DoWRKY26 positively regulating flavonoid biosynthesis in Dendrobium officinale

Dendrobium officinale is renowned as the foremost among the "Nine Immortal Herbs of China". Our previous research showed enhanced flavonoid accumulation following induction by the endophyte Wickerhamomyces sp. KLBMPSYLp8. Transcriptome data analysis identified multiple upregulated transcription factor (TF) genes. We conducted transient overexpression analysis of 10 significantly upregulated TF genes in D. officinale leaves. The results demonstrated that transient overexpression of the DoWRKY26 significantly enhanced flavonoid accumulation, with a 33 % increase compared to the empty vector control group. Furthermore, DoWRKY26 overexpression also upregulated the expression levels of key enzyme genes implicated in the flavonoid biosynthesis pathway. Subcellular localization confirmed its nuclear presence. DoWRKY26 expression was induced by salicylic acid (SA), abscisic acid (ABA), 1-Aminocyclopropane-1-carboxylic Acid (ACC) and methyl jasmonate (MeJA). Yeast one-hybrid (Y1H), Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assays verified that DoWRKY26 could bind and activate the promoter of DoCCoAOMT. These findings provide a foundational basis for further exploring the biosynthesis and transcriptional regulation mechanisms of flavonoids in D. officinale.