Uncovering Carbohydrate Metabolism and Endogenous Hormone Regulation during Flush Phenology in Citrus Trees using Proteomics and Metabolomics

Rapid shoot growth (flushing) phenology is a fundamental developmental process in perennial woody plants such as citrus. In a separate study, we identified physiological shifts from photosynthesis to mobilization of nitrogen and carbohydrate to support new shoot growth. However, the underlying molecular and biochemical signals remain largely unknown. Here, we integrated proteomic and metabolomic analyses to investigate carbohydrate and hormone dynamics across three flush stages in Citrus sinensis: quiescent period (stage 1), new shoot initiation (stage 2), and full expansion (stage 3). Sucrose, maltose, and trehalose accumulated in apical leaves during early shoot initiation and declined during subsequent shoot expansion, indicating depletion of carbohydrate reserves and enhanced resource remobilization. These changes were accompanied by coordinated regulation of starch-metabolizing enzymes, including ADP-glucose pyrophosphorylase, -amylase, and isoamylase, supporting a transition from carbon storage to carbon export during active shoot growth. Indole-3-acetic acid increased continuously across stages, while trans-zeatin and gibberellin A{square} showed opposite trends in apical versus basal leaves before jointly increasing at stage 3. Hormone analysis revealed dynamic and coordinated signaling changes during flush development. Abscisic acid declined from stage 1 to 2, whereas jasmonoyl-isoleucine and salicylic acid increased from stage 2 to 3. Some hormone-responsive proteins, including Gretchen Hagen 3 and Gibberellin-insensitive dwarfing 1, exhibited expression patterns consistent with hormonal fluctuations. Together, these results support a stage-specific regulatory framework in which carbohydrate metabolism and hormone signaling are tightly coordinated to regulate rapid source-sink transitions during citrus flush development. HighlightWe reveal how carbohydrate metabolism and hormone signaling are spatiotemporally coordinated during citrus shoot growth phenology, and we develop an integrated metabolic-hormonal model that connects carbon allocation to developmental transitions.