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

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.