Comprehensive characterization of the bHLH transcription factor family in Curcuma wenyujin and functional elucidation of CwbHLH27 in jasmonate-regulated sesquiterpenoid biosynthesis.

Curcuma wenyujin is acknowledged as a crucial medicinal plant containing essential oils, primarily composed of sesquiterpenoids. While numerous sesquiterpenoids exhibit versatile physiological activities, their levels in C. wenyujin are generally low, particularly the pivotal anti-cancer component elemene. Our previous research demonstrated that basic helix-loop-helix (bHLH) is involved in modulating jasmonate-mediated sesquiterpenoid biosynthesis. In this study, a total of 106 CwbHLHs were identified and systematically analyzed. Under MeJA treatment, the expression levels of CwbHLH15, CwbHLH27, CwbHLH58, CwbHLH73, and CwbHLH89 were significantly upregulated, whereas CwbHLH81 was downregulated. Subsequently, CwbHLH27 was selected for further functional characterization. CwbHLH27 overexpression resulted in increased levels of β-elemene, γ-elemene, β-caryophyllene, and curzerene in C. wenyujin leaves. The expression levels of CwHMGS, CwHMGR, CwDXS, CwDXR, CwFPPS, and CwHDR, key enzyme genes in sesquiterpenoid biosynthesis, were upregulated in transgenic lines. Conversely, CwbHLH27 silencing resulted in the opposite effects. Further analysis revealed that CwbHLH27 activated the transcription of CwHMGS, CwHMGR, and CwDXS by directly binding to the E-box cis-elements within their promoters. Moreover, CwbHLH27 interacts with CwJAZ1/17, thereby executing JA signal transduction and regulating sesquiterpenoid biosynthesis in C. wenyujin. Finally, we elucidated the molecular mechanism by which the CwJAZs-CwbHLH27 regulatory module regulates sesquiterpenoid biosynthesis in response to JA signaling. Our research provides a molecular foundation for biotechnological-assisted breeding of varieties with enhanced active ingredient content.