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In vivo transgenic studies confirm the critical acylation function of LeBAHD56 for shikonin in Lithospermum erythrorhizon

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Abstract

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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.

Abstract

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.

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Funding

This research was supported by the National Natural Science Foundation of China (31970321, U1903201, 31670298), the Natural Science Foundation of Jiangsu Bureau of Science and Technology (BK20191254), and the Program for Changjiang Scholars and Innovative Research Team in University from the Ministry of Education of China (IRT_14R27).

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JQ and YY conceived and designed the experiments. LY, CW, XL, and SJ performed the experiments. LY, CW, XL, SJ, YD, and ZL analyzed the data. XW, ZW, MY, AF, JC, GL, HL, and HH contributed to resources. LY wrote the draft of the manuscript. YY, JQ, and LY contributed to review and edit the manuscript. All authors carefully checked and approved the final manuscript.

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Correspondence to Yonghua Yang or Jinliang Qi.

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Communicated by Kunpeng Jia.

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Yang, L., Wang, C., Lai, X. et al. In vivo transgenic studies confirm the critical acylation function of LeBAHD56 for shikonin in Lithospermum erythrorhizon. Plant Cell Rep 43, 160 (2024). https://doi.org/10.1007/s00299-024-03242-7

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