Casein Kinase ? Is Involved In The Regulation Of Jasmonate Signaling In Arabidopsis

Protein phosphorylation pathways have been previously implicated in the regulation of JA signaling, and MYC2 that regulates JA resonse has been shown to be phosphorylated in vivo. However, the identity of kinases involved in the phosphorylation of MYC2 and possible consequences of this posttranslational modification on the MYC2-regulated transcription is still largely unknown.In our studies, CK2 was indentified as the upstream kinase of MYC2. CK2 can phosphorylate MYC2 and regulate its DNA binding activity, thereby modulate JA signaling response. Meanwhile, we characterized CKA4 knocked-out mutant, and revealed the roles of CKA4 during seedling development in Arabidopsis. Detailed results are present briefly as below:1. We used a yeast two-hybrid (Y2H) test and a biomolecular fluorescence complementation (BiFC) assay to show that CK2 regulatory subunits can interact with MYC2 in vitro and in vivo. Recombinant CK2 was purificated, and showed kinase activity toward MYC2.2. The CK2 specific inhibitor TBB could significantly repress the induction of JA response genes by JA. Furthermore, this induction was also attenuated in CK2 dominant-negative line CK2mut, indicating that the kinase activity of CK2 is required for the activation of JA response.3. Overexpression of CKB4 enhances JA-mediated inhibition of root elongation and increases the expression of JA response genes. And these positive roles of CKB4 were MYC2-dependent, as the induction of these genes by JA in CKB4ox jinl-9 cross lines was weakened to the level in jin1-9.4. In ChIP (Chromatin immunoprecipitation) assays, JA-stimulated binding of MYC2 to the promoter of JAZ1, JAZ3, LOX3 and COIR3, was inhibited in CK2mut, and was enhanced in CKB4ox line, indicating that CK2 regulates the DNA-binding activity of MYC2.5. The CKA4 T-DNA mutant was identified. The cka4-1 shows delayed expansion of leaves, reduction in hypocotyl and primary root length, indicating that CKA4 plays essential roles during seedling development. The cka4-1 also showed increased anthocyanin content and delayed flowering in long-day condition.6. The 35::CKA4-GFP construct rescued the abnormal phenotype of cka4-1, and showed GFP fluorescence not only in cytoplasm but also in the nuclei in the root of the transgenic line.Our studies proved that CK2 is required for the activation of JA response, and its role in JA signaling is MYC2-dependent. We identified the first kinase that can phosphorylate MYC2 and regulate its function. Meanwhile, the essential roles of CKA4 during seedling development were first revealed using previously uncharacterazied cka4 mutant. We also showed that CKA4 is a ubiquitously localizied protein, but not plasmid-specific.Our studies revealed the regulation mechanism of JA signaling response by protein phosporylation pathway, enriched the knowledge about CK2 biology functions, and provided a good foundation for the further research of CK2 functions.