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Taxol is a rare secondary metabolite that accumulates considerably in  Taxus  species under salicylic acid (SA) and methyl jasmonate treatment. However, the molecular mechanism of its accumulation remains unclear. We investigated  TcWRKY33 , a nuclear-localized group I WRKY transcription factor, as an SA-responsive regulator of taxol biosynthesis. Overexpression and RNA interference of  TcWRKY33  confirmed that  TcWRKY33  regulates the expression of most taxol biosynthesis genes, especially  10-deacetylbaccatin III-10-O-acetyltransferase  ( DBAT ) and  taxadiene synthase  ( TASY ), which were considered as key enzymes in taxol biosynthesis. Transient overexpression of  TcWRKY33  in  Taxus chinensis  leaves resulted in increased taxol and 10-deacetylbaccatin accumulation by 1.20 and 2.16 times compared with the control, respectively. Furthermore,  TcWRKY33, DBAT , and  TASY  were confirmed to respond positively to SA signals. These results suggested that  TcWRKY33  was the missing component of taxol biosynthesis that responds to SA. The sequence analysis identified two W-box motifs in the promoter of  DBAT  but not in the  TASY . Yeast one-hybrid and dual-luciferase activity assays confirmed that  TcWRKY33  can bind to the two W-boxes in the promoter of  DBAT , upregulating its expression level. Hence,  DBAT  is a direct target of  TcWRKY33 . Furthermore,  TcERF15 , encoding a  TASY  activator, also contains two W-boxes in its promoter. Yeast one-hybrid and dual-luciferase activity assays further confirmed that  TcWRKY33  can upregulate  TASY  expression through the activation of  TcERF15 . In summary,  TcWRKY33  transmits SA signals and positively regulates taxol biosynthesis genes in two ways: directly and through the activation of other activators. Therefore,  TcWRKY33  is an excellent candidate for genetically engineering regulation of taxol biosynthesis in  Taxus  plants.

Salicylic Acid-Responsive Factor TcWRKY33 Positively Regulates Taxol Biosynthesis in Taxus chinensis in Direct and Indirect Ways