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

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

doi:10.3389/fpls.2021.697476

Abstract

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.

Highlights

Taxol, known as paclitaxel, is a specialized metabolite originally isolated from Taxus species that functions as an anticancer drug (Wani et al, 1971; Schiff et al, 1979)
We evaluated the potential functions of the group I WRKY transcription factor TcWRKY33 in the regulation of taxol biosynthesis
Analyzing the hormonal response patterns of TcWRKY33 will enrich our understanding of the regulatory network of taxol biosynthesis

Summary

Introduction

Known as paclitaxel, is a specialized metabolite originally isolated from Taxus species that functions as an anticancer drug (Wani et al, 1971; Schiff et al, 1979). Similar to other secondary plant metabolites, such as artemisinin and vinblastine, taxol is very rare in vivo (Kwak et al, 1995; White, 2008; Abdul Rahim et al, 2018). Salicylic acid (SA) is an important endogenous hormone in plants. It is involved in the regulation of the biosynthesis of secondary metabolites including terpenoids, alkaloids, and flavonoids (Kang et al, 2004; Tounekti et al, 2013). Mairei, the addition of 20 mg/L of SA induces taxol biosynthesis (Wang et al, 2007). Many transcription factors function by responding to SA signals. SA-induced transcription factors that regulate taxol biosynthesis remain to be discovered

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Conclusion