Analysis of BpbHLH Gene Family Responsive to MeJA Signalling in Betula platyphylla Suk. and Functional Mechanisms of BpbHLH42/44 in Genetic Improvement and Triterpenoid Biosynthesis

The basic helix-loop-helix (bHLH) transcription factor family regulates plant secondary metabolism, development and stress responses. Although triterpenoids such as betulinic acid (BA), betulin (BT) and oleanolic acid (OA) from Betula platyphylla Suk. are of pharmacological importance, the methyl jasmonate (MeJA)-induced regulation of IVa bHLHs remains unclear. A total of 131 BpbHLH genes were identified, and functional characterisation was performed for two IVa members, BpbHLH42 and BpbHLH44, in yeast, tobacco, birch cells and transgenic plants. Both enhanced triterpenoid biosynthesis but exhibited distinct specificities: BpbHLH42 primarily promoted OA accumulation by activating BpHMGR, BpSE1/3, BpW and BpY6, while BpbHLH44 favoured BT production through BpW, BpY6 and BpY11. Both indirectly regulated BpY9 via BpMYB21, forming a bHLH-MYB complex, and the MeJA repressor BpJAZ2 disrupted the BpbHLH42-MYB21 interaction. Transgenic birch lines further revealed divergent phenotypes: BpbHLH42 overexpression enlarged and smoothed leaves and increased OA levels, whereas BpbHLH44 enhanced alkali stress tolerance by elevating secondary metabolite accumulation. These results uncover the differential regulatory roles of BpbHLH42 and BpbHLH44 in triterpenoid biosynthesis and demonstrate their potential for metabolic engineering and genetic improvement in woody plants.