Phytohormone and transcriptome analyses unravel the molecular mechanisms of heterophylly in Pinellia ternata

Pinellia ternata (Thunb.) Breit. exhibits significant leaf shape variation, which is closely associated with its growth, development, and medicinal quality. However, the physiological and molecular mechanisms underlying heterophylly remain poorly understood. In this study, we systematically investigated the relationship between leaf shape variation and phytohormone regulation as well as gene expression. Four distinct leaf types of P. ternata (peony-leaf type, peach-leaf type, bamboo-leaf type, and willow-leaf type) were used as materials. Leaf traits were measured, and the contents of auxin (IAA), cytokinin (CTK), and gibberellin (GA₃) were quantified using ELISA. Transcriptome sequencing was performed to identify differentially expressed genes (DEGs) among the leaf types, followed by GO and KEGG enrichment analyses. Candidate genes related to hormone synthesis and signal transduction were validated by RT-qPCR. Significant differences were observed in leaf length, leaf width, and length-to-width ratio among the four leaf types, with the willow-leaf type having the longest leaves and the peony-leaf type the widest leaves. IAA content showed a strong positive correlation with leaf length and length-to-width ratio, and a strong negative correlation with leaf width, leaf base angle, and leaf tip angle, suggesting that IAA may be a key hormone influencing leaf shape formation. Transcriptome analysis identified a large number of DEGs, with the willow-leaf type showing the most distinct gene expression profile. GO and KEGG enrichment analyses indicated that the DEGs were mainly involved in pathways such as secondary metabolite biosynthesis and starch and sucrose metabolism. Several candidate genes, including PtIAA13, PtGID2, PtARF19, and PtGA3ox1, were screened and validated. Leaf shape variation in P. ternata is regulated by endogenous hormones, especially IAA, and is closely associated with the expression of related genes. The peony-leaf type exhibited the most stable gene expression pattern. This study provides a theoretical basis for understanding the physiological and molecular mechanisms underlying leaf shape formation in P. ternata and offers references for variety breeding and quality control of this medicinal plant.