Crosstalk between H2O2 and Ca2+ signaling is involved in root endophyte-enhanced tanshinone biosynthesis of Salvia miltiorrhiza.

Tanshinones are bioactive ingredients derived from the herbal plant Salvia miltiorrhiza and are used for treating diseases of the heart and brain, thus ensuring quality of S. miltiorrhiza is paramount. Applying the endophytic fungus Trichoderma atroviride D16 can significantly increase the content of tanshinones in S. miltiorrhiza, but the potential mechanism remains unknown. In the present study, the colonization of D16 effectively enhanced the levels of Ca2+ and H2O2 in the roots of S. miltiorrhiza, which is positively correlated with increased tanshinones accumulation. Further experiments found that the treatment of plantlets with Ca2+ channel blocker (LaCl3) or H2O2 scavenger (DMTU) blocked D16-promoted tanshinones production. LaCl3 suppressed not only the D16-induced tanshinones accumulation but also the induced Ca2+ and H2O2 generation; nevertheless, DMTU did not significantly inhibit the induced Ca2+ biosynthesis, implying that Ca2+ acted upstream in H2O2 production. These results were confirmed by observations that S. miltiorrhiza treated with D16, CaCl2, and D16+LaCl3 exhibit H2O2 accumulation and influx in the roots. Moreover, H2O2 as a downstream signal of Ca2+ is involved in D16 enhanced tanshinones synthesis by inducing the expression of genes related to the biosynthesis of tanshinones, such as DXR, HMGR, GGPPS, CPS, KSL and CYP76AH1 genes. Transcriptomic analysis further supported that D16 activated the transcriptional responses related to Ca2+ and H2O2 production and tanshinones synthesis in S. miltiorrhiza seedlings. This is the first report that Ca2+ and H2O2 play important roles in regulating fungal-plant interactions thus improving the quality in the D16-S. miltiorrhiza system.

A Muti-Substrate Flavonol O-glucosyltransferases from Safflower.

To explore the complete biosynthesis process of flavonoid glycosides in safflower, specifically the key glycosyltransferase that might be involved, as well as to develop an efficient biocatalyst to synthesize flavonoid glycosides, a glycosyltransferase CtUGT4, with flavonoid-O-glycosyltransferase activity, was identified in safflower. The fusion protein of CtUGT4 was heterologously expressed in Escherichia coli, and the target protein was purified. The recombinant protein can catalyze quercetin to form quercetin-7-O-glucoside, and kaempferol to form kaempferol-3-O in vitro, and a series of flavones, flavonols, dihydroflavones, chalcones, and chalcone glycosides were used as substrates to generate new products. CtUGT4 was expressed in the tobacco transient expression system, and the enzyme activity results showed that it could catalyze kaempferol to kaempferol-3-O-glucoside, and quercetin to quercetin-3-O-glucoside. After overexpressing CtUGT4 in safflower, the content of quercetin-3-O-rutinoside in the safflower florets increased significantly, and the content of quercetin-3-O-glucoside also tended to increase, which preliminarily confirmed the function of CtUGT4 flavonoid-O-glycosyltransferase. This work demonstrated the flavonoid-O-glycosyltransferase function of safflower CtUGT4 and showed differences in the affinity for different flavonoid substrates and the regioselectivity of catalytic sites in safflower, both in vivo and in vitro, providing clues for further research regarding the function of UGT genes, as well as new ideas for the cultivation engineering of the directional improvement of effective metabolites in safflower.

Both Two CtACO3 Transcripts Promoting the Accumulation of the Flavonoid Profiles in Overexpressed Transgenic Safflower.

The unique flavonoids, quinochalcones, such as hydroxysafflor yellow A (HSYA) and carthamin, in the floret of safflower showed an excellent pharmacological effect in treating cardiocerebral vascular disease, yet the regulating mechanisms governing the flavonoid biosynthesis are largely unknown. In this study, CtACO3, the key enzyme genes required for the ethylene signaling pathway, were found positively related to the flavonoid biosynthesis at different floret development periods in safflower and has two CtACO3 transcripts, CtACO3-1 and CtACO3-2, and the latter was a splice variant of CtACO3 that lacked 5' coding sequences. The functions and underlying probable mechanisms of the two transcripts have been explored. The quantitative PCR data showed that CtACO3-1 and CtACO3-2 were predominantly expressed in the floret and increased with floret development. Subcellular localization results indicated that CtACO3-1 was localized in the cytoplasm, whereas CtACO3-2 was localized in the cytoplasm and nucleus. Furthermore, the overexpression of CtACO3-1 or CtACO3-2 in transgenic safflower lines significantly increased the accumulation of quinochalcones and flavonols. The expression of the flavonoid pathway genes showed an upward trend, with CtCHS1, CtF3H1, CtFLS1, and CtDFR1 was considerably induced in the overexpression of CtACO3-1 or CtACO3-2 lines. An interesting phenomenon for CtACO3-2 protein suppressing the transcription of CtACO3-1 might be related to the nucleus location of CtACO3-2. Yeast two-hybrid (Y2H), glutathione S-transferase (GST) pull-down, and BiFC experiments revealed that CtACO3-2 interacted with CtCSN5a. In addition, the interactions between CtCSN5a and CtCOI1, CtCOI1 and CtJAZ1, CtJAZ1 and CtbHLH3 were observed by Y2H and GST pull-down methods, respectively. The above results suggested that the CtACO3-2 promoting flavonoid accumulation might be attributed to the transcriptional activation of flavonoid biosynthesis genes by CtbHLH3, whereas the CtbHLH3 might be regulated through CtCSN5-CtCOI1-CtJAZ1 signal molecules. Our study provided a novel insight of CtACO3 affected the flavonoid biosynthesis in safflower.