development Plan of Jilin Province of China (20210302008NC) and National Natural Science Foundation of China (32101664).

Availability of data and materials

All data generated or analysed during this study are included in this published article and its Additional files.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

All the authors declare no competing interests.

Author details

Jilin Province Engineering Laboratory of Plant Genetic Improvement, College of Plant Science, Jilin University, Changchun, China

Supplementary Information

Additional file 1: Figure S1. OsWRKY76 positively regulates rice drought stress tolerance. Table S1. List of primers used in this study. Table S2. List of the accession numbers of genes used in this study.

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No competing interests reported.

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OsWRKY76 positively regulates drought stress via OsbHLH148-mediated jasmonate signaling in rice

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Abstract

Figures

Background

Results

Expression of the OsWRKY76 gene in response to drought stress and MeJA treatment

Oswrky76 Confers Drought Tolerance In Rice

Oswrky76 Positively Regulates Ja Signaling

Oswrky76 Interacts With Osjaz12

Oswrky76 Competes With Osjaz12 To Bind Osbhlh148

Discussion

Conclusions

Materials And Methods

Plant materials and growth conditions

Drought-tolerance Assays And Treatments

Rt-qpcr Analysis

Dab Staining

Yeast Two- And Three-hybrid Assays

Bifc

Gal4-dependent Chimeric Transactivation Assay

Yeast One-hybrid Assay

EMSA

Dual-luciferase Assay

Primers And Accession Numbers

Abbreviations

Declarations

Acknowledgements

Author contributions

Funding

Availability of data and materials

Ethics approval and consent to participate

Consent for publication

Competing interests

Author details

Supplementary Information

References

Additional Declarations

Supplementary Files

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