Flavonoids play multiple roles in plant coloration and stress resistance and are closely associated with human health. Flavonoids and non-flavonoids (such as phenolic acids) are produced via the phenylpropanoid-derived pathway. Anthocyanidin synthase (ANS) catalyzes the synthesis of anthocyanins from leucoanthocyanidin in the flavonoids branched pathway. In this study, SmANS from Salvia miltiorrhiza was cloned and mainly localized in the endoplasmic reticulum (ER), plastids, Golgi, plasma membrane, and nucleus of tobacco epidermal cells, and was most highly expressed in purple petals in S. miltiorrhiza, whereas it showed almost no expression in white petals, green calyxes, and pistils in S. miltiorrhiza Bge f. alba. Overexpressed SmANS enhanced anthocyanin accumulation but reduced salvianolic acid B (SAB) and rosmarinic acid (RA) biosynthesis in S. miltiorrhiza and S. miltiorrhiza Bge f. alba plantlets, meanwhile, it restored the purple-red phenotype in S. miltiorrhiza Bge f. alba. These changes were due to reallocation of the metabolic flow, which was influenced by the SmANS gene. These findings indicate that SmANS not only plays a key role in anthocyanin accumulation in S. miltiorrhiza, but also acts as a “switch” for the coloration of S. miltiorrhiza Bge f. alba. This study provides baseline information for further research on flavonoids metabolism and improvement of anthocyanin or phenolic acid production by genetic engineering.

Flavonoid secondary metabolites can treat and prevent many diseases, but systematic studies on regulation of the biosynthesis of such metabolites in aboveground parts of Salvia miltiorrhiza are lacking. In this study, metabonomic and transcriptomic analyses of different S. miltiorrhiza phenotypes were conducted to explore pathways of synthesis, catalysis, accumulation, and transport of the main flavonoid secondary metabolites regulating pigment accumulation. Tissue localization and quantitative analysis of flavonoid secondary metabolites were conducted by laser scanning confocal microscopy (LSCM). A total 3090 differentially expressed genes were obtained from 114,431 full-length unigenes in purple and green phenotypes, and 108 functional genes were involved in flavonoid biosynthesis. Five key phenylpropane structural genes (PAL, 4CL, ANS, 3AT, HCT) were highly differentially expressed, and four transcription factor genes (MYB, WRKY, bHLH, bZiP) were identified. In addition, six GST genes, nine ABC transporters, 22 MATE genes, and three SNARE genes were detected with key roles in flavonoid transport. According to LSCM, flavonoids were mainly distributed in epidermis, cortex, and collenchyma. Thus, comprehensive and systematic analyses were used to determine biosynthesis, accumulation, and transport of flavonoids in stems and leaves of different S. miltiorrhiza phenotypes. The findings will provide a reference for flavonoid production and cultivar selection.

Introduction Salvia miltiorrhiza is an important model medicinal plant for secondary metabolism research. There is a leaf color variation (purple) in the aboveground part of S. miltiorrhiza in the process of growth and development, which is mostly caused by the loss or variation of structural genes regulating a certain pigment. Objectives In order to screen the key genes related to purple traits and explore the differences of their metabolites. In addition to further explore the synthesis, accumulation and transport pathway of flavonoids secondary metabolites in aboveground of S. miltiorrhiza. Methods The above ground tissues were analyzed by metabolomics and transcriptomics. And tissue localization and quantitative analysis of flavonoids secondary metabolites were carried out by laser scanning confocal microscope (LSCM). Results The results showed PAL、CYP73A/C4H、4CL、ANS、DFR、3AT、HCT and MYB、bHLH、WADS、WRKY transcription families involved in anthocyanin synthesis were highly differentially expressed. In addition, six GST genes, nine ABC transporters, twenty-two MATE genes and three SNARE genes were detected to play a key role in metabolite transport. A variety of flavonoids and glycosidic carbohydrates were found in the metabolites. Finally, tissue localization and quantitative analysis showed that flavonoids were mainly distributed in epidermis, cortex and collenchyma. Conclusion The dynamic accumulation model of flavonoids biosynthesis pathway was established, and the biological transport pathway of flavonoids was explored. These findings provide insights into the changes of purple phenotype during the development of S. miltiorrhiza, and provide valuable resources for the study of metabolic regulatory networks.