Angelica sinensis, a perennial herb that produces ferulic acid and phthalides for the treatment of cardio-cerebrovascular diseases, prefers growing at an altitude of 1800–3000 m. Geographical models have predicted that high altitude, cool temperature and sunshade play determining roles in geo-authentic formation. Although the roles of altitude and light in yield and quality have been investigated, the role of temperature in regulating growth, metabolites biosynthesis and gene expression is still unclear. In this study, growth characteristics, metabolites contents and related genes expression were investigated by exposing A. sinensis to cooler (15 °C) and normal temperatures (22 °C). The results showed that plant biomass, the contents of ferulic acid and flavonoids and the expression levels of genes related to the biosynthesis of ferulic acid (PAL1, 4CLL4, 4CLL9, C3H, HCT, CCOAMT and CCR) and flavonoids (CHS and CHI) were enhanced at 15 °C compared to 22 °C. The contents of ligustilide and volatile oils exhibited slight increases, while polysaccharide contents decreased in response to cooler temperature. Based on gene expression levels, ferulic acid biosynthesis probably depends on the CCOAMT pathway and not the COMT pathway. It can be concluded that cool temperature enhances plant growth, ferulic acid and flavonoid accumulation but inhibits polysaccharide biosynthesis in A. sinensis. These findings authenticate that cool temperature plays a determining role in the formation of geo-authentic and also provide a strong foundation for regulating metabolites production of A. sinensis.

Sinopodophyllum hexandrum is a perennial alpine herb producing the anti-cancer metabolite podophyllotoxin (PPT). Although the adaptation of S. hexandrum to high altitudes has been demonstrated and the effects of temperature, precipitation, and UV-B light on plant growth and metabolite accumulation have been studied, knowledge on the role of flavonoid biosynthesis in adapting to high altitudes is limited. In this study, light intensity, amount and type of flavonoids, and differentially expressed proteins (DEPs) and genes (DEGs) at 2300 and 3300 m were analyzed by HPLC, proteomic, transcriptomic, and qRT-PCR analysis. We found that higher light intensity correlated with greater flavonoid, flavonol, and anthocyanin content as well as higher anthocyanin to total flavonoid and flavonol ratios observed at the higher altitude. Based on proteomic and transcriptomic analyses, nine DEPs and 41 DEGs were identified to be involved in flavonoid biosynthesis and light response at 3300 m. The relative expression of nine genes (PAL, CHS1, IFRL, ANS, MYB4, BHLH137, CYP6, PPO1, and ABCB19) involved in flavonoid biosynthesis and seven genes (HSP18.1, HSP70, UBC4, ERF5, ERF9, APX3, and EX2) involved in light stress were observed to be up-regulated at 3300 m compared with 2300 m. These findings indicate that light intensity may play a regulatory role in enhancing flavonoid accumulation that allows S. hexandrum to adapt to elevated-altitude coupled with high light intensity.

Angelica sinensis is a traditional Chinese medicinal plant that has been primarily used as a blood tonic. It largely relies on its bioactive metabolites, which include ferulic acid, volatile oils, polysaccharides and flavonoids. In order to improve the yield and quality of A. sinensis, the two cultivars Mingui 1 (M1), with a purple stem, and Mingui 2 (M2), with a green stem, have been selected in the field. Although a higher root yield and ferulic acid content in M1 than M2 has been observed, the differences of flavonoid biosynthesis and stem-color formation are still limited. In this study, the contents of flavonoids and anthocyanins were determined by spectrophotometer, the differences of flavonoids and transcripts in M1 and M2 were conducted by metabolomic and transcriptomic analysis, and the expression level of candidate genes was validated by qRT-PCR. The results showed that the contents of flavonoids and anthocyanins were 1.5- and 2.6-fold greater in M1 than M2, respectively. A total of 26 differentially accumulated flavonoids (DAFs) with 19 up-regulated (UR) and seven down-regulated (DR) were obtained from the 131 identified flavonoids (e.g., flavonols, flavonoid, isoflavones, and anthocyanins) in M1 vs. M2. A total 2210 differentially expressed genes (DEGs) were obtained from the 34,528 full-length isoforms in M1 vs. M2, and 29 DEGs with 24 UR and 5 DR were identified to be involved in flavonoid biosynthesis, with 25 genes (e.g., CHS1, CHI3, F3H, DFR, ANS, CYPs and UGTs) mapped on the flavonoid biosynthetic pathway and four genes (e.g., RL1, RL6, MYB90 and MYB114) belonging to transcription factors. The differential accumulation level of flavonoids is coherent with the expression level of candidate genes. Finally, the network of DAFs regulated by DEGs was proposed. These findings will provide references for flavonoid production and cultivars selection of A. sinensis.

Introduction: Cistanche deserticola is an important traditional Chinese herbal medicine. The fresh cistanche squamous stem is typically yellow-white and brown after drying. Oil cistanche is a cistanche variant with a purple squamous stem that turns black after drying. The color difference between oil cistanche and cistanche is obvious, and the former has a higher market price. However, the mechanism underlying the color difference of oil cistanche and cistanche remains unclear.Methods: This study evaluated the total flavone contents in oil cistanche and cistanche and compared the differential metabolites and differentially expressed genes (DEGs) and the contents of iridoid of dried oil cistanche and cistanche samples were determined by high-performance liquid chromatography, and finally the polysaccharides contents of them were determined to comprehensively analyze the formation mechanism of color difference between oil cistanche and cistanche.Results: The results showed that the total flavonoid content in oil cistanche was significantly higher than that in cistanche. Metabolomic analysis identified 50 differentially accumulated metabolites (DAMs) (34 up-regulated and 16 down-regulated), including carbohydrates, terpenoids, and flavonoids. Moreover, 3,376 DEGs were selected, among which significant up-regulated of IGS1 and CYP84A1 and down-regulated of 4CLL1, F6H2-2-1 and 5MAT1 genes jointly regulated flavonoid biosynthesis and affected the accumulation of differentially accumulated metabolites. Significant up-regulated of the CCD7 gene affected carotenoid component production, and significant up-regulated of the UGT85A24 gene promoted the accumulation of geniposidic acid. In addition, the contents of iridoid and polysaccharide in oil cistanche were significantly higher than those in cistanche.Discussion: The differential expression of flavonoids and terpenoid differential metabolites and CYP84A1, 5MAT1, FLS, UGT85A24 and CCD7 mainly caused the purple color of fresh oil cistanche. Dried samples of oil cistanche were darker in color than those of cistanche, due to the higher content of iridoids and polysaccharides in the former. This study preliminarily revealed the causes of the color differences between oil cistanche and cistanche, and provided references for the systematic study of cistanche and its germplasm resources, as well as for the breeding of C. deserticola.