CRISPR/Cas9-mediated CHS2 mutation provides a new insight into resveratrol biosynthesis by causing a metabolic pathway shift from flavonoids to stilbenoids in Vitis davidii cells

Abstract

Resveratrol is an important phytoalexin that adapts to and responds to stressful conditions and plays various roles in health and medical therapies. However, it is only found in a limited number of plant species in low concentrations, which hinders its development and utilization. Chalcone synthase (CHS) and stilbene synthase (STS) catalyze the same substrates to produce flavonoids and resveratrol, respectively. However, it remains unclear how CHS and STS compete in metabolite synthesis. In this study, two CHS2 mutant cell lines (MT1 and MT2) were generated using CRISPR/Cas9 genome editing. These CHS2 mutant cell lines exhibited abundant mutations in CHS2, leading to the premature termination of protein translation and subsequent CHS2 knockout. Amplicon sequencing confirmed comprehensive CHS2 knockout in MT1, whereas the wild-type sequence remained predominant in the MT2 cell line. Transcriptome and RT-qPCR results showed a significant downregulation of genes involved in flavonoid biosynthesis, including CHS2, CHS3, F3H, F3’H, DFR, FLS, LDOX, among others, resulting in decreased flavonoid accumulation, such as anthocyanins, proanthocyanidins, quercetin, and kaempferol. Conversely, STS genes involved in stilbenoid biosynthesis were upregulated competing with the flavonoid pathway. Consequently, there was a marked increase in stilbenoids, including resveratrol, piceatannol, piceid, and pterostilbene, with a 4.1-fold increase in resveratrol and a 5.3-fold increase in piceid (a derivative of resveratrol) observed in CHS2 mutant cell lines. This research demonstrates that CHS2 mutation induces a shift from flavonoid biosynthesis towards stilbenoid biosynthesis, offering new insights into metabolite biosynthesis and regulation, as well as an alternative solution for natural resveratrol production, and a novel breeding approach for eliminating non-target agronomic traits using CRISPR-Cas9.

Acknowledgements

This work was founded by National Natural Science Foundation of China (32302284), Natural Science Foundation of Fujian Province, China (2021 J05091), High Quality Development “5511” Collaborative Innovation Project between Fujian and Chinese Academy of Agricultural Sciences (XTCXGC2021014), and Fujian Provincial Department of Science and Technology of Special Public-funded Projects (2021R1032009). We thank Prof. Yaoguang Liu for kindly providing the CRISPR/Cas9 plasmids, Prof. Hongliang Zhu and Dr. Rui Li for providing genome editing assistance.

Author contributions

GL, JL, and CL supervised the project. GL and PL obtained the funding. GL, PF, and LH performed the experiments. GL, PF, JC, and QW performed the data analysis. GL and CL wrote the original draft. JL, QW, and JC revised the manuscript. All authors read and approved the final manuscript.

Data availability

Data supporting the results are available in the article and its supplementary data.

Conflict of interest statement

The authors declare no competing interests.

Supplementary Data

Supplementary data is available at Horticulture Research online.

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