ABA-responsive transcription factor CbNAC63 is involved in modulating biosynthesis of triterpenoid saponins in Conyza blinii

Conyza blinii, a traditional Chinese medicinal herb, accumulates triterpenoid saponins. NAC transcription factors (TFs) are pivotal for plant stress responses and hormone-mediated secondary metabolism, while abscisic acid (ABA) regulates terpenoid biosynthesis in medicinal plants. However, how NAC TFs mediate ABA-regulated triterpenoid saponin synthesis in C. blinii remains unclear. In order to address this gap, the focus was directed towards CbNAC63--a NAC TF identified from the nocturnal low-temperature transcriptome of C. blinii. Phylogenetic and motif analyses showed it belongs to the stress-responsive NAC subclade with conserved domains. Y2H assays revealed its self-interaction, enhancing transcriptional activity; subcellular localization confirmed nuclear localization. RT-qPCR results indicated CbNAC63 exhibits tissue-specific expression under nocturnal low-temperature stress, accompanied by increased triterpenoid saponin accumulation. ABA signaling primarily regulates this process: exogenous ABA enhanced CbNAC63 expression and saponin content, while fluridone suppressed both. Overexpression (OE) and virus-induced gene silencing (VIGS) demonstrated CbNAC63 positively regulates saponin biosynthesis. Y1H and dual-luciferase reporter assays confirmed CbNAC63 directly binds to the promoters of Cb{beta}AS and CbHMGR and activates their transcription. In summary, the study shows CbNAC63 integrates ABA and low-temperature signals, activating MVA pathway genes to regulate saponin synthesis. This unveils a novel "ABA-CbNAC63-MVA" module, enriches the classic regulatory network, and provides a target for improving medicinal plant quality. HighlightsIdentifies CbNAC63 as a novel regulator that integrates ABA and cold signals to boost saponin production in medicinal plants.