Comparative transcriptome analysis elucidates species-specific expression patterns of steroid biosynthesis genes in four Physalis species

The genus Physalis of Solanaceae plants is rich in steroidal compounds with various biological activities, such as physalins, which exhibit significant pharmacological potential. However, the genetic underpinnings and interspecific variation in their biosynthesis remain poorly understood. This study presents a comprehensive comparative transcriptome analysis of four Physalis species— P. angulata (PA), P. minima (PM), P. philadelphica (PP), and P. alkekengi var. franchetii (PAF)—to delineate the expression landscape of steroid biosynthesis genes. LC-MS/MS quantification revealed substantial interspecific disparity in physalin F content, with PAF exhibiting the highest accumulation. De novo assembly of leaf transcriptomes generated 88,732 unigenes, of which 51,492 were functionally annotated. Differential gene expression analysis uncovered significant transcriptional divergence in both the upstream terpenoid backbone (MVA and MEP) pathways and the downstream steroid biosynthetic pathway. Notably, PM displayed pronounced upregulation of most MVA and MEP pathway genes, while key steroid pathway enzymes, including CYP51G1, DWF1, DWF5, and SMT1, were predominantly expressed in PM. Furthermore, we identified 31 cytochrome P450s and four 3β-hydroxysteroid dehydrogenases (3β-HSDs), with several isoforms showing species-specific expression patterns, suggesting their potential roles in the structural diversification of steroidal lactones. Our findings provide crucial insights into the evolutionary diversification of steroid biosynthesis in Physalis and establish a valuable genetic resource for the metabolic engineering of high-value phytosteroids. Future functional validation of candidate CYP450s and 3β-HSDs, combined with spatiotemporal multi-omics analysis, will be essential to fully decipher the regulatory mechanisms of species-specific physalin accumulation.