The Molecular Mechanism Of Leaf-derived Caffeine Systmetic Transport Regulating Nitrogen Deficiency Response In The Root Of The Tea Plant(Camellia Sinensis L.)

Caffeine（1,3,7-trimethylxanthine）is the most abundant alkaloids in tea plant and one of the most important metabolites conferring the sensory quality and health benefits of tea infusion.However,the biological function of caffeine in tea plant remains unclear.Caffeine is mainly synthesized and accumulated in young buds and leaves,and is distributed in all tissues except seeds in tea plants.It’s reported that there is no caffeine de novo synthesis activity in the root,although root contains low amount of caffeine,implying that caffeine in the root is transported from shoot.Long-distance transport of alkaloids in other plants is responsive to nitrogen levels,and is mediated by Multidrug and Toxic Compound Extrusion（MATE）transporters.In tea plant,it remains unclear whether there is caffeine long-distance transport,and whether the long-distance transport is related to nitrogen supply.The molecular mechanism of caffeine transport is also to be studied.In this study,we analyzed the content of caffeine in the roots of tea plant under normal and nitrogen-deficient conditions,and feed the young leaves with stable isotope-labeled caffeine.We explored the long-distance transport activities of caffeine and its responses to nitrogen deficiency.To further explore the underlying molecular mechanism,we cloned CsMATE genes,and functionally characterized these CsMATEs in caffeine transport.The tissue-specific expression and subcellular localization of CsMATEs were further examined.The main results of this study are as follows.（1）The content of caffeine in the roots increased under nitrogen deficiency.Caffeine was detected in the phloem sap.These results suggested that caffeine transports via the phloem and responds to nitrogen deficiency in tea plant.（2）Stable-isotope-labeled caffeine(¹⁵N₂-CAF)was feed to the young leave,and the¹⁵N/¹⁴N,¹⁵N₂-CAF,endogenous caffeine were examined in other tissues.The results showed that nitrogen deficiency significantly induced the transport of ¹⁵N to stem,other leaves and roots,and induced the transport of ¹⁵N₂-CAF to stem.The contents of ¹⁵N₂-CAF and endogenous caffeine increased under nitrogen deficiency treatment.（3）The transcriptome results of root showed that nitrogen deficiency response in the roots was significantly inhibited by leaf-derived ¹⁵N₂-CAF.（4）Four nitrogen deficiency-responsive CsMATE genes were identified through analyzing the transcriptome data of roots under normal and nitrogen-deficient conditions.These CsMATEs were cloned and named as CsMATE1,CsMATE2,CsMATE3,CsMATE4.（5）The expression of CsMATE1,CsMATE3 and CsMATE4 in yeast cells improved the resistance to the toxic level of caffeine.This result suggested that these CsMATEs have caffeine export activity.（6）CsMATE1 mainly expressed in 5^th leaf,the 5^th main veins and vascular bundles.CsMATE3 specifically expressed in the root.CsMATE4 expressed constitutively in all tissues.The subcellular localization showed that both CsMATE1 and CsMATE4 localize in the plasma membrane.