Mechanism Of Exogenous 2,4-epibrassinolide(EBR) In Regulating Drought Tolerance Of Tea Plants

Tea plant[Camellia sinensis（L.）O.Kuntze]is one of the important cash crops in China,whose growth prefers a humid environment.However,due to complex geographical environment and climate change,rainfall is not evenly distributed in tea producing areas.As a result,tea plants often suffer from drought stress,which severely limits tea production.Therefore,exploring effective ways to improve drought tolerance in tea plants and understanding the mechanisms involved can be helpful for tea cultivation and production.Exogenous application of EBR can improve drought tolerance of plants by regulating a series of physiological and biochemical processes.However,whether EBR could contribute to drought tolerance in tea plants and the underlying mechanisms has not yet been investigated.In this study,the effect of exogenous EBR on the drought tolerance of tea plants was investigated by comparing the physiological changes of EBR-treated and non-treated tea seedlings under drought stress.Moreover,transcriptome sequencing was employed to explore the underlying molecular mechanisms.Base on the transcriptome results,the role of EBR-regulated genes under drought stress and their transcriptional regulation mechanisms in response to EBR are further validated.The main results are as listed as follows:1.EBR treatment benefits to drought tolerance of tea plants.Fv/Fm,PSII and q P are important indicators to evaluate drought tolerance of plants.In this study,the high concentration（1.0μM）of EBR had no significant effect on Fv/Fm,PSII and q P of tea plants under drought conditions.However,the low concentrations（0.1μM,0.25μM and 0.5μM）of EBR treatment could alleviate the decrease of Fv/Fm,PSII and q P under drought conditions,therein,0.5μM EBR was the most effective and could alleviate the decrease of Fv/Fm,PSII and q P by 61.2%,76.5%and 80.6%,respectively,after drought treatment for 24 h.In addition,0.5μM EBR treatment could increase the relative water content,relieve the wilting and reduce the MDA contents of tea leaves under drought conditions,suggesting that exogenous EBR treatment could increase drought tolerance of tea plants.2.Transcriptome analysis revealed that the EBR could regulate multiple physiological metabolic processes to increase the drought tolerance of tea plants.The EBR treatment increased the activity of APX,CAT,POD and SOD by 22.1%,20.6%,7.8%and 17.9%respectively in tea leaves under drought conditions,which is supported by the higher expression of Cs APX,Cs CAT,Cs POD and Cs SOD in EBR-treated seedlings under drought conditions.As a result,this resulted in a 31.9%and 22.5%decrease in H₂O₂ and O₂^-in leaves,respectively.These results suggest that EBR may increase the drought tolerance of tea plants by enhancing the activity of antioxidant enzymes.Moreover,the expression of genes related to photosystem I,photosystem II,light trapping complex and Rubisco small subunits were induced by EBR,which led to an increase of Rubisco activity and net photosynthetic rate（Pn）of tea plants under drought conditions,suggesting that EBR could promote the photosynthesis of tea plants to maintain energy supply for physiological activities under drought stress.In addition,the enrichment of KEGG pathway with EBR-upregulated genes suggests that EBR may also have an effect on phenylpropanoid biosynthesis,ABA synthesis and signal transduction,and galactose metabolism in tea plants,which have been found to be beneficial for plant drought tolerance.3.The EBR treatment may increase the drought tolerance of tea plants by modulating the ABA signal.ABA has been reported to increase drought tolerance by promoting stomatal closure.In the present study,the exogenous EBR treatment promotes the expressions of Cs NCED1 and Cs NCED2,which encode the rate-limiting enzymes for ABA synthesis,and thus promotes ABA accumulation in tea leaves under drought conditions.In addition,EBR treatment promotes expression of genes associated with ABA signal transduction and ABA-dependent stomatal closure regulation in drought conditions,and therefore further promotes stomatal closure in drought conditions.These results suggest that the exogenous EBR may increase the drought tolerance of tea plants by promoting ABA synthesis and signaling.Moreover,in the current study,the EBR-responsive transcription factor Cs MYC2-2 was found to be involved in the regulation of EBR-mediated ABA synthesis in tea plants by directly binding to the promoters of Cs NCED1 and Cs NCED2 and activating their expression.4.Galactinol accumulation is involved in drought tolerance induced by exogenous EBR in tea plants.EBR treatment has an effect on galactose metabolism in current study,therein,multiple galactinol synthetase genes（Gol S）was found to be induced by EBR treatment under drought conditions,and resulting in an additional accumulation of galactinol in tea leaves under drought conditions.It was found that the expression levels of Cs Gol S2-1 and Cs Gol S2-2 were highly correlated with the accumulation of galactinol in the leaves;Moreover,Cs Gol S2-1 and Cs Gol S2-2 proteins showed Gol S activity in vitro and in tobacco leaves.These results suggest that exogenous EBR may promote the accumulation of galactinol by modulating the expression of Cs Gol S2-1 and Cs Gol S2-2.In addition,the overexpression of Cs Gol S2-1 and Cs Gol S2-2 in Arabidopsis reduces the ROS content,MDA content,and relative electrical conductivity in drought conditions,suggesting that Cs Gol S2-1 and Cs Gol S2-2 mediated synthesis of galactinol is beneficial for plant drought tolerance.Moreover,in the current study,the EBR-responsive transcription factor Cs Dof1.1 was found to be involved in the regulation of EBR-mediated galactinol synthesis in tea plants by directly binding to the promoters of Cs Gol S2-1 and Cs Gol S2-2 and activating their expression.