Tripterygium wilfordii, Hook., Hook.

Zhou, Jiawei, Hu, Tianyuan, Liu, Yuan, Tu, Lichan, Song, Yadi, Lu, Yun, Zhang, Yifeng, Tong, Yuru, Zhao, Yujun, Su, Ping, Wu, Xiaoyi, Huang, Luqi & Gao, Wei, 2021, Cytochrome P 450 catalyses the 29 - carboxyl group formation of celastrol, Phytochemistry (112868) 190, pp. 1-10 : 2

2.1. CYP450 candidates from T. wilfordii

Based on the annotation of transcriptomic data, we identified approximately two hundred full-length P450s in the T. wilfordii transcriptomic library. To find an enzyme that could catalyse the formation of the carboxyl group of celastrol at the C-29 position, we used CYP72A154 from G. uralensis and CYP72A63 from M. truncatula as templates to search the CYP450 candidates of T. wilfordii by homologybased searches of the transcriptomic library. This analysis revealed seven CYP72 candidates with 55.49–69.03% amino acid identity with CYP72A154 and CYP72A63 (Supplementary Fig. S1 ). Considering that celastrol was mainly distributed in the root periderm of T. wilfordii , we also selected a set of candidate P450 genes according to the transcript level in the root periderm from the T. wilfordii transcriptome.

The analysis of pathway gene expression in different tissues and MeJA treated cells could aid candidate selection. Therefore, we analysed the transcript level of these candidates in different tissues expressing Tw OSC 1-3. The results showed that although some of the candidates showed a high transcript level in the root periderm, some had a higher transcript level in other tissues ( Fig. 2a ). In our previous study, we found that genes in the MVA pathway and TwOSC1-3 can be induced by MeJA treatment with the accumulation of celastrol ( Liu et al., 2016; Zhou et al., 2019). We further analysed the expression levels of these P450 candidates and Tw OSC 1-3 in T. wilfordii cell suspensions treated with MeJA. We found that some of the candidates could be induced by MeJA and showed a similar expression pattern to Tw OSC, while other CYP450 enzymes showed no significant change in gene expression compared to the control group ( Fig. 2b ).

Based on the gene expression analysis, the candidates that showed specific expression in the root and could also be induced by MeJA, as well as CYP72 candidates, were chosen for gene cloning. We finally cloned 17 candidates from the complementary DNA library of T. wilfordii for further functional identification. All the cloned candidates were named by the nomenclature committee (David Nelson: dnelson@uthsc. edu).

Figures

Fig. 1. The proposed carboxyl group formation of celastrol at the C-29 position and carboxyl group formation of β-amyrin at C-30 position by other cytochrome P450s. The red dashed arrows indicate one or multiple proposed step reactions, and the black solid arrow indicates a biosynthetic reaction catalysed by known CYP450 genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. Hierarchical clustering of RNA-Seq expression data and heat map depicting the expression profile of candidate P450 genes. (a) The gene expression of candidate P450 genes in different tissues of T. wilfordii. (b) The gene expression of candidate P450 genes in MeJA-induced suspension cells of T. wilfordii. Colour key: candidate CYP72 family genes (blue), candidate CYP450 genes showing specific expression in the root and induced by MeJA (red), and other candidate CYP450 genes with high expression levels in the root and TwOSC1-3 (black). RX, root xylem; RP, root phloem; RB, root periderm; PS, stem vascular bundle; SB, stem periderm; F, Flowers; and L, Leaves. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)