Isolation of mesophyll protoplasts from tea ( Camellia sinensis ) and localization analysis of enzymes involved in the biosynthesis of specialized metabolites

Specialized metabolites in tea (Camellia sinensis) are fundamental quality factors. It is important to characterize gene function in vivo to identify key enzymes and reactions involved in the biosynthesis of such metabolites. Here we report a transient expression method to analyze gene function in isolated tea mesophyll protoplasts. This method is an alternative approach to traditional genetic transformation for studies on gene function in vivo. We screened several tea cultivars and different digestion conditions to optimize protoplast isolation. Digestion of newly emerged leaves of C. sinensis ‘Zhongbai 4’ with 3% cellulase R-10 and 0.3% macerozyme R-10 for about 12 h yielded approximately 107 mesophyll protoplasts. Genes encoding enzymes involved in secondary metabolite synthesis were transiently expressed in the protoplasts, and their subcellular locations were determined. With further improvements in the transfection efficiency, this transient expression system will contribute greatly to the analyses of in vivo gene function in tea.