Spatial multi-omics reveals the putative biosynthetic sites and distribution patterns of key flavor-contributing metabolites in tea leaves

The quality of tea (Camellia sinensis) is determined by key metabolites, including catechins, caffeine, theanine, and volatiles, but their biosynthesis and spatial distribution at the cellular level remain poorly understood. Here, we constructed a cell-type-resolved spatial multi-omics atlas of tea leaves from C. sinensis var. sinensis 'Jinxuan' and C. sinensis var. assamica 'Yinghong No. 9' by integrating single-nucleus RNA sequencing, physically isolated cell-type multi-omics, and matrix-assisted laser desorption/ionization mass spectrometry imaging. Multi-omics analyses identified mesophyll cells as the major site of catechin biosynthesis, particularly for terminal esterification steps. Theanine biosynthesis is predominantly associated with mesophyll cells, but theanine accumulates in both mesophyll and vascular bundle cells, consistent with the distribution of its precursors, glutamate and glutamine. Caffeine biosynthesis and accumulation are mainly associated with mesophyll cells, whereas other alkaloids show vascular-associated accumulation patterns. Benzenoid and fatty acid-derived volatiles show broader cellular distributions, whereas monoterpene biosynthesis is mainly associated with mesophyll and epidermal cells, and glycosylated monoterpene derivatives accumulate preferentially in vascular tissues. Further comparison of the two cultivars showed that linalool and geraniol display distinct cultivar-dependent accumulation patterns associated with cell density, synthase expression, and enzyme catalytic properties. Overall, key flavor-related metabolites are mainly synthesized in mesophyll cells, whereas vascular bundle cells may contribute to their accumulation and redistribution. These findings reveal the cellular organization of tea flavor metabolism and provide a cellular and molecular framework for future evaluation of tea germplasm with desirable flavor traits.