Ectopic expression of BpbHLH9 suggested the presence of a self-activating loop mechanism of clade Ia bHLHs to enhance betulinic acid biosynthesis in Lotus japonicus hairy roots

For the optimal production of specialized (secondary) metabolites in plant hosts, a comprehensive understanding of their regulatory mechanisms is imperative. Bioactive C-28-oxidized triterpenes, such as oleanolic, ursolic, and betulinic acids, are metabolites ubiquitously found across the plant kingdom; however the precise regulatory mechanisms governing their biosynthesis remain elusive. Previously, we demonstrated that the clade Ia bHLH transcription factor, LjbHLH50, plays a pivotal role in the upregulation of betulinic acid biosynthesis in Lotus japonicus. However, inconsistent outcomes have been observed in transient effector-reporter assays, which are commonly employed in transcription factor studies. Thus, in the present study, we sought to further characterize LjbHLH50 by examining the ectopic expression of BpbHLH9, a homolog of LjbHLH50 in Betula platyphylla, in L. japonicus hairy roots. Remarkably, BpbHLH9 expression elicited metabolic and transcriptomic alterations almost similar to those induced by LjbHLH50 overexpression, highlighting the conserved function of clade Ia bHLHs. Through RNA-sequencing analysis, we found that LjbHLH50 was upregulated by ectopic BpbHLH9 expression, implying the existence of a self-activating loop in clade Ia bHLHs that facilitates enhanced betulinic acid biosynthesis. Notably, among the clade Ia bHLHs homologous to BpbHLH9, LjbHLH50 and two LjbHLH50 paralogs were upregulated upon BpbHLH9 induction, underscoring the central role of these clade Ia bHLHs in betulinic acid biosynthesis regulatory networks in L. japonicus hairy roots.