Integrated single-nucleus transcriptomic and metabolomic insights into bud-to-leaf development and metabolite synthesis in tea plant

The tea plant is an important nonalcoholic beverage crop known for its abundant secondary metabolites, particularly in buds and leaves. However, the coordinated regulation of bud-to-leaf development and metabolism remains poorly understood. Here, we applied single-nucleus RNA sequencing (snRNA-Seq), bulk RNA sequencing (RNA-Seq), and metabolomics to comprehensively profile the developmental trajectory and metabolic characteristics of tea plant buds and leaves. The snRNA-Seq analysis revealed 17 cell clusters and 8 cell types in buds and leaves, respectively. Notably, the proportion of palisade mesophyll (PM) cells increased progressively during development, while proliferating cells (PC) decreased. Interestingly, key enzymes in the flavonoid biosynthetic pathway were specifically localized to PM cells. Metabolomic analyses demonstrated dynamic accumulation patterns of various metabolites, including phytohormones, flavonoids, and amino acids, as the buds transitioned to mature leaves. Using multi-omics profiling, we identified <i>CsmiRNA396b</i>, <i>CsUGT94P1</i>, <i>CsTCP3</i>, and <i>CsTCP14</i> as critical regulatory components. Enzyme activity assays confirmed that CsUGT94P1 catalyzes the conversion of flavonols into flavonol glycosides <i>in vitro</i>. Furthermore, <i>CsmiRNA396b</i> was found to regulate leaf development by inhibiting <i>CsGRF3</i> expression, while <i>CsTCP3</i> and <i>CsTCP14</i> played antagonistic roles in leaf development and flavonoid biosynthesis. Our findings provide novel insights into the regulatory mechanisms underlying bud-to-leaf development and metabolite production in tea plants.