Uncovering the auxin regulatory mechanism underlying the development of single-leaf buds in Cremastra appendiculata via integrated transcriptomic and proteomic analyses

Cremastra appendiculata, a medicinal orchid of rare occurrence, exhibits a single-leaf bud growth pattern that results in extremely low production efficiency and consequently substantially restricts the development of the associated industry. However, the regulatory basis underlying this growth strategy remains elusive. UPLCMS/MS detection analysis showed that free IAA (indole-3-acetic acid) levels in sprouting buds and growing buds were significantly greater than those in dormant buds and growth-inhibited buds. To investigate the basis of this uneven distribution of free IAA, transcriptomic analysis demonstrated that auxin homeostasis in leaf buds is regulated by key genes involved in its biosynthesis (CaTAA1, CaYUCCA, CaALDH), metabolism (CaDAO, CaGH3) and transport (CaPIN, CaLAX). In contrast, proteomic analysis revealed that leaf bud growth is mainly regulated by key enzymes involved in auxin biosynthesis and metabolism, which maintain appropriate free IAA homeostasis to promote or suppress leaf bud growth. Further analyses at three levels, gene transcription, protein expression, and signaling molecules, revealed that C. appendiculata finely regulates endogenous auxin homeostasis through bidirectional control of auxin biosynthesis and catabolism. Actively sprouting buds maintain a high steady-state concentration of endogenous auxin through strong auxin biosynthetic capacity and weak catabolic activity, which continuously activate growth-related signaling pathways and promote bud growth. In contrast, growth-inhibited buds stabilize endogenous auxin at a low threshold through coordinated regulation of downregulated biosynthesis and persistently high catabolism, thereby blocking the transmission of bud growth signals and inhibiting bud growth. This study provides insights into the development of high-efficiency production technologies for C. appendiculata and lays a foundation for future theoretical studies of rhizome branching.