Salicylic acid regulates anthesis via CmWRKY25/CmWRKY53-CmSOC1 model in Clivia miniate

Flowering time is a vital trait in Clivia miniata, yet underlying regulatory mechanisms remain poorly understood. Integrated metabolomic, physiological and molecular analyses were performed to characterize salicylic acid (SA)-dependent flowering regulation. Metabolite profiling across four tissues yielded 787 tissue-specific metabolites, and abundant accumulation of lipids, phenolic acids and SA was detected in scapes. Anthesis was accelerated by exogenous SA in a concentration-dependent pattern, and flowering was advanced by 34 d under 100mg/L SA treatment. Two WRKY transcription factors, CmWRKY25 and CmWRKY53, were markedly upregulated by SA, and close homology to Arabidopsis flowering-related WRKY proteins was validated via phylogenetic analysis. Flower bud differentiation was promoted by transient overexpression of CmWRKY25 and CmWRKY53. Physical interaction between CmWRKY25 and CmWRKY53 to form a functional complex was confirmed by BiFC and Y2H assays. SA-responsive cis-elements were identified within promoter sequences of CmWRKY25, CmWRKY53 and CmSOC1. Cooperative transcriptional activation of CmSOC1 by the two proteins was further verified through Y1H and dual-luciferase assays. Collectively, a regulatory pathway was uncovered: the CmWRKY25-CmWRKY53 complex was induced by SA to activate CmSOC1 and accelerate flowering in C. miniata. Novel insights into SA-mediated floral transition were provided for this ornamental species.