Insights into the Diversification of Subclade IVa bHLH Transcription Factors in Fabaceae

Abstract

Background: Fabaceae plants appear to contain larger numbers of subclade IVa basic-helix-loop-helix (bHLH) transcription factors than other plant families, and some members of this subclade have been identified as saponin biosynthesis regulators. We aimed to systematically elucidate the diversification of this subclade and obtain insights into the evolutionary history of saponin biosynthesis regulation in Fabaceae.Results: In this study, we collected sequences of subclade IVa bHLH proteins from 40 species, including fabids and other plants, and found greater numbers of subclade IVa bHLHs in Fabaceae. We confirmed conservation of the bHLH domain, C-terminal ACT-like domain, and exon-intron organisation among almost all subclade IVa members in model legumes, supporting the results of our classification. Phylogenetic tree-based classification of subclade IVa revealed the presence of three different groups. Interestingly, most Fabaceae subclade IVa bHLHs fell into group 1, which contained all legume saponin biosynthesis regulators identified to date. These observations support the co-occurrence and Fabaceae-specific diversification of saponin biosynthesis regulators. Comparing the expression of orthologous genes in Glycine max, Medicago truncatula, and Lotus japonicus, orthologues of MtTSAR1 (the first identified soyasaponin biosynthesis regulatory transcription factor) were not expressed in the same tissues, suggesting that group 1 members have gained different expression patterns and contributions to saponin biosynthesis during their duplication and divergence. On the other hand, groups 2 and 3 possessed fewer members, and their phylogenetic relationships and expression patterns were highly conserved, indicating that their activities may be conserved across Fabaceae.Conclusions: This study suggests subdivision and diversification of subclade IVa bHLHs in Fabaceae plants. The results will be useful for candidate selection of unidentified saponin biosynthesis regulators. Furthermore, the functions of groups 2 and 3 members are interesting targets for clarifying the evolution of subclade IVa bHLH transcription factors in Fabaceae.

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

“…4D). Fabaceae clade IVa bHLH TFs were further subdivided into three subclades, and subclade IVa1 included all Fabaceae triterpene saponin biosynthesis activators identified in clade IVa (Suzuki et al ., 2021). Interestingly, LjbHLH32 was a member of subclade IVa2 but not IVa1 (Fig.…”

“…4D). The orthologs in subclade IVa2 were highly conserved in Fabaceae plants and were commonly expressed in roots and nodules of model Fabaceae plants (Suzuki et al ., 2021). However, no subclade IVa2 bHLHs have been functionally characterized to date.…”

“…Sequences were collected as described previously (Suzuki et al ., 2021). The full-length peptide sequence of CjbHLH1 (BAJ40865.1; Yamada et al ., 2011) was retrieved from GenBank (https://www.ncbi.nlm.nih.gov/genbank/).…”

“…A phylogenetic tree of lateral organ boundaries domain (LBD) proteins was generated using Clustal Omega v. 1.2.3 (Sievers et al, 2011), FastTree v. 2.1.10 (Price et al, 2010), and MEGA X (Kumar et al, 2018) following our previous report (Suzuki et al, 2021).…”

“…Plant genomes possess a large number of basic helix-loop-helix (bHLH)-type transcription factors (TFs), which are classified into approximately 25 clades based on sequence similarity in the bHLH domain and other conserved domains (Heim et al ., 2003). Fabaceae plants possess more clade IVa bHLH genes than related plant families, which are subdivided into three subclades (IVa1-3) based on a phylogenetic tree of the full-length proteins (Suzuki et al ., 2021). Some members of subclade IVa1 are regulators of Fabaceae triterpene saponin biosynthesis.…”