Characterization of AP2/ERF and bHLH transcription factors and association with terpenoid metabolism in Phlomoides rotata from the Qinghai-Tibet Plateau

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Phlomoides rotata , a Qinghai–Tibet Plateau endemic perennial valued for its hemostatic and anti-inflammatory effects, derives part of these bioactivities from terpenoid secondary metabolism. While AP2/ERF and bHLH transcription factors are established regulators of plant terpenoid biosynthesis, their roles in mediating P. rotata ’s adaptation to natural high-altitude gradients remain uncharacterized. Here, we integrated transcriptomic and metabolomic profiles from four wild P. rotata populations spanning the 3,540-4,270 m altitudinal gradient to investigate the associations of AP2/ERF and bHLH with terpenoid biosynthesis. Genome-wide identification retrieved 169 AP2/ERF and 212 bHLH family members, of which 71 AP2/ERF and 50 bHLH genes displayed significant differential expression across four populations. Phylogenetic analysis resolved AP2/ERF members into 15 subfamilies and bHLH members into 21 subfamilies, including a P. rotata -specific clade (subgroup XXII). We further focused on differential expression of AP2/ERF and bHLH. Bioinformatic profiling revealed most encoded proteins are nuclear-localized, acidic, and unstable, with phytohormone-responsive cis-elements dominating AP2/ERF promoters (39.31%) and abiotic stress-responsive elements enriched in bHLH promoters (41.55%). Weighted gene co-expression network analysis (WGCNA) identified eight modules, with the green module showing the strongest correlation with terpenoid metabolites (12 of 52 significant associations, | r | > 0.7, p < 0.01). Intra-modular analysis revealed distinct correlation patterns: specific AP2/ERF and bHLH TFs exhibited strong positive correlations with iridoid glycosides, such as shanzhiside methyl ester, and key structural genes, including IO and GGR , but strong negative correlations with triterpenoid saponins, such as azukisaponin IV. qRT-PCR validation confirmed the reliability of the transcriptomic profiles. This study offers insights into the correlative framework linking AP2/ERF and bHLH transcription factors with terpenoid secondary metabolism in P. rotata .