| Catechin （flavan-3-ols）, as the main secondary metabolites in tea plant[Camelliasinensis （L.） O. Kuntze], shows decisive effects on the quality of tea and health functions.The aim of this study was to elucidate pathways of galloylated catechins biosynthesis andmetabolism, and to focus the tissue and developmental specificity of biosynthesis andaccumulation of phenolic compounds in tea plant. The main research achievements were asfollows.1. Established an important platform for the study of secondary metabolism in teaplant---The callus culture system of tea plant.The callus culture system of tea plant was established based on callus induction,screening of callus and callus conservation research, which provide a good platform for theregulation of catechins metabolism in tea plant.The results of our study showed that among H, S and B induction mediums, only Bmedium was fit for inducing and screening calluses which grow quickly, loosely organizedand even textured. In terms of explant, the plant regeneration ability of callus inducedfrom seed was stronger; calluses induced from tender stems and leaves grow quickly andloosely organized with weaker regeneration ability.Cell lines “yunjing63Y”,“yunjing63X” with different catechin contents wereobtained by visual method and dichotomy, combining with TLC and HPLC analyses. Theresults showed that catechin components of the two cell lines were similar to that of freshleaves, but the catechin contents of cell lines “yunjing63Y”and “yunjing63X” differedobviously. The catechin contents of the former was7.77mg g-1Dw, the latter was0.13mgg-1DW.To preserve cell lines with stable metabolism, the cryopreservation of callus byvitrification in tea plant has been preliminarily investigated. It showed that the preculturefor four days was best, loading with60%PVS2in freezing bath for20minutes was optimal.Treating with100%PVS2dehydration in freezing bath for60minutes was suggested. Thecryopreservation of callus warmed in40℃water bath had the highest survival rate. The cells’ survival rate was as high as76%when these parameters were adopted through thewhole trial.2. Investigated biosynthesis pathways of galloylated catechins and its related keyenzymes.Synthesis and hydrolysis pathways of galloylated catechins were investigatedthrough enzymatic assays in vitro, combining TLC, HPLC, LC-MS,1H NMR,13C NMRand enzyme purification. The reaction assay of enzyms involved in galloylated catechinsmetabolism was established. The synthetic substrate of galloylated catechins:1-O-galloyl-β-D-glucose was isolated, purified and identified. Besides, galloylatedcatechins synthetase （epicatechin:1-O-galloyl-β-D-glucose O-galloyltransferase） waspurified.The results of the study presented pathways of galloylated catechins biosynthesis andmetabolism for the first time. The biosynthesis way of galloylated catechins containedtwo-step reactions: In the first-step reaction, the galloylated acyl donor β-glucogallin （βG） wasbiosynthesized by （UDP-glucose:galloyl-1-O-β-D-glucosyltransferase, UGGT） from thesubstrates gallic acid （GA） and uridine diphosphate glucose （UDPG）. In the second-stepreaction, galloylated group was transferred to the3-postion of the C-ring in catechin andformed galloylated catechins by action of （epicatechin:1-O-galloyl-β-D-glucoseO-galloyltransferase, ECGT）. Besides, the galloylated catechins could be hydrolyzed toungalloylated catechins and gallic acid with the （galloylated catechins hydrolase, GCH）action.To obtain further evidence for the existence of ECGT and UGGT in the tea plant, βG wasextracted and identified from tea plant; the optimal detection systems of UGGT, ECGT andGCH were established based on crude enzyme extract; ECGT was purified effectively byammonium sulfate grading precipitation, hydrophobic interaction chromatography, affinitycolumn chromatography and SDS-PAGE electrophoresis analysis technology, whichactivity was1420times higher than before.3. The tissue and developmental specificity of biosynthesis and accumulation ofphenolic compounds in tea plant were studied. The accumulation pattern of the main phenolic compounds in different organs andleaves at different developmental stages were investigated by the LC-TOF/MS andspectrophotometer method. The expression patterns of the main synthetase and genesinvolved in phenolic compounds metabolism in tea plant in different organs and leaves atdifferent developmental stages were studied through qRT-PCR technology. Thesite-specific accumulation of catechins in organs and tissues of tea plant was studied withvanillin-HCL staining method.The result showed the content and components of phenolic compounds existedsignificant differently in different tissues and organs. The high content and components ofphenolic compounds were abundant and diverse in both leaves and stems; on contrary, inroots, only catechin and flavonols with di-hydroxy in B-ring were lowly deteced,galloylated catechins were absent. Procyanidins in tea plant mainly existed in the form ofdipolymer and tripolymer of catechins. In terms of content, the content of procyanidins inroot was obviously higher than that of fresh leaf and stem; In terms of components,procyanidins in root were mainly catechins with double hydroxies in B-ring, while inleaves procyanidins coexisted on catechins with mono-to tri-hydroxyl in B-ring. In termsof gene expression, from fresh leaves, stems to roots, gene expressions decreased in turnfrom CHI, F3H, to F3’5’H, which may be related to the missing of tri-hydroxyl in B-ringand the low content of catechins, flavonols. Data of enzymatic experiment showed fromfresh leaves, stems to roots, enzymatic activity of GCH decreased in turn. There is noobvious difference of enzymatic activities of UGGT and EGCT in fresh leaves and stems,while in root the enzymatic activities coud not be detected, this may be one possible reasonfor why galloylated catechins was missed in roots.The results also showed that with the development of fresh leaves, catechins,flavonols, phenolic acids, anthocyanins and procyanidins displayed different changingpatterns. Among the developmental stages of fresh leaves, the content of catechin was thehighest in the first leaf, followed by bud, while reduced obviously in mature leaves. Withfresh leaves developed futher, the contents of monomer GC, C and EC and polymers werelow and relatively stable, but the content of EGC in turn increased, and the content of EGCG and ECG declined successively. The content of flavonols in leaf was relatively highin the first and second leaf. The content of anthocyanins in fresh leaves was relatively lowand reduced successively with the development of fresh leaves. Results of qRT–PCRshowed that gene expression patterns of PAL, F3H, LAR, DFR were consistent withaccumulation patterns of catechins and flavonols in fresh leaf at different developmentalstages. Data of enzymatic experiment showed that with the development of the fresh leaf,the activity of UGGT and ECGT reduced, in contrast, the activity of GCH increasedsuccessively. This changing pattern was consistent with the rule that the content of EGCGand ECG decline in turn while the content of EGC rises.The results of the vanillin-HCL staining showed that catechins existed ubiquitously inall inspected tissues in young tea leaf, but the distribution was concentrated in the vascularbundle and palisade tissue, whereas the large parenchyma cells of the main vein containedlower amounts of catechins. At the subcellular level, we found that catechins were locatedmainly in the chloroplasts of mesophyll cells and in the vessel wall. In young stems,catechins could be detected in most cells except the parenchyma cells of the pith and thecortex, whereas in roots, catechins could only be detected in those cells surrounding thepericycle. Moreover, we found differing distributions of catechins in calluses cultivated indarkness and light. |
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