AUTHOR=Chen Ying , Zhang Hua , Zhang Meng , Zhang Wenli , Ou Ziqi , Peng Zehang , Fu Chunhua , Zhao Chunfang , Yu Longjiang 

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

JOURNAL=Frontiers in Plant Science

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.697476

DOI=10.3389/fpls.2021.697476

ISSN=1664-462X

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