The goal of the present study was to compare the structural and compositional differences of cuticle between tender leaf and fully-expanded leaf in Camellia sinensis, and provide metabolic base for the further characterization of wax biosynthesis in this economically important crop species. The tender second leaf and the fully-expanded fifth leaf from new twig were demonstrated to represent two different developmental stages, their cuticle thickness were measured by transmission electron microscopy. The thickness of the adaxial cuticle on the second and fifth leaf was 1.15 µm and 2.48 µm, respectively; the thickness of the abaxial cuticle on the second and fifth leaf was 0.47 µm and 1.05 µm, respectively. The thickness of the epicuticular wax layer from different leaf position or different sides of same leaf were similar. However, the intracuticular wax layer of the fifth leaf was much thicker than that of the second leaf. Total wax lipids were isolated from the second leaf and the fifth leaf, respectively. Gas chromatography-mass spectrometry analysis identified 51 wax constituents belonging to 13 chemical classes, including esters, glycols, terpenoids, fatty acids and their derivatives. Wax coverage on the second and fifth leaf was 4.76 µg/cm² and 15.38 µg/cm², respectively. Primary alcohols dominated in the tender second leaf. However, triterpenoids were the major components from the fully-expanded fifth leaf. The predominant carbon chains varied depending on chemical class. These data showed that the wax profiles of Camellia sinensis leaves are development stage dependent, suggesting distinct developmental dependent metabolic pathways and regulatory mechanisms.

Lipid droplets (LDs) have been widely found from diverse species and exhibit diverse functions. It remains unexplored what potential roles they played in tea. To address this question, we analyzed the chemical composition and the dynamic changes of cytosolic LDs during leaf growth and diurnal cycle. Using TopFluor cholesterol and Nile Red staining we demonstrated that cytosolic LDs were heterogeneous in tea tree (Camellia sinensis cv. Tieguanyin); the size and number of LDs increased with leaf growth. Compositional analysis showed that terpenoid esters and diacylglycerol are the major components of cytosolic LDs. The contents of total sterol esters (SEs) and β-amyrin esters increased with leaf expansion and growth; individual SE also showed diurnal changes. Our data suggest that cytosolic LDs from tea tree leave mainly serve as storage site for free sterols and triterpenoids in the form of esters. Cytosolic LDs were not the major contributors to the aroma quality of made tea.