VdCHS2 Overexpression Enhances Anthocyanin Biosynthesis, Modulates the Composition Ratio, and Increases Antioxidant...

Anthocyanins are significant secondary metabolites that are essential for plant growth and development, possessing properties such as antioxidant, anti-inflammatory, and anti-cancer activities and cardiovascular protection. They offer significant potential for applications in food, medicine, and cosmetics. However, since anthocyanins are mainly obtained through plant extraction and chemical synthesis, they encounter various challenges, including resource depletion, ecological harm, environmental pollution, and the risk of toxic residuals. To address these issues, this study proposes a plant cell factory approach as a novel alternative solution for anthocyanin acquisition. In this study, the VdCHS2 gene was successfully transformed into spine grape cells, obtaining a high-yield anthocyanin cell line designated as OE1. Investigations of the light spectrum demonstrated that white light promoted spine grape cell growth, while short-wavelength blue light significantly boosted anthocyanin production. Targeted metabolomics analysis revealed that the total anthocyanin content in the OE1 cell line reached 11 mg/g, representing a 60% increase compared to the WT. A total of 54 differentially accumulated metabolites were identified, among which 44 were upregulated. Overexpression of the CHS gene enhanced the expression of downstream genes involved in anthocyanin biosynthesis, resulting in the differential expression of CHI, F3Hb, F3'5'H, DFR4, and LDOX. This led to the differential accumulation of anthocyanin monomers, predominantly consisting of 3-O-glucosides and 3-O-galactosides, thereby causing alterations in anthocyanin levels and composition. Furthermore, the OE1 cell line increased the activity of various antioxidant enzymes, improved the clearance of reactive oxygen species, and reduced the levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA). The subsequent cultivation of the transformed OE1 cell line, in conjunction with cell suspension culture, established a plant cell factory for anthocyanin production, significantly increasing anthocyanin yield while shortening the culture duration. This study elucidates the molecular mechanisms through which the VdCHS2 gene influenced anthocyanin accumulation and compositional variations. Additionally, it established a model for a small-scale anthocyanin plant cell factory, thereby providing a theoretical and practical foundation for the targeted synthesis of anthocyanin components and the development and utilization of plant natural products.