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.
The K-homology (KH) domain is a nucleic acid binding domain present in many proteins. Recently we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding single-stranded (ss)DNA and ssRNA. Electrophoretic mobility shift assay (EMSA) and nuclear magnetic resonance (NMR) revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG-loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. SELEX (systematic evolution of ligands by exponential enrichment), chromatin immunoprecipitation (ChIP)-seq, and crosslinking immunoprecipitation (CLIP)-seq showed that the KH domain binds C/T rich DNA and U rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-HSQC NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single strand tails, and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.
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