Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Mar 25;12(7):uhaf096.
doi: 10.1093/hr/uhaf096. eCollection 2025 Jul.

The SmWRKY32-SmbHLH65/SmbHLH85 regulatory module mediates tanshinone biosynthesis in Salvia miltiorrhiza

Affiliations

The SmWRKY32-SmbHLH65/SmbHLH85 regulatory module mediates tanshinone biosynthesis in Salvia miltiorrhiza

Xiumin Nie et al. Hortic Res. .

Abstract

Tanshinones are valuable compounds found in Salvia miltiorrhiza, and gaining a deeper understanding of their transcriptional regulation mechanisms is a key strategy for increasing their content. Previous research revealed that SmWRKY32 acts as a repressor of tanshinone synthesis. This study identified the SmbHLH65 transcription factor, whose expression was significantly reduced in the SmWRKY32 overexpression transcriptome. Overexpression of SmbHLH65 stimulated tanshinone accumulation, while its silencing resulted in a decrease in tanshinone content. However, SmbHLH65 does not directly target the key enzyme genes involved in tanshinone synthesis. Subsequently, we discovered the SmbHLH65-interacting protein SmbHLH85. SmbHLH85 facilitates tanshinone biosynthesis by directly upregulating SmDXS2 and SmCPS1. Further investigation demonstrated that SmbHLH65 not only promotes the expression of SmbHLH85 but also enhances its binding to the promoters of SmDXS2 and SmCPS1, thereby amplifying the activation of these biosynthetic genes. Additionally, SmWRKY32 directly binds to the SmbHLH65 promoter to suppress its activity. In summary, these findings reveal that the regulatory module SmWRKY32-SmbHLH65/SmbHLH85 controls tanshinone synthesis in S. miltiorrhiza. This study uncovers a novel transcriptional regulatory mechanism, offering fresh insights into the complex network controlling tanshinone biosynthesis.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Molecular characterization and subcellular localization of SmbHLH65. (A) Multiple sequence alignment of SmbHLH65 with other plant species bHLH proteins, including AtbHLH30 (AT1G68810.1), OsbHLH30 (XP_015648851.1), GmbHLH30 (XP_006573268.1), and SibHLH30 (XP_011077316.1). The bHLH domain is indicated by lines, and the HER motif (His5-Glu9-Arg13) is marked by a pentagram. (B) The SmbHLH65 relative expression levels in leaf, flower, stem, and root tissues of S. miltiorrhiza. Leaf samples served as the experimental control. Error bars represent standard deviation (SD) (Student’s t-test, **P < 0.01). (C) Subcellular localization of SmbHLH65 in tobacco. SmbHLH65-GFP was cotransformed with the nuclear marker SmWRKY2-mCherry. Scale bar = 20 μm.
Figure 2
Figure 2
SmbHLH65 promotes tanshinone biosynthesis by upregulating key biosynthetic genes. (A) Content of tanshinones in EV, ATCC, and SmbHLH65 transgenic hairy roots. DW refers to dry weight. DT, CT, TI, and TIIA. TT was the sum of these four types of tanshinones. (B) Transcription levels of key enzyme genes associated with tanshinone biosynthesis in SmbHLH65 transgenic lines. Error bars indicate SD. Ordinary one-way ANOVA with multiple comparisons, **P < 0.01, *P < 0.05.
Figure 3
Figure 3
SmbHLH65 interacts with SmbHLH85. (A) Y2H experiments demonstrated the interaction of SmbHLH65 with SmbHLH85. (B) Pull-down assays confirmed the interaction of SmbHLH65 with SmbHLH85. (C) Split-luciferase complementation assays indicated that SmbHLH65 interacts with SmbHLH85 in tobacco leaves.
Figure 4
Figure 4
SmbHLH85 actively regulates tanshinone biosynthesis by modulating biosynthetic pathway genes. (A) Tanshinone concentration in overexpressing and silencing SmbHLH85. DT, CT, TI, and TIIA. TT was the sum of DT, CT, TI, and TIIA. (B) Transcript levels of key tanshinone synthesis enzyme genes in EV, ATCC, and SmbHLH85 transgenic lines. Error bars indicate SD. Ordinary one-way ANOVA with multiple comparisons, **P < 0.01, *P < 0.05.
Figure 5
Figure 5
SmbHLH85 activates the transcription of SmDXS2 and SmCPS1 through direct binding to their respective promoters. (A) Y1H assays displayed that SmbHLH85 binds to SmDXS2 and SmCPS1 promoters. (B) EMSA experiments showed that SmbHLH85 binds to the biotin-labeled probes derived from the promoter of SmDXS2 and SmCPS1. m, mutant. (C) Dual-LUC assays exhibited that SmbHLH85 activates the transcription of both SmDXS2 and SmCPS1. Error bars represent SD (Student's t-test, **P < 0.01).
Figure 6
Figure 6
SmbHLH65 positively regulates the expression of SmbHLH85 and enhances the activation of SmDXS2 and SmCPS1 mediated by SmbHLH85. (A) EMSA assays demonstrated that SmbHLH65 strengthens the binding of SmbHLH85 to the SmDXS2 and SmCPS1 promoters. (B) Dual-LUC assays indicated that SmbHLH65 amplifies the transcriptional activation of SmDXS2 and SmCPS1 by SmbHLH85. (C) Relative expression levels of SmbHLH85 in SmbHLH65 overexpression and RNAi lines. (D) Transcription levels of SmbHLH65 in SmbHLH85 transgenic hair roots. Error bars represent SD (Student’s t-test, **P < 0.01).
Figure 7
Figure 7
SmWRKY32 binds to the SmbHLH65 promoter and suppresses its transcription activity. (A) Transcript levels of SmbHLH65 in the EV, ATCC, SmWRKY32O, and SmWRKY32R lines. (B and C) Y1H and EMSA experiments validated the binding between SmWRKY32 and the promoter of SmbHLH65. m, mutant. (D) Dual-LUC assays showed that SmWRKY32 represses the transactivation activity of the SmbHLH65 promoter. Error bars represent SD (Student’s t-test, **P < 0.01).
Figure 8
Figure 8
A working model of the SmWRKY32-SmbHLH65/SmbHLH85 regulation of tanshinone biosynthesis. SmWRKY32 binds to the SmbHLH65 promoter, suppressing its expression. SmbHLH65 positively regulates tanshinone biosynthesis by forming a complex with SmbHLH85, which promotes synthesis by increasing SmDXS2 and SmCPS1 transcription. Additionally, SmbHLH65 stimulates the expression of SmbHLH85. SmWRKY32 indirectly inhibits tanshinone biosynthesis by suppressing the activation complex formed by SmbHLH65 and SmbHLH85.

Similar articles

References

    1. Jiang Z, Gao W, Huang L. Tanshinones, critical pharmacological components in Salvia miltiorrhiza. Front Pharmacol. 2019;10:202. - PMC - PubMed
    1. Li Z, Xu S, Liu P. Salvia miltiorrhiza Burge (Danshen): a golden herbal medicine in cardiovascular therapeutics. Acta Pharmacol Sin. 2018;39:802–24 - PMC - PubMed
    1. Mei X, Cao Y, Che Y. et al. Danshen: a phytochemical and pharmacological overview. Chin J Nat Med. 2019;17:59–80 - PubMed
    1. Dai Z, Cui G, Zhou S. et al. Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme a reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation. Plant Physiol. 2011;168:148–57 - PubMed
    1. Wang Z, Peters RJ. Tanshinones: leading the way into Lamiaceae labdane-related diterpenoid biosynthesis. Curr Opin Plant Biol. 2022;66:102189 - PMC - PubMed

LinkOut - more resources