Roles Of Purine Permeases In Caffeine Transport In Camellia Sinensis

Caffeine is an important functional metabolite in plants,which has effects of excitement and diuresis on human body.Caffeine in tea plants(Camellia sinensis)not only participate in plant development and growth,but also determines tea flavor and quality along with other secondary metabolites,such as theanine and catechins.The accumulation of caffeine in tea plants is much higher than those in other plants,even for the coffee plants.Moreover,different from coffee and cacao plants,the synthesis and accumulation of caffeine in tea plants mainly occur in leaves,rather than in seeds and fruits.The pathway of caffeine biosynthesis has been generally established.However,studies about its degradation and transport process have not been reported.In this research,F1 strains of longjing43♂ × baihaozao♀ and different tea cultivars and were used as materials to study the caffeine metabolism and function of purine permease(PUP).High performance liquid chromatography(HPLC),real-time quantitive PCR(q RT-PCR),subcellular localization,yeast and Arabidopsis heterologous expression systems were applied to explore the gene function of Cs PUPs and molecular mechanism in caffeine transport.The main conclusions are as follows:1.‘zhongcha108’ and its offspring cultivar ‘zhongming7’ were used to investigate the variation in characteristic compounds and related gene expression at different leaf positions.An integrated correlation analysis of secondary metabolites and relative gene expression was conducted to identify the regulatory networks.Competition for substrates among theanine,caffeine,and catechins biosynthesis was identified.Moreover,these data revealed different regulatory mechanisms of secondary metabolism within two genetically similar tea cultivars,especially for caffeine pathway.2.To explore the molecular mechanism of caffeine hyper-accumulation in tea plants,Cs PUPs were selected for further study.Eight Cs PUPs members were screened base on the ‘shuchazao’(Camellis sinensis var.sinensis)genome database.Bioinformatic analysis showed that the Cs PUPs contained 813-1299 bp and encoded270-432 amino acids,which randomly distributed on six chromosomes.Ten transmembrane structures were found in all Cs PUPs,except Cs PUP1 and Cs PUP10.2.The expression modes of Cs PUPs in different tissues were further investigated.The results showed that Cs PUPs mainly expressed in aerial part of tea plants.Cs PUP1,Cs PUP3.1,Cs PUP3.3,Cs PUP10.1 and Cs PUP10.2 expressed higher in leaves.While Cs PUP3.2 and Cs PUP4 showed the highest expression level in stems.3.Young,mature,and old leaves in different tea cultivar and F1 strains of longjing43 × baihaozao were used for caffeine content determination.Further analysis about Cs PUPs gene expression and correlation showed that Cs PUP1,Cs PUP3.1,Cs PUP10.1,and Cs PUP10.2 expressed higher in mature and old leaves,and showed negative correlation with caffeine in different materials.While Cs PUP3.2,Cs PUP3.3and Cs PUP4 showed higher expression levels in young leaves than in mature and old leaves,and positively related to caffeine in different materials.4.Yeast fcy2 mutant(caffeine transport defective)was further applied for Cs PUPs functional identification.The results showed that Cs PUP1,Cs PUP3.1,Cs PUP3.2,Cs PUP4,Cs PUP10.1 and Cs PUP10.2 could partly or completely rescue the caffeine transport function in fcy2 mutant.Transgenic yeast of Cs PUP10.1 exhibited the strongest capacity of caffeine transport.In contrast,Cs PUP3.3 function as caffeine efflux transporter in yeast cells.5.Subcellular localization and transgenic technology in Arabidopsis were conducted for further gene functional verification of Cs PUP1,Cs PUP3.1 and Cs PUP10.1.The results showed that Cs PUP1,Cs PUP3.1 and Cs PUP10.1 were located in plasma membrane,and also widely distributed as vesicles in cells.Over-expression lines of Cs PUP1,Cs PUP3.1 and Cs PUP10.1 in Arabidopsis showed longer root and better growth conditions than control.Higher caffeine accumulation in transgenic plants than wild type arabidopsis was also observed.Transgenic arabidopsis of Cs PUP10.1 exhibited the strongest capacity of caffeine transport.It revealed that Cs PUP1,Cs PUP3.1 and Cs PUP10.1 could transport caffeine to different subcellular fractions by vesicle trafficking.Then partial of caffeine in transgenic ababidopsis could be converted into a form of nitrogen source for further absorption and utilization.Thus,tolerance of transgenic plants to caffeine were enhanced.6.Based on the above results,it suggested that Cs PUP1,Cs PUP3.1,Cs PUP10.1and Cs PUP10.2 were involved in the transport of caffeine in mature leaves.Cs PUP3.3may function as a caffeine transporter in young leaves.Cs PUP3.2 and Cs PUP4 may play important roles in intra-organ transport of caffeine.These specific models in different tissues improved the transport efficiency of caffeine,which determined abundant caffeine accumulation in tea plants.