| The tea plant(Camellia sinensis)is one of the most important commercial plant,which contains many healthy components including polyphenols,amino acids,vitamins and alkaloids,and is beneficial for the prevention and treatment of cancers,cardiovascular and cerebrovascular diseases.Studies have shown that the major healthy components of tea are catechins.O-methylated catechin,especially(-)-epigallocatechin 3-O-(3-O-methyl)gallate(EGCG3?Me),has been reported recently to have stronger antiallergic and antihypertensive effects than epigallocatechin 3-gallate(EGCG).Another biologically important characteristic of EGCG3?Me that it is absorbed more easily than EGCG.Previously,two tea cultivars ‘Jinmudan' and ‘Jinguanyin' with high EGCG3?Me content were identified,but the molecular mechanism(s)concerning EGCG3?Me biosynthesis in these two cultivars remain unclear.Which restricts the understanding of these specific tea resources.Therefore,on the basis of the previous research,this studys are aiming to explore the regulatory mechanisms of EGCG3 "Me biosynthesis and provide a theoretical basis for the breeding of high EGCG3" Me tea cultivars that contribute to further promote the development and utilization of specific tea resources.Catechin and EGCG3 "Me are thought to be synthesized through the flavonoid biosynthesis pathway and are a complex and highly ordered biological process.The content of catechins and EGCG3" Me might be controlled by the flavonoid biosynthetic genes,such as CsLAR,CsDFR,CsANS,CsANR and CCoAOMT.However,the transcription regulation of Omethylated catechin biosynthesis remain elusive.Recently,many transcription regulators,including MYB,b HLH,MADS,and WD40,were found to be associated with catechin metabolic,but there are few systematic functional studies.As major transcription factor(TF)familys in plants,WRKYs participate in many physiological processes and play an important regulatory role in plants development,biotic and abiotic stress,and metabolism.WRKY transcription factors are reported to participate in the flavonoid biosynthesis pathway in many plants such as Arabidopsis and Rice.However,knowledge regarding the involvement of WRKY TFs in catechins and EGCG3?Me biosynthesis is far from complete.In this study,two tea cultivars ‘Jinmudan' and ‘Jinguanyin' with high levels of EGCG3?Me were used,and a tea cultivar Fudingdabai without EGCG3 "Me was setted as control.The possible molecular mechanisms of WRKY transcription factor regulating catechin and EGCG3" Me biosynthesis were fully investigated.The major results are showed as following:1.Three different tea cultivars including ‘Fudingdabai',‘Jinmudan',and ‘Jinguanyin' with different EGCG3"Me content were used as experimental materials,the transcriptome datas between these different cultivars were analyzed.Results showed that 12457 and 9908 differentially expressed genes were detected between "JMD vs FD" and "JGY vs FD",respectively.More importantly,the expression level of WRKYs and EGCG3"Me biosynthesisrelated genes CsLAR,CsDFR,CsANS,CsANR and CCoAOMT were further performed.The results of real-time quantitative PCR(RT-qPCR)showed that the expression of CsLAR,CsDFR and CCoAOMT was highly consistent with the accumulation pattern of EGCG3"Me content,consistent with the results of RNA-seq,indicating that the datas obtained by RNAseq are reliable.2.The promoter sequences of CsLAR,CsDFR,and CCoAOMT were amplified by PCR.The potential cis-acting elements presented in the promoters were predicted and identified through the plant-CARE online software.The results showed that the promoters of CsLAR CsDFR and CCoAOMT contain the core cis-acting elements,such as TATA box and CAAT box,as well as many other important cis-acting elements such as photoreactive elements,stress response elements and transcription factors binding elements including MYB,MYC and WRKY,suggesting the possible involvement of WRKY TFs in regulating CsLAR CsDFR and CCoAOMT.3.Two down-regulated WRKY genes Cs WRKY31 and CsWRKY48 in ‘Jinguanyin' and‘Jinmudan' were cloned,and their possible mechanisms involved in the metabolism of EGCG3"Me in tea leaves were analyzed.Similar to the RNA-seq data,RT-qPCR analysis exhibited that CsWRKY31 and CsWRKY48 transcript levels were remarkably lower in ‘Jinguanyin' and ‘Jinmudan' than in ‘Fudingdabai',showing the opposite pattern with CsLAR,CsDFR,and CCoAOMT expression,and EGCG3?Me accumulation.Further characterization indicated that CsWRKY31 and CsWRKY48 are nuclear proteins and possessed transcriptional inhibition activity.More importantly,W-box(C/T)TGAC(T/C)elements were located in the promoter of CsLAR,CsDFR,and CCoAOMT,and further assays revealed that CsWRKY31and CsWRKY48 were capable of repressing the transcription of CsLAR,CsDFR,and CCoAOMT via the attachment of their promoters to the W-box elements.These founds indicate that CsWRKY31 and CsWRKY48 act as transcriptional repressors and might be negatively affecting the EGCG3?Me biosynthesis in tea plants through directly repressing catechins biosynthetic genes including CsLAR,CsDFR and CCoAOMT.4.A WRKY gene Cs WRKY57 like,which was up-regulated in ‘Jinguanyin' and ‘Jinmudan'was cloned,and its possible mechanisms involved in EGCG3 "Me biosynthesis was analyzed.RT-qPCR analysis revealed CsWRKY57 like were obviously higher in ‘Jinguanyin' and ‘Jinmudan' than in ‘Fudingdabai',which is consistent with EGCG3" Me content.Further subcellular localization and transcriptional activity analysis indicated that CsWRKY57 like was a nucleus protein and has transcriptional activation activity.EMSA and ChIP-qPCR analysis showed that CsWRKY57 like could directly bind to the labeled CsLAR,CsDFR,and CCoAOMT fragments containing the W-box and caused mobility shifts.Dual luciferase transient expression analysis indicated that CsWRKY57 like activated the promoter activity of CsLAR,CsDFR,and CCoAOMT.The results indicated that CsWRKY57 like acts as a transcriptional activator,and regulates EGCG3?Me biosynthesis through transcriptional activating the expression of EGCG3?Me biosynthetic genes including Cs LAR,CsDFR and CCoAOMT in different tea plants.5.Seven VQ motifs contained genes were identified and selected from the C.sinensis genome.Yeast two-hybrid(Y2H)assay and bimolecular fluorescence complementation(BiFC)techniques were used to analyze the interaction between CsWRKY31,CsWRKY48 and CsWRKY57 like with the VQ proteins.The results clearly demonstrate the direct protein interaction between CsWRKY48 and CsVQ10,CsWRKY48 and CsVQlike2,CsWRKY57 like and CsVQ4.The transcriptional activities with a GAL4-responsive reporter system in yeast cells indicates that CsVQlike2 growth well in the SD plates showed ?-galactosidase activity and possess trans-activation ability,while CsVQ4 and CsVQ10 showed no activation activity.These results suggest that they might participate in the regulation of downstream genes by activating or inhibiting the activity of interaction proteins.These results indicate that CsWRKY48 and CsWRKY57 like could individually or interacted with VQ proteins to coregulate EGCG3?Me biosynthesis-related genes. |
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