Identification of basic helix-loop-helix transcription factors that activate betulinic acid biosynthesis by RNA-sequencing of hydroponically cultured Lotus japonicus

Although triterpenes are ubiquitous in plant kingdom, their biosynthetic regulatory mechanisms are limitedly understood. Here, we found that hydroponic culture dramatically activated betulinic acid (BA) biosynthesis in the model Fabaceae Lotus japonicus, and investigated its transcriptional regulation. Fabaceae plants develop secondary aerenchyma (SA) on the surface of hypocotyls and roots during flooding for root air diffusion. Hydroponic culture induced SA in L. japonicus and simultaneously increased the accumulation of BA and the transcript levels of its biosynthetic genes. RNA-sequencing of soil-grown and hydroponically cultured plant tissues, including SA collected by laser microdissection, revealed that several transcription factor genes were co-upregulated with BA biosynthetic genes. Overexpression of LjbHLH32 and LjbHLH50 in L. japonicus transgenic hairy roots upregulated the expression of BA biosynthesis genes, resulting in enhanced BA accumulation. However, transient luciferase reporter assays in Arabidopsis mesophyll cell protoplasts showed that LjbHLH32 transactivated promoters of biosynthetic genes in the soyasaponin pathway but not the BA pathway, like its homolog GubHLH3, a soyasaponin biosynthesis regulator in Glycyrrhiza uralensis. This suggested the evolutionary origin and complex regulatory mechanisms of BA biosynthesis in Fabaceae. This study sheds light on the unrevealed biosynthetic regulatory mechanisms of triterpenes in Fabaceae plants.