Ectopic expression of BpbHLH9 suggested the presence of a self-activating loop mechanism of clade Ia bHLHs to enhance betulinic acid biosynthesis in <i>Lotus japonicus</i> 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 <i>Lotus japonicus</i>. 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 <i>BpbHLH9</i>, a homolog of <i>LjbHLH50</i> in <i>Betula platyphylla</i>, in <i>L. japonicus</i> hairy roots. Remarkably, <i>BpbHLH9</i> expression elicited metabolic and transcriptomic alterations almost similar to those induced by <i>LjbHLH50</i> overexpression, highlighting the conserved function of clade Ia bHLHs. Through RNA-sequencing analysis, we found that <i>LjbHLH50</i> 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 <i>BpbHLH9</i>, <i>LjbHLH50</i> and two <i>LjbHLH50</i> paralogs were upregulated upon BpbHLH9 induction, underscoring the central role of these clade Ia bHLHs in betulinic acid biosynthesis regulatory networks in <i>L. japonicus</i> hairy roots.