DataSheet_1_Functional identification of purine permeases reveals their roles in caffeine transport in tea plants (Camellia sinensis).xlsx

Caffeine is a characteristic secondary metabolite in tea plants. It confers tea beverage with unique flavor and excitation effect on human body. The pathway of caffeine biosynthesis has been generally established, but the mechanism of caffeine transport remains unclear. Here, eight members of purine permeases (PUPs) were identified in tea plants. They had diverse expression patterns in different tissues, suggesting their broad roles in caffeine metabolism. In this study, F1 strains of "Longjing43" ♂ × "Baihaozao" ♀ and different tea cultivars were used as materials to explore the correlation between caffeine content and gene expression. The heterologous expression systems of yeast and Arabidopsis were applied to explore the function of CsPUPs. Correlation analysis showed that the expressions of CsPUP1, CsPUP3.1, and CsPUP10.1 were significantly negatively correlated with caffeine content in tea leaves of eight strains and six cultivars. Furthermore, subcellular localization revealed that the three CsPUPs were not only located in plasma membrane but also widely distributed as circular organelles in cells. Functional complementation assays in yeast showed that the three CsPUPs could partly or completely rescue the defective function of fcy2 mutant in caffeine transport. Among them, transgenic yeast of CsPUP10.1 exhibited the strongest transport capacity for caffeine. Consistent phenotypes and functions were further identified in the CsPUP10.1-over-expression Arabidopsis lines. Taken together, it suggested that CsPUPs were involved in caffeine transport in tea plants. Potential roles of CsPUPs in the intracellular transport of caffeine among different subcellular organelles were proposed. This study provides a theoretical basis for further research on the PUP genes and new insights for caffeine metabolism in tea plants.

咖啡因是茶树特有的次生代谢产物，赋予茶饮料独特风味，并对人体产生兴奋作用。目前，咖啡因的生物合成途径已基本明确，但咖啡因的转运机制仍不清楚。本研究在茶树中鉴定出8个嘌呤通透酶（purine permeases，PUPs）家族成员，它们在不同组织中呈现多样化的表达模式，提示其在咖啡因代谢中发挥广泛作用。本研究以“龙井43”（♂）ד白毫早”（♀）的F1代品系及不同茶树品种为材料，探究咖啡因含量与基因表达之间的相关性；并通过酵母和拟南芥异源表达系统，解析CsPUPs的功能。相关性分析结果显示，CsPUP1、CsPUP3.1及CsPUP10.1的表达量与8个F1品系和6个茶树品种的茶叶咖啡因含量呈显著负相关。进一步的亚细胞定位实验表明，这3个CsPUP蛋白不仅定位于质膜，还广泛分布于细胞内的环状细胞器中。酵母功能互补实验显示，这3个CsPUP可部分或完全恢复fcy2突变体的咖啡因转运缺陷功能；其中，CsPUP10.1的转基因酵母对咖啡因的转运能力最强。在CsPUP10.1过表达拟南芥株系中进一步验证得到了一致的表型与功能结果。综上，本研究表明CsPUPs参与茶树的咖啡因转运过程，并提出CsPUPs可能在不同亚细胞细胞器间的咖啡因胞内转运中发挥作用。本研究为PUP基因的后续研究提供了理论基础，也为茶树咖啡因代谢研究提供了新的见解。