Regulation Mechanism Of Exogenous Melatonin On Photosynthesis And Related Secondary Metabolism Of Phenylpropanoid Pathway In Tea Plant

Tea plant[Camellia sinensis（L.）O.Kuntze],known as a leaf-type beverage crop,its tender buds and leaves are usually picked for producing tea beverage.The chemical components of tea include tea polyphenols,alkaloids and theanine,etc,which are beneficial to human health.Melatonin（MT）,an endogenous indolamine found in many plants,has been shown to be involved in metabolic,physiological,cellular responses,and affecting growth and development,such as regulating seed germination,circadian rhythm,photosynthesis and senescence process.In this study,‘Zhongcha 108’was used as the test material and sprayed with various concentrations（0,0.2,0.3 and 0.4 m M）of MT at 0,8and 16 d.Its photosynthetic characteristics（include photosynthetic gas exchange parameters,stomatal characteristics and chlorophyll fluorescence level）,anthocyanin and lignin contents were measured.The samples of control and 0.2 m M MT treatment groups at8 and 16 d were selected for transcriptome analysis.The differentially expressed genes（DEGs）involved in photosynthesis,chlorophyll（Chl）and related secondary metabolism of phenylpropanoid pathway were identified,and RT-q PCR was performed to verify the expression level of corresponding genes.Preliminary exploration is being made of regulation mechanism of exogenous MT on photosynthesis and related secondary metabolism of phenylpropanoid pathway in tea plant.The main results are set as follows:1.The samples of the control（CK1 and CK2）and 0.2 m M MT treatment groups（MT1and MT2）at 8 and 16 d were sequenced by transcriptome,and obtain 4 c DNA libraries.The percentage of bases with Phred value>30（Q30）of clean data of each library is between 93.17%and 94.24%,indicating that sequencing results were reliable.The sequence was aligned to tea reference genome（CSS＿Chr Lev）,and the mapped reads were spliced to complete the transcriptional assembly process.The gene sequences were compared with 6 databases（NR,Swiss-Prot,Pfam,Egg NOG,GO and KEGG）to obtain gene annotation information.There were 5273（4368 genes showed upregulation and 905genes showed downregulation relative to that CK1）and 3019 DEGs（922 genes showed upregulation and 2097 genes showed downregulation relative to that CK2）were identified among the two comparisons,CK1 vs MT1 and CK2 vs MT2,respectively.GO enrichment analysis showed that DEGs were significantly enriched in GO terms related to Chl metabolism,photosynthesis and secondary metabolism process of phenylpropanoid pathway.GO enrichment analysis showed that DEGs were significantly enriched in GO terms related to chlorophyll metabolism,photosynthesis,and biosynthesis of phenylpropanes.The significant enrichment of the top 20 KEGG pathways showed that the comparative transcriptome were enriched in photosynthesis and phenylpropanoid biosynthesis pathways.2.The photosynthetic rate（Pn）,stomatal conductance（Gs）,intercellular CO₂concentration（Ci）and transpiration rate（Tr）of each sample were measured,respectively.Treatment at 0.2 m M MT significantly enhanced Pn,and the change of Ci was consistent with that of Gs.Stomatal characteristics showed that stomatal density decreased and stomatal area increased under 0.2 m M MT treatment,which indicated that MT could improve photosynthetic efficiency by regulating stomata.Chl contents of the control decreased over time,while MT increased Chl accumulation（0.2 and 0.3 m M）or delayed Chl degradation（0.4 m M）.The Chl fluorescence levels were detected under various treatments.The maximum photochemical quantum yield of PSII（Fv/Fm）of the control decreased at 16 d,and 0.2 m M MT significantly increased Fv/Fm level at 8 d,decreased the contents of H₂O₂ and MDA,and enhanced the activities of SOD and POD,indicating that MT alleviated the oxidative damage of PSII.Under 0.2 and 0.3 m M MT treatments,the levels of photochemical quenching（q P）and non-photochemical quenching（NPQ,q N）increased.In addition,0.2 m M MT increased the percentage of Y（II）,reducing passive energy dissipation.The results of the light response curve showed that the electron transfer rate（ETR）decreased over time of the control,but increased under the treatment of 0.2 m M MT,and 0.3 and 0.4 m M MT kept the ETR at a relatively stable level.Hence,MT can enhance the photochemical efficiency in tea plants in a dose-dependent manner,and 0.2m M was better.Based on the results of RNA-seq,genes involved in photosynthesis and Chl metabolism pathways were identified,and the expression levels of some genes were verified.The application of 0.2 m M MT upregulated genes related to Chl biosynthesis and cycle.MT improved the photosynthetic capacity of tea leaves by modifing the expression of genes related to photosynthesis.3.In order to explore the regulatory mechanism of exogenous MT on related secondary metabolism of phenylpropanoid pathway,the contents of lignin and anthocyanin of each sample were determined.The results showed that 0.2 m M MT increased the anthocyanin and lignin contents at 8 and 16 d in comparison with 0 d,respectively.The anthocyanin content at 16 d was significantly lower than that at 8 d.The anthocyanin accumulation was reduced at 8 d under 0.3 m M MT treatment,and gradually recovered at16 d.In 0.3 m M MT-treated group,lignin ceaselessly deposited over time,but the increase extent was less than 0.2 m M MT-treated group.Under 0.4 m M MT treatment,the anthocyanin content decreased significantly,while the lignin content increased.The results of histochemical staining were consistent with quantitative analysis of lignin.RNA-seq and RT-q PCR showed that the 0.2 m M MT significantly activated key genes involved in related secondary metabolism of phenylpropanoid pathway,such as Cs PAL,Cs4CL,Cs CHS,Cs CHI,Cs F3H,Cs F3’H,Cs FLS、Cs LAR,Cs ANS,Cs ANR,Cs UFGT,Cs HCT,Cs CAD,Cs CCR,Cs CCo AOMT and Cs POD,which were considered as candidates for the regulation of anthocyanin and lignin biosynthesis.MT might change the contents of products of related secondary metabolism of phenylpropanoid pathway by promoting the up-regulation of endogenous synthetic genes.