Transcription factors TgbHLH42-1 and TgbHLH42-2 positively regulate anthocyanin biosynthesis in Tulip (Tulipa gesneriana L.)

The floral coloration of tulip flowers is one of the most prominent traits contributing to its high ornamental value. The molecular mechanisms of petal coloration remain elusive in tulip species. In this study, we performed comparative metabolome and transcriptome analyses using four tulip cultivars with distinguished petal colors. Four types of anthocyanins were identified, including cyanidin derivatives and pelargonidin derivatives. Comparative transcriptome analysis identified 22,303 differential expressed genes (DEGs) from the four cultivars, and 2589 DEGs were commonly regulated in three comparison groups (colored vs. white cultivar), including anthocyanins biosynthesis-related genes and regulatory transcription factors. Two basic helix-loop-helix (bHLH) transcription factors, TgbHLH42-1 and TgbHLH42-2, with differential expression levels among cultivars and petal developmental stages, have high homology to TRANSPARENT TESTA 8 (AtTT8) of Arabidopsis. The anthocyanins accumulation in TgbHLH42-1 overexpressing (OE) seedlings was markedly greater than that in wild-type seedlings in the presence of methyl jasmonate (MeJA), but not for TgbHLH42-2 OE seedlings. Both TgbHLH42-1 and TgbHLH42-2 restored pigmentation defects in tt8 mutant seeds after complementation assay. TgbHLH42-1 could interact with MYB protein AtPAP1 to synergistically activate the transcription of AtDFR, whereas TgbHLH42-2 failed to. Silencing TgbHLH42-1 or TgbHLH42-2 individually could not, but simultaneously silencing both TgbHLH42 could reduce the anthocyanin in tulip petals. These results indicate that TgbHLH42-1 and TgbHLH42-2 function partially redundantly to positively regulate anthocyanin biosynthesis during tulip petal coloration.