The INDUCER OF CBF EXPRESSION (ICE)–C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 (CBF/DREB1) transcriptional pathway plays a critical role in modulating cold stress responses in Arabidopsis thaliana. Dissecting crucial upstream regulatory signals or components of the ICE-CBF/DREB1 cascade will enhance our understanding of plant cold-tolerance mechanisms. Here, we show that jasmonate positively regulates plant responses to freezing stress in Arabidopsis. Exogenous application of jasmonate significantly enhanced plant freezing tolerance with or without cold acclimation. By contrast, blocking endogenous jasmonate biosynthesis and signaling rendered plants hypersensitive to freezing stress. Consistent with the positive role of jasmonate in freezing stress, production of endogenous jasmonate was triggered by cold treatment. In addition, cold induction of genes acting in the CBF/DREB1 signaling pathway was upregulated by jasmonate. Further investigation revealed that several JASMONATE ZIM-DOMAIN (JAZ) proteins, the repressors of jasmonate signaling, physically interact with ICE1 and ICE2 transcription factors. JAZ1 and JAZ4 repress the transcriptional function of ICE1, thereby attenuating the expression of its regulon. Consistent with this, overexpression of JAZ1 or JAZ4 represses freezing stress responses of Arabidopsis. Taken together, our study provides evidence that jasmonate functions as a critical upstream signal of the ICE-CBF/DREB1 pathway to positively regulate Arabidopsis freezing tolerance.

Leaf senescence is regulated by diverse developmental and environmental factors. Exogenous jasmonic acid (JA) can induce leaf senescence, whereas auxin suppresses this physiological process. Crosstalk between JA and auxin signaling has been well studied, but not during JA-induced leaf senescence. Here, we found that upon methyl jasmonate treatment, Arabidopsis thaliana wrky57 mutants produced typical leaf senescence symptoms, such as yellowing leaves, low chlorophyll content, and high cell death rates. Further investigation suggested that senescence-associated genes were upregulated in the wrky57 mutants. Chromatin immunoprecipitation experiments revealed that WRKY57 directly binds to the promoters of SENESCENCE4 and SENESCENCE-ASSOCIATED GENE12 and represses their transcription. In vivo and in vitro experiments suggested that WRKY57 interacts with JASMONATE ZIM-DOMAIN4/8 (JAZ4/8) and the AUX/IAA protein IAA29, repressors of the JA and auxin signaling pathways, respectively. Consistent with the opposing functions of JA and auxin in JA-induced leaf senescence, JAZ4/8 and IAA29 also displayed opposite functions in JA-induced leaf senescence and competitively interacted with WRKY57. Our results suggested that the JA-induced leaf senescence process can be antagonized by auxin via WRKY57. Moreover, WRKY57 protein levels were downregulated by JA but upregulated by auxin. Therefore, as a repressor in JA-induced leaf senescence, WRKY57 is a common component of the JA- and auxin-mediated signaling pathways.

SNF 1-RELATED PROTEIN KINASE 2 (SnRK2) is a family of plant-specific protein kinases which is the key regulator of hyper-osmotic stress signalling and abscisic acid (ABA)-dependent development in various plants. Among the rice subclass-I and -II SnRK2s, osmotic stress/ABA–activated protein kinase 2 (SAPK2) may be the primary mediator of ABA signaling. However, SAPK2 has not been comprehensively characterized. In this study, we elucidated the functional properties of SAPK2 using loss-of-function mutants produced with the CRISPR/Cas9 system. The SAPK2 expression level was strongly upregulated by drought, high-salinity, and polyethylene glycol treatments. The sapk2 mutants exhibited an ABA-insensitive phenotype during the germination and post-germination stages, suggesting that SAPK2 had a pivotal role related to ABA-mediated seed dormancy. The sapk2 mutants were more sensitive to drought stress and reactive oxygen species than the wild-type plants, indicating that SAPK2 was important for responses to drought conditions in rice. An additional investigation revealed that SAPK2 increased drought tolerance in the following two ways: (i) by reducing water loss via the accumulation of compatible solutes, promoting stomatal closure, and upregulating the expression levels of stress-response genes such as OsRab16b, OsRab21, OsbZIP23, OsLEA3, OsOREB1 and slow anion channel (SLAC)-associated genes such as OsSLAC1 and OsSLAC7; (ii) by inducing the expression of antioxidant enzyme genes to promote ROS-scavenging abilities that will ultimately decrease ROS damages. Moreover, we also observed that SAPK2 significantly increased the tolerance of rice plants to salt and polyethylene glycol stresses. These findings imply that SAPK2 is a potential candidate gene for future crop improvement studies.

Summary: The WRKY transcription factors have been demonstrated to play crucial roles in regulating stress responses; however, the exact mechanisms underlying their involvement in stress responses are not fully understood. Arabidopsis WRKY8 was predominantly expressed in roots and was highly upregulated by salt treatment. Disruption of WRKY8 rendered plants hypersensitive to salt, showing delayed germination, inhibited post‐germination development and accelerated chlorosis. Further investigation revealed that WRKY8 interacted with VQ9, and their interaction decreased the DNA‐binding activity of WRKY8. The VQ9 protein was exclusively localized in the nucleus, and VQ9 expression was strongly responsive to NaCl treatment. Mutation of VQ9 enhanced tolerance to salt stress, indicating that VQ9 acts antagonistically with WRKY8 to mediate responses to salt stress. The antagonist functions of WRKY8 and VQ9 were consistent with an increased or reduced Na⁺/K⁺ concentration ratio, as well as contrasting expression patterns of downstream stress‐responsive genes in salt‐stressed wrky8 and vq9 mutants. Moreover, chromatin immunoprecipitation (ChIP) assays showed that WRKY8 directly bound the promoter of RD29A under salt conditions. These results provided strong evidence that the VQ9 protein acts as a repressor of the WRKY8 factor to maintain an appropriate balance of WRKY8‐mediated signaling pathways to establish salinity stress tolerance.