is a member of a bHLH gene cluster regulating terpenoid indole alkaloid biosynthesis in .

Basic helix-loop-helix (bHLH) transcription factors (TFs) are key regulators of plant specialized metabolites, including terpenoid indole alkaloids (TIAs) in . Two previously characterized subgroup-IVa bHLH TFs, BIS1 (bHLH Iridoid Synthesis 1) and BIS2 regulate iridoid biosynthesis in the TIA pathway. We reanalyzed the recently updated genome sequence and discovered that and are clustered on the same genomic scaffold with a previously uncharacterized bHLH gene, designated as . Only a few bHLH gene clusters have been studied to date. Comparative analysis of 49 genome sequences from different plant lineages revealed the presence of analogous bHLH clusters in core angiosperms, including the medicinal plants (giant milkweed) and (yellow jessamine), but not in the analyzed basal angiosperm and lower plants. Similar to the iridoid pathway genes, is highly expressed in roots and induced by methyl jasmonate. BIS3 activates the promoters of iridoid branch genes, (), (), (), (), (), and (), but not (). Transactivation of the promoters was abolished when BIS3 is converted to a dominant repressor by fusing with the ERF-associated amphiphilic repression (EAR) sequence. In addition, BIS3 acts synergistically with BIS1 and BIS2 to activate the promoter in tobacco cells. Mutation of the known bHLH TF binding motif, G-box (CACGTG) in the promoter significantly reduced but did not abolish the transactivation by BIS3. Promoter deletion analysis of suggests that the sequences adjacent to the G-box are also involved in the regulation by BIS3. Overexpression of in flower petals significantly upregulated the expression of iridoid biosynthetic genes and increased loganic acid accumulation. expression was significantly induced by BIS3 although BIS3 did not directly activate the promoter. Our results advance our understanding of the regulation of plant specialized metabolites by bHLH TF clusters.