State Key Laboratory of Pharmaceutical Biotechnology, Institute for Plant Molecular Biology, School of Life Sciences, Nanjing University, Nanjing, 210023, China.
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, 210037, China.
Plant Cell Rep. 2024 Jun 3;43(6):160. doi: 10.1007/s00299-024-03242-7.
LeBAHD56 is preferentially expressed in tissues where shikonin and its derivatives are biosynthesized, and it confers shikonin acylation in vivo. Two WRKY transcriptional factors might regulate LeBAHD56's expression. Shikonin and its derivatives, found in the roots of Lithospermum erythrorhizon, have extensive application in the field of medicine, cosmetics, and other industries. Prior research has demonstrated that LeBAHD1(LeSAT1) is responsible for the biochemical process of shikonin acylation both in vitro and in vivo. However, with the exception of its documented in vitro biochemical function, there is no in vivo genetic evidence supporting the acylation function of the highly homologous gene of LeSAT1, LeBAHD56(LeSAT2), apart from its reported role. Here, we validated the critical acylation function of LeBAHD56 for shikonin using overexpression (OE) and CRISPR/Cas9-based knockout (KO) strategies. The results showed that the OE lines had a significantly higher ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than the control. In contrast, the KO lines had a significantly lower ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than controls. As for its detailed expression patterns, we found that LeBAHD56 is preferentially expressed in roots and callus cells, which are the biosynthesis sites for shikonin and its derivatives. In addition, we anticipated that a wide range of putative transcription factors might control its transcription and verified the direct binding of two crucial WRKY members to the LeBAHD56 promoter's W-box. Our results not only confirmed the in vivo function of LeBAHD56 in shikonin acylation, but also shed light on its transcriptional regulation.
LeBAHD56 优先在合成紫草素及其衍生物的组织中表达，并在体内赋予紫草素酰化。两个 WRKY 转录因子可能调节 LeBAHD56 的表达。紫草素及其衍生物存在于紫草的根部，在医学、化妆品和其他行业有广泛的应用。先前的研究表明，LeBAHD1（LeSAT1）负责紫草素酰化的生化过程，无论是在体外还是体内。然而，除了其已证实的体外生化功能外，除了其报道的作用外，没有体内遗传证据支持高度同源基因 LeSAT1 的酰化功能，即 LeBAHD56（LeSAT2）。在这里，我们使用过表达（OE）和 CRISPR/Cas9 基于敲除（KO）策略验证了 LeBAHD56 对紫草素酰化的关键作用。结果表明，OE 系的乙酰紫草素、异丁酰紫草素或异戊酰紫草素与紫草素的比值明显高于对照。相比之下，KO 系的乙酰紫草素、异丁酰紫草素或异戊酰紫草素与紫草素的比值明显低于对照。至于其详细的表达模式，我们发现 LeBAHD56 优先在根部和愈伤组织细胞中表达，这些是紫草素及其衍生物的生物合成部位。此外，我们预计广泛的假定转录因子可能控制其转录，并验证了两个关键 WRKY 成员直接结合到 LeBAHD56 启动子的 W-box 上。我们的研究结果不仅证实了 LeBAHD56 在紫草素酰化中的体内功能，还揭示了其转录调控。
