Regulation Mechanism Of Theanine Synthesis Mediated By Gibberellins In Tea(Camellia Sinesis.L)

Gibberellins（GAs）,as a kind of important signal molecules,regulate seed dormancy,bud germination and growth,secondary metabolism and other important biological processes through a series of signal transduction pathways.GAs also played a crucial role in the"green revolution"that dramatically increased crop yields.At present,gibberellin and its inhibitors have been widely used in agricultural production as plant growth regulators.However,the application of gibberellin in the improvement of tea quality and yield is insufficient,and the mechanism of gibberellin regulating tea quality,especially theanine biosynthesis is unclear.We carried out systematic studies on GAs regulation of tea plant growth&development,theanine biosynthesis and Cs TS1 expression.The physiological and molecular mechanism of gibberellin regulating theanine synthesis was preliminarily explored.The results obtained are as follows.1.Gibberellin significantly promoted the growth of tea shoots and the increase of tea yield.GA₃ treatment promoted the germination rate of tea shoots,increased the shoot yield by56%（W/W）significantly compared with mock,and significantly promoted the elongation of tea shoots.The negative control,Uniconazole（UZ）,significantly inhibited the germination and elongation of tea shoots,and the yield decreased by 12%（W/W）.The development stage of tea shoots in GA₃ treatment group was 7 days earlier than that of mock,which means that the plucking stage of tea was effectively earlier,and was conducive to the promotion of economic value of tea.2.Gibberellin significantly increased the synthesis and accumulation of theanine in tea shoots.Gibberellin significantly increased theanine content in tea shoots.The dynamic changes of gibberellin and theanine content in growth stages of tea shoot showed that the change trend of GA₃ and theanine was significantly positively correlated.Exogenous GA₃ increased the theanine content by 27%and up-regulated the expression of Cs TS1.Nine transcription factors related to gibberellin signal transduction were identified by RNA-seq analysis.The expression patterns of Cs TS1,Cs GSII-1.1,Cs GSII-1.2,Cs GSII-1.3 and Cs GSII-2 genes in theanine biosynthesis pathway were analyzed,and the transcription factors regulating gibberellin signal transduction pathway were enriched.ERF,BPBF,GAMYB and WRKY71 were identified as potential key transcription factors involved in the regulation of theanine synthesis.3.Gibberellin promotes the synthesis of theanine in tea shoots by reducing the rate of carbon assimilation.Gibberellin reduced the carbon assimilation rate of tea shoots.Gibberellin reduced chlorophyll content,photochemical quenching coefficient（q P）and relative electron transfer rate（r ETR）of photosystem II in tender leaves of tea plant,and reduced the carbon assimilation rate in tea shoots,which made carbon/nitrogen cycle more favorable to nitrogen metabolism,and promoted nitrogen synthesis and theanine accumulation in tea shoots.At the same time,the content of theanine in tea shoots treated with Uniconazole（UZ）was relatively lower than that of mock,and the photosynthesis related parameters were relatively higher,which confirmed the positive regulation of gibberellin on theanine synthesis.4.The expression pattern and biological function of the theanine synthase gene（Cs TS1）in tea plant were elucidated.The coding sequence of Cs TS1 in Baojinghuangjincha 1#（HJ1）was cloned and transformed into Arabidopsis thaliana.The gene was heterologously expressed in Arabidopsis thaliana and the transgenic gene plants were fed with substrate of theanine synthesis,the function of Cs TS1 was consistent with that reported in the genome database of Camellia sinensis.The promoter of Cs TS1（pro Cs TS1）was fused with GFP and transferred into Arabidopsis thaliana.It was found that GA₃ could regulate the expression of GFP protein,indicating GA₃ could also up regulate the expression of Cs TS1 in tea shoots through the interaction of transcription factors and pro Cs TS1.Bioinformatics analysis of pro Cs TS1showed that the binding sites of ERF,BPBF and WRKY71 were found in the range of-600bp,which further suggested that these three transcription factors regulated the expression of Cs TS1.5.The regulatory pathway of theanine synthesis mediated by gibberellin was preliminarily analyzed.The interaction between WRKY71/ERF and Cs TS1 was found through yeast one-hybrid interaction experiment.The predicted sites of element were verified by EMSA and WRKY71could combine with TGAC element in promoter to regulate the expression of Cs TS1.Combined with the functions of WRKY71 and ERF in gibberellin signal transduction,GA₃released the inhibition of WRKY71 on gibberellin signal,and up regulated Cs TS1 expression together with its positive regulator ERF,thus clarifying the regulatory pathway of gibberellin on theanine synthesis in tea plants.In this paper,the physiological and molecular mechanism of gibberellin regulating theanine synthesis was preliminarily explored,which provided a theoretical support for improving tea quality and creating new varieties of Camellia sinensis with high theanine content.Meanwhile,in tea production,the application of gibberellin is of great practical significance to improve the yield of tea plant,extend the period of tea plucking period,and improve the economic value of tea.