Mechanisms Of Cold Stress Responses Regulated By The Protein Kinas OST1in Arabidopsis

Plants are usually suffered from cold stress in natural conditions. As one of the most important environmental factors, cold stress greatly restrains plant growth, development, and geographic distribution. Plants have evolved sophisticated and fine-tuned mechanisms to adapt to the low temperatures. Upon exposure to cold stress, a set of cold responsive (COR) genes are induced to help plants adapt to cold stress. ICE1-CBF transcriptional regulatory cascade has been extensitively studied to modulate COR genes. CBF genes are rapidly up-regulated by cold stress and confer enhanced freezing tolerance in plants. The expression of CBFs is regulated by several transcription factors, including ICE1and CAMTA3. ICE1is a bHLH transcription factor that binds to the CBF3promoter and activates its expression. ICE1has been shown to be ubiquitinated by the E3ligase HOS1and degraded by the26S-proteasome pathway, whereas sumoylation of ICE1mediated by SIZ1stabilizes ICE1. Therefore, modifications of ICE1are important for its functions. Protein phosphorylation plays pivotal roles in plant development and responses to environmental stress. Some evidence has shown that ubiquitination and sumoylation of proteins are often associated with their phosphorylation. However, specific proten kinases involved in cold signailing remain largely unknown.By genetic screening with a freezing tolerance assay, ostl muants were isolated from a set of Arabidopsis T-DNA insertion mutants of genes encoding protein kinases. OST1(open stomatal1)/SnRK2.6/SnRK2E is a Ser/Thr protein kinase in ABA signaling and belongs to SnRK2protein kinases family which has ten members in Arabidopsis. When ABA accumulates under stress conditions, the PYR/PYL/RCAR ABA receptors bind to ABA and subsequently interact with and inhibit PP2C phosphatases, thus releasing the inhibition of OST1by PP2Cs. Activated OST1can phosphorylate ABRE-binding proteins/factors (AREBs/ABFs) to regulate the expression of stress-responsive genes. Morever, OST1can phosphorylate S-type anion channel SLAC1to control stomatal movement in plants. In this study, we found that ostl kncokout mutants, ostl-3and ostl-4, showed freezing hypersensitivity, whereas OST1overexpressing plants exhibited enhanced freezing tolerance. The basal level and cold-induction of CBFs and their target genes were down-reguated in ostl mutants, while up-regulated in OST1overexpressing plants. These results suggest that OST1acts upstream of CBFs to regulate CBF-dependent gene expression.Intriguingly, OST1protein kinase activity was induced by cold in an in-gel kinase assay. The similar cold-induced OST1activities were observed in both wild type and aba2-21mutant plants. And this activity was not even compromised in aba2-21mutant treated with ABA inhibitor nordihydroguaiaretic acid (NDGA). Most importantly, ABA content was moderatly decreased after cold treatment for0.5h when OST1was activated. Taken together, these results suggest that activation of OST1by cold and ABA may be through two distinct pathways. In ABA signaling, OST1kianse activity is regulated by PP2C proteins. To study whether OST1acitivity is regulated by PP2Cs under cold stress, we detected OST1kianse acitivity in abil-1(C) gain-of-function mutant plants and found that OST1was not activated in abil-1(C). Consistently, abil-1(C) mutant and ABI1overexpressing plants showed freezing hypersensitivity, whereas abil abi2habl loss-of-function plants exhibited enhanced freezing tolerance. Accordingly, CBF genes were down-reguated in abil-1(C) mutant and ABI1overexpressing plants. These results suggest that ABI1represses cold-induced OST1activity, which in turn negatively controls freezing tolerance.We further found that OST1interacted with ICE1protein in vitro using yeast two hybrid and pull down assays and in vivo using coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays. OST1was able to directly phosphorylate ICE1in vitro. Morever, ICE1phosphorylation was enhanced by cold in plants, which was nearly abolished in ostl mutant plants. These results suggest that the phosphorylation of ICE1is predominantly mediated by OST1under cold conditions. Further study demonstrated that OST1phosphorylated ICE1and in turn enhanced the transcriptional activity and protein stability of ICE1. Strikingly, we found that Ser278of ICE1was a major phosphorylation site by OST1. The stability of ICE1S278D (the phosphorylated active form of ICE1carrying a serine to aspartate mutation) was dramatically enhanced in transgenic plants. Furthermore, the interaction of ICE1S278D with HOS1was dramatically compromised. The freezing sensitive phenotype of icel-2mutant could be rescued by wild-type ICE1, but not ICE1S278A (non-phosphorylation mutated form of ICE1carrying a serine278to alanine mutation). These data indicate that phosphorylation of ICE1at Ser278is required for its function in cold signaling. Genetic analysis indicated that ostl icel double mutant showed freezing sensitive phenotype, whereas overexpression of ICE1can restore freezing hypersensitive phenotype of ostl mutant, implying that OSTl acts upstream of ICE1in the CBF dependent pathway.Additionally, OSTl physically interacted with HOS1and interfered with the interaction between HOS1and ICE1, thereby enhancing the stability of ICE1.In summary, we demonstrate that OST1is a crucial protein kinase in the CBF dependent pathway to regulate cold responses. Under cold stress, OST1is activated and then phosphorylates ICE1, which in turn inhibits the degradation of ICE1mediated by HOS1and also enhances the transcriptional acitivity of ICEL Meanwhile, the OSTl protein also interacts with HOS1and competes with HOS1for binding to ICE1, thus liberating ICE1from the HOS1-ICE1complex. The dual role of OST1contributes to the enhancement of ICE1stability to increase CBFs expression and freezing tolerance. This study will shed new light on our understanding of cold signaling and cold tolerance in plants.