Table_1_Salicylic Acid-Responsive Factor TcWRKY33 Positively Regulates Taxol Biosynthesis in Taxus chinensis in Direct and Indirect Ways.XLSX

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.

紫杉醇（Taxol）是一种罕见的次生代谢产物，在水杨酸（salicylic acid, SA）与茉莉酸甲酯（methyl jasmonate）处理下，可在红豆杉属（Taxus）植物中大量积累。然而其积累的分子机制仍未明确。本研究针对TcWRKY33展开探究：该蛋白是一类定位于细胞核的第I组WRKY转录因子（WRKY transcription factor），可作为响应水杨酸信号的紫杉醇生物合成调控因子。通过对TcWRKY33进行过表达与RNA干扰（RNA interference）实验，证实其可调控多数紫杉醇生物合成相关基因的转录，其中尤以紫杉醇生物合成关键酶——10-去乙酰巴卡亭III-10-O-乙酰转移酶（10-deacetylbaccatin III-10-O-acetyltransferase, DBAT）与紫杉二烯合酶（taxadiene synthase, TASY）的调控效应最为显著。在中国红豆杉（Taxus chinensis）叶片中瞬时过表达TcWRKY33后，紫杉醇与10-去乙酰巴卡亭的积累量较对照组分别提升1.20倍与2.16倍。进一步实验证实，TcWRKY33、DBAT与TASY均可正向响应水杨酸信号。上述结果表明，TcWRKY33是紫杉醇生物合成通路中响应水杨酸信号的关键缺失组分。序列分析显示，DBAT的启动子区域存在两个W-box基序（W-box motif），而TASY的启动子中未检测到该基序。酵母单杂交（yeast one-hybrid）与双荧光素酶活性实验（dual-luciferase activity assay）证实，TcWRKY33可结合DBAT启动子中的两个W-box，从而上调其转录水平，因此DBAT是TcWRKY33的直接靶标基因。此外，编码TASY激活因子的TcERF15，其启动子区域同样含有两个W-box基序。后续酵母单杂交与双荧光素酶活性实验进一步证实，TcWRKY33可通过激活TcERF15，间接上调TASY的表达。综上，TcWRKY33通过两种途径传递水杨酸信号并正向调控紫杉醇生物合成相关基因：一是直接结合靶基因启动子以调控其表达，二是通过激活其他转录激活因子实现间接调控。因此，TcWRKY33可作为调控红豆杉属植物紫杉醇生物合成的优质基因工程候选靶点。