Resveratrol is an important phytoalexin that adapts to and responds to stressful conditions, 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.

中文翻译：

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CRISPR/Cas9 介导的 CHS2 突变通过引起 Vitis davidii 细胞中从类黄酮转变为二苯乙烯类化合物，为白藜芦醇的生物合成提供了新的见解


白藜芦醇是一种重要的植物抗毒素，可适应和应对压力条件，在健康和医学治疗中发挥各种作用。然而，它仅存在于有限数量的低浓度植物物种中，这阻碍了其开发和利用。查尔酮合酶 （CHS） 和二苯乙烯合酶 （STS） 催化相同的底物，分别产生类黄酮和白藜芦醇。然而，目前尚不清楚 CHS 和 STS 如何在代谢物合成中竞争。在这项研究中，使用 CRISPR/Cas9 基因组编辑生成了两种 CHS2 突变细胞系 （MT1 和 MT2）。这些 CHS2 突变细胞系在 CHS2 中表现出丰富的突变，导致蛋白质翻译过早终止和随后的 CHS2 敲除。扩增子测序证实了 MT1 中 CHS2 的全面敲除，而野生型序列在 MT2 细胞系中仍然占主导地位。转录组和 RT-qPCR 结果显示，参与类黄酮生物合成的基因显著下调，包括 CHS2、CHS3、F3H、F3'H、DFR、FLS、LDOX 等，导致类黄酮积累减少，如花青素、原花青素、槲皮素和山奈酚。相反，参与二苯乙烯生物合成的 STS 基因与类黄酮途径竞争上调。因此，在 CHS2 突变细胞系中观察到白藜芦醇、白皮杉醇、白藜芦和紫檀芪等二苯乙烯类化合物显著增加，其中白藜芦醇增加 4.1 倍，白藜芦醇（白藜芦醇衍生物）增加 5.3 倍。 这项研究表明，CHS2 突变诱导了从类黄酮生物合成向类二苯乙烯生物合成的转变，为代谢物的生物合成和调节提供了新的见解，以及天然白藜芦醇生产的替代解决方案，以及一种使用 CRISPR-Cas9 消除非靶标农艺性状的新型育种方法。