Study On The Tea (Camellia Sinensis) Quality Components And Their Underlying Mechanisms By Short-term Moderate Drought On Pre-harvest Shoots

Camellia sinensis is the most important genus in the family Theaceae,and a perennial evergreen woody plant.Tea is rich in various primary and secondary metabolites such as amino acids,catechins,and caffeine.Drought is one of the most common environmental stresses that affects the accumulation of specialized compounds.Given the importance of drought on tea quality,metabolic and genetic changes of tea plants under depleting soil moisture have been a concern for a long time.To avoid serious growth damage to plants,moderate drought was proposed by researchers and has been shown to be an appropriate water condition that can maintain plant growth,active tolerance,and enhance specialised metabolites.In this experiment,the genome sequenced Yunkang 10（Camellia sinensis var.assamica）was used as the experimental material.A short term drought stress was set according to the Standard of Classification for drought severity SL424-2008 in China,as FW（full watered）,CK（Control）,MI（mild drought）,MO（moderate drought）and SE（severe drought）.Non-targeted metabolite profiling and second generation high-throughput sequencing technology were adopted to analyze the quality components and their metabolic pathways.With combined omics analysis,the key quality components and their metabolic pathways,as well as the key regulatory DEGs were unearthed.There are 8 RLs were identified by bioinformatics analysis and genic cloning.q RT-PCR was used to analyze their tissue profiling.Subcellular localization technique was adopted to determine the location of the expression products of CSA027066 and CSA018425.Transient expression was used to verify the regulatory functions of CSA027066 and CSA018425 on physiological indicators,flavonoids biosynthesis and lipid metabolism.The main contents and conclusions are as follows:1.Effect of drought stress on quality components in tea shootsNon-targeted metabolomics was used to analyze the metabolites of tea shoots under different soil moisture,and a total of 550 substances were detected,of which 208were differentially accumulated.Most of differentialy accumulated metabolites are grouped into amino acids and their derivatives,nucleotides and their derivatives,flavonoids,organic acids,and lipids.Flavonoids,nucleotides and their derivatives increased with the decrease of soil moisture under MI and MO,and then began to decrease under SE.Amino acids and their derivatives increased sharply under MI,and remained unchanged under MO and SE.Orgnic acids increased with the aggravation of drought stress.Lipid decreased as the drought enhanced to the lowest level under MO,and then recovered to MI under SE.KEGG annotation and pathway enrichment were performed on the differential metabolites,and group CK vs.MO,compared with group CK vs.MI and group CK vs.SE,involves the most abundant secondary metabolic pathways,especially the flavonoid biosynthesis pathway,flavonoid glycosylation pathway and lipid metabolism pathways.Drought had little impact on the accumulations of theanine and caffeine,however,catechins decreased by 11.01%under MO.Trace amino acids and their derivatives are the main contributors to the total contents of amino acids and their derivatives.Nucleotides and their derivatives are umami and sweet taste enhancers,which were greatly increased under MI and MO.Glycosylflavonoids and isoflavones which have antioxidant and anti-tumor functions greatly increased under MO.Lipids that are considered to be detrimental to the aroma quality of tea are significantly reduced under MO.Organic acids are flavor substances,and their content continues to increase with drought stress aggravated.Through the above work,combined with previous studies on flavor and health functions,it can come to the conclusion that drought can improve the quality components of tea,and short-term moderate drought is the best treatment condition.MO increased the accumulation of flavonoid glycosides,isoflavones,organic acids,amino acids and their derivatives,nucleotides and their derivatives,but reduced lipid content.It was speculated that glycosylflavonoids,isoflavones and lipids are the key factors affecting the quality of tea.2.Response of tea shoots to drought stressContents of H₂O₂,MDA,Pro and POD,and contents of stress hormones ABA,JA,SA and the activity of Eth synthetase were tested.The results showed that the osmotic adjustment substances and antioxidant enzymes in tea plants can be timely reduce the cell water potential and clear the reactive oxygen species to maintain the intracellular biological macromolecular structure.Drought stress activates stress hormones to co-stimulates signal transduction,and to jointly regulate the response of tea shoots to environmental changes.The results of physiological indicators and stress hormones show that short-term drought will not significantly inhibit the growth of tea shoots.3.The effect of drought stress on the metabolic pathways of quality componentsTwelve transcriptome sequencing libraries were constructed and yielded 113Gbp raw data,which were deposited in the NCBI Sequence Read Archive under accession SRA:PRJNA564414.The quality assessment showed the data quality was high with a deep sequencing depth.687 DEGs were screened and q RT-PCR of 15 DEGs showed the reliability of the transcriptome data.The Venn diagram showed that 648 DEGs were specific DEGs,accounting for 94.32%,indicating that tea shoots responded to different drought stresses.GO analysis was used to analyze the biological processes of DEGs in each group and found that CK vs.MO enriched a variety of biological processes associated with primary and secondary metabolites,such as organic acid metabolism,nucleobase compound metabolism,lipid metabolism,amino acid metabolism and transport,flavonoid metabolism and glycosylation,indicating that MO can significantly induce the metabolic pathways of quality components.Drought moderately increased the expression of xanthine dehydrogenase（XDH）gene.MO moderately inhibited the expression of theanine synthase（TS）gene and significantly promoted the expression of UGFT that negatively correlated with catechins.These DEGs may be related to the slight decrease of caffeine precursors,theanine and catechins.MO significantly promoted flavonoid glycosylation,isoflavonoid biosynthesis,amino acid metabolism and organic acid metabolism,however,significantly inhibited anthocyanin biosynthesis and lipid metabolism.Nucleotides and their derivatives related DEGs had the largest number in group CK vs.MO,compared with groups of CK vs.MI and CK vs.SE.These DEGs explained the large increase of isoflavones and flavonoid glycosides,amino acids and their derivatives,nucleotides and their derivatives,and organic acids,and the obvious decrease of anthocyanins and lipids in tea shoots under MO treatment.Drought did not significantly affect the expressions of synthase genes of phenylacetaldehyde and phenylethyl alcohol.Drought had no dignificant effects on LOX but MO significantly inhibited ADH in green leaf volatiles（GLVs）pathway.MO obviously upregulated the expressions of DXS,FPS and GGPPS of terpene backbone biosynthesis,as well as biosynthesis genes ofβ-carotene,lutein and zeaxanthin.Β-carotene.Through above work,how drought affects quality components in tea shoots has been revealed from the molecular level.Transcriptome analysis showed that MO significantly promote flavonoid glycosylation,isoflavone synthesis,and aroma precursor biosynthesis in the carotenoid pathway,but significantly inhibit lipid metabolism and the production of cis-3-hexenol.It is speculated that these metabolic changes are key important to improve the quality of tea under drought stress.4.Combined analysis of metabolomics and transcriptomicsThe differentially accumulated metabolites and DEGs were map into the KEGG pathway,and Pearson correlation coefficient（PCC）>0.8 was used to carry out a preliminary analysis.Group CK vs.MO was found to be involved in abundant of quality-assciated DEGs,especially in biosynthesis of flavanone,flavonoids,glycosylflavonoids,isoflavonoids,fatty acids,glycerides and phosphoglyceride metabolic.PCC>0.9 was used to scale down the potential targets,and a batch of key structural genes and transcription factors of flavonoid biosynthesis pathway and lipid metabolism were identified and drawn into correlation network diagrams.Meanwhile,29 and 18 DEGs that are respectively and closely related to flavonoids and lipid metabolism were discovered,mainly of which encodes transcription factors,transporters,and hydrolases.5.Identification bioinformatics analysis of Radialis-likes in yunkang 108 Protein RADIALIS-likes were identified and cloned,including CSA027066,CSA011543,CSA018425,CSA032285,CSA017695,CSA017034,CSA017368 and CSA011662.Analysis of phylogenetic tree and tissue expression profiling,combined with transcriptome profiling,revealed that RADIALIS-likes are specifically expressed in flowering plants and have very conservative sequences in tea plants.Both CSA027066 and CSA018425 are drought stress response genes.CSA027066 mainly plays roles in leaf tissue,while CSA018425 may be related to the formation of xylem.6.Functional identification of CSA027066 and CSA018425Green fluorescent proteins fused with CSA027066 and CSA018425 were constructed separately,and transiently expressed in N.benthamiana for subcellular localization.It was found that both of CSA027066 and CSA018425 were localized in cytoplasm and nucleus.GUS reporter fused with CSA027066 and CSA018425 were constructed separately,and transiently expressed in leaves of N.benthamiana.It was found that both CSA027066 and CSA018425 could affect the physiological indexes.CSA027066 and CSA018425 can differentially accumulate the total flavonoids and lipids.According to previous studies and our bioinformatic analysis,a batch of tobacco homologous genes related to biosynthesis of flavonoids,lipids,ABA synthesis,etc.,were selected to undergone q RT-PCR detecting.The result shows that both CSA027066and CSA018425 can significantly regulate flavonoid biosynthesis genes and lipid hydrolase genes.Both CSA027066 and CSA018425 are involved in ABA biosynthesis,strangolactone biosynthesis and signal transduction,xylem formation and terpenoid biosynthesis.Through the above work,the tea plant RADIALIS-like family genes were identified for the first time and carried out by bioinformatics analysis.CSA027066 and CSA018425 were identified to play roles in the accumulation of flavonoids and free fatty acids.RADIALIS-likes were also speculated to be involve in the biosynthesis of ABA and strigolactone.Protein RADIALIS-likes are stress-responsive genes,and it is likely to regulate in the upstream of flavonoid biosynthesis and lipid metabolism.Their specific regulatory networks are worthy of further exploration.