A bHLH Transcription Factor Regulates Bisbibenzyl Biosynthesis in the Liverwort Plagiochasma appendiculatum

Liverworts, a section of the bryophyte plants which pioneered the colonization of terrestrial habitats, produce cyclic bisbibenzyls as secondary metabolites. These compounds are generated via the phenylpropanoid pathway, similar to flavonoid biosynthesis, for which basic helix-loop-helix (bHLH) transcription factors have been identified as one of the important regulators in higher plants. Here, a bHLH gene homolog (PabHLH) was isolated from the liverwort species Plagiochasma appendiculatum and its contribution to bisbibenzyl biosynthesis was explored. Variation in the abundance of PabHLH transcript mirrored that of tissue bisbibenzyl content in three different liverwort tissues. A phylogenetic analysis based on the bHLH domain sequence suggested that the gene encodes a member of bHLH subgroup IIIf, which clusters proteins involved in flavonoid synthesis. The gene's transient expression in onion epidermal cells implied that its product localized to the nucleus, and a transactivation assays in yeast showed that it was able to activate transcription. In both callus and thallus, the overexpression of PabHLH boosted bisbibenzyl accumulation, while also up-regulating PaPAL, Pa4CL1, PaSTCS1 and two genes encoding P450 cytochromes, and its RNA interference (RNAi)-induced suppression down-regulated the same set of genes and reduced the accumulation of bisbibenzyls. The abundance of PaCHS and PaFNSI transcript was related to flavonoid accumulation in transgenic thallus. PabHLH represents a candidate for the metabolic engineering of bisbibenzyl content.