Discovery Of Leaf Development-Related MiRNAs And Transcription Factors And Analysis Of The Proteome Differences Of Postharvest Leaves Under Different Treatments In Tea Plant(Camellia Sinensis)

Tea plant,Camellia sinensis(L.)O.Kuntze,which belongs under the genus Camellia of the family Theaceae,is a perennial evergreen woody plant.Tea leaves are important raw materials for non-alcoholic beverage processing.Tea is rich in a variety of beneficial secondary metabolites,such as tea polyphenols(especially catechins),theanine and caffeine.The specific substances accumulated in tea leaves can effectively remove free radicals,prevent cancer,cardiovascular disease and neuropathy,relieve fatigue and enhance immunity.The content of functional substances in tea leaves is related to tea quality and health care.It was found that the accumulation of specific metabolites in tea leaves was affected by factors such as tea leaf development stages and seasonal climate.In plants,the development of leaves and the metabolism of substances are regulated by complex molecules.Leaf morphology is formed from the continuous development processes consist of the cell division and differentiation of the shoot apical meristem(SAM),the formation of leaf base,leaf polarity and leaf size regulation.Leaf growth and development are strictly regulated by the molecules in the transcriptional,post-transcriptional and protein levels.It was found that some genes were simultaneously involved in the formation of leaf morphology and substance accumulation.It indicates that leaf development and substance metabolism are uniformly regulated in a molecular network.In this study,the leaves of the tea cultivars 'Longjing 43'are selected as the material.The reference genes used for tea leaf development(the first leaf,the second leaf,the third leaf,the fourth leaf and the older leaf)were identified.miRNAs in tea leaves of the five developmental stages were sequenced by high throughput and were revealed the roles in leaf development.In addition,based on homology analysis,three leaf development-related family genes,namely,GRF,GIF and TCP family,were identified by tea plant transcriptome.This paper provides a molecular basis for studying the formation of tea leaves and the association of substance formation.The picking and processing of tea leaves involves the research field of postharvest science of agricultural products.The physiological processes of the leaf cells after the postharvest are similar to the natural senescence.Postharvest storage techniques for crops always extend this senescence process to improve shelf life.The technology by controlling the temperature,humidity and light to evaporate moisture,adjust enzyme activity,and convert characteristic components in postharvest tea plant leaves is called withering,which is conducive to the shape of dry tea and enhance the inherent quality.Tea withering is the anthropic process of controlling the physiological,biochemical and molecular changes in the postharvest tea leaves to promote the formation of beneficial fragrance,taste and color.Withering process generally is the first process of tea processing,such as white tea,black tea and oolong tea.In the withering process,the factors such as withering time,temperature and humidity conditions and light conditions affect the formation of tea quality,thus affecting the final sensory evaluation of dry tea.In this study,we investigated the changes of the proteomes in three withering treatments(i.e.,different time,different temperature and shade/light withering)by iTRAQ technique,and revealed the influences of withering treatments on physiological and biochemical characteristics of tea leaves at protein level.The major results are listed as following:1.Transcriptome database of four tea cultivars of'Yunnanshilixiang','Chawan sanhao','Ruchengmaoyecha' and 'Anjibaicha' were constructed by high-throughput sequencing technique.A total of 146,342 unigenes were obtained,which provided data for the study of the molecular mechanism of leaf development.Unigenes are annotated by Nt,Nr,GO,KEGG,COG,and UniProtKB databases.One hundred and fifty unigenes encoding 18 enzymes are involved in catechin biosynthesis.The contents of C,GC,EC and EGC in four tea cultivars were detected by RP-HPLC.The expression profiles of three key enzyme genes(ANR,ANS and LAR)in catechin biosynthesis were detected by qRT-PCR.The results showed that the expression levels of three key enzyme genes may affect catechin differences among different tea cultivars.2.Based on the homology analysis With Arabidopsis thaliana,nine reference genes(i.e.,CsACT7,CsEF-1?,CseIF-4a,CsGAPDH,CsPP2A,CsSAND,CsTBP,CsTIP41 and CsTUB)were identified from the tea plant transcriptome.The full length of the nine genes was cloned from tea cultivars 'Longjing 43'.The expression levels of nine candidate genes in the five developmental stages(the first leaf,the second leaf,the third leaf,the fourth leaf and the older leaf)and response to five hormone treatments(ABA,GA3,IAA,MeJA and SA)were detected by qRT-PCR.Based on the Cq values,the expression stabilities of nine candidate genes were calculated and evaluated using three common softwares,i.e.,geNorm,NormFinder and BestKeeper.CsTBP and CsTIP41 are the most suitable reference genes in tea leaf developmental stages,CsTBP is the best reference gene under different hormonal stimuli,and CsTBP and CsTIP41 genes can be used as the most suitable reference genes in total sample.Quantitative validation of CsNAM gene showed that the stability evaluation of the three software calculations was reliable.The more stable reference gene causes the more reliable results.3.Leaves are organs for photosynthesis and formation of major secondary metabolites in tea plant.The microstructures of the five developmental stages(the first leaf,the second leaf,the third leaf,the fourth leaf and the older leaf)of the tea leaves('Longjing 43')were obtained by resin microtomy and microscopic observation.Photosynthetic characteristics(i.e.,photosynthetic rate,conductance to H2O,intercellular CO2 concentration,and transpiration rate)of tea plant leaves from five development stages were detected using a LI-6400XT portable photosynthesis system.Leaf structure and photosynthetic characteristics showed that the development of tea plant leaves is a continuous change process in morphological and physiological characteristics.The third leaf and the fourth leaf may be important functional leaves.The small RNAs of five leaf developmental stages from tea cultivars 'Longjing 43' were obtained by high throughput sequencing.Based on miRNA database of other species and tea leaf transcriptome,a total of 111 conserved miRNAs and 68 novel miRNAs were identified.A total of 1,379 potential target genes were predicted by 155 miRNAs.Functional annotation showed that the target gene with the GO term "binding" is the most.It indicates that the target genes of miRNAs in tea plant may play an important role in transcriptional regulation.A total of 50 target genes encoding the transcription factor were identified.miRNAs that are complementary to transcripts of transcription factors are usually conserved miRNAs.It suggests that miRNAs are highly conserved in the regulation of plant.Many miRNAs are involved in the formation of leaf morphology in the up-and-down relationship with the target genes.4.Based on homology analysis,six GRF and two GIF genes were identified from the tea plant transcriptome.Sequence alignment showed that CsGRF and CsGIF proteins were highly consistent with Arabidopsis and rice homologous proteins.Based on the motif analysis,it was found that the CsGRF proteins have five conserved motifs of QLQ,WRC,FFD,TQL and GGPL.QLQ may have a binding function that interacts with the CsGIF protein,and the WRC may have a nuclear localization function.CsGIF proteins have a conserved SNH domain,which may interact with the QLQ structure of CsGRF protein.miR396 and the CsGRF sequences encoding the C-terminals of the WRC domains are almost complementary.CsGRF and CsGIF protein interactions and CsGRF-miR3 96 may co-regulate leaf development.Quantitative experiments showed that CsGRF and CsGIF genes were involved in leaf development(first leaf,second leaf,third leaf,fourth leaf and older leaf)and response to hormones(ABA,GA3,IAA,MeJA and SA).The expression levels of CsGRF gene decreased gradually with the leaf development.It indicates that the CsGRF gene may play an important role in the early development of tea leaves.In addition,17 TCP family genes were identified from the tea plant transcriptome on the basis of homology analysis.The TCP domains of CsTCP proteins were highly similar to that of Arabidopsis thaliana and rice.The TCP domains of CsTCP proteins may have functions in nuclear localization and protein interactions.Subcellular localization experiments showed that the CsTCP 15 protein was localized on the nuclei of the onion epidermal cells.It indicates that the CsTCP 15 protein has a nuclear localization function and may regulate the downstream genes by nuclear localization transcription.miRNA target site prediction showed that miR319 target site is present in the the C-terminal of protein encoded by CsTCP15 transcript.It indicates that miR319 may regulate leaf development by regulating the post-transcriptional level of CsTCP15 gene.Quantitative experiments showed that CsTCP genes were involved in leaf development(first leaf,second leaf,third leaf,fourth leaf and older leaf)and response to hormones(ABA,GA3,IAA,MeJA and SA).By integrating phylogenetic relationship and expression clustering analysis,CsTCP7-CsTCP8 and CsTCP11-CsTCP12-CsTCP14 may be functional redundant pairs.5.Based on the iTRAQ technique,we investigated three proteomes of tea postharvest witherings:different times,i.e.,0 h(CK),1 h(S1),4 h(S2)and 12 h(S3);different temperatures,i.e.,room temperature(25?,RT),high temperature(38?,HT)and low temperature(4 0C,LT)for 4 h;Shade/light treatments,i.e.,1 h light(G1),4 h light(G2),1 h shade(Z1)and 4 h shade(Z2).Three groups of results received a total of 2,486 proteins.A total of 863 DEPs were obtained during withering duration(S1/CK,S2/CK and S3/CK).GO,KEGG and PPI functional annotation and protein interaction analysis showed that DEPs were involved in the regulation of cell senescence,the inhibition of primary metabolism and regulation of the secondary metabolic pathway.A total of 787 DEPs were obtained in different temperatures(HTURT and LT/RT).GO,KEGG and PPI functional annotation and protein interaction analysis showed that DEPs were involved in the response to extreme temperature,the formation of tea-specific compounds,the regulation of endogenous hormones and the inhibition of cell development.In the shade/light witherings,a total of 863 DEPs were obtained(Zl/G1 and Z2/G2).GO,KEGG and PPI functional annotation and protein interaction analysis showed that DEPs were involved in photosynthesis and photosynthetic carbon sequestration and the formation of secondary metabolites.Different time,different temperature and shade/light treatments in the tea withering processes caused different stress responses of postharvest tea leaves.DEPs regulated various metabolic pathways.Substance degradation or biosynthesis in a short time may affect the final fragrance,taste and color of dry tea.