茶树酯型儿茶素合成途径及酚类物质积累特异性研究
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摘要
儿茶素是茶树的主要次生代谢产物，对决定茶叶品质及其健康功效具有重要作用。本文重点探索了茶树酯型儿茶素的合成代谢途径，研究了茶树酚类物质合成积累的组织特异性和发育特异性。主要研究结果如下：
1.建立了茶树次生代谢研究的重要平台---茶树愈伤组织培养体系。
开展了愈伤组织诱导、筛选、保存等方面研究，在此基础上，建立了茶树愈伤组织培养体系，为开展茶树儿茶素合成代谢及调控研究建立了良好平台。
研究结果表明，在H、S和B三种诱导培养基中，只有B培养基适合于诱导生长迅速、组织疏松、质地均一的愈伤组织。从外植体上看，种子诱导出的愈伤组织具有较强的植株再生能力；幼茎和叶片诱导出的愈伤组织，其生长速度快、组织疏松，但再生能力弱。
采用目视法和二分法，结合TLC和HPLC分析，对愈伤组织进行了筛选，得到含不同儿茶素含量的细胞系“yunjing63Y”和“yunjing63X”。分析结果显示，细胞系“yunjing63Y”和“yunjing63X”的儿茶素组分虽与鲜叶相似，但儿茶素含量差异较大，前者为7.77mg g~(-1)DW，后者仅为0.13mg g~(-1)DW。
为保存具有稳定代谢能力的优良茶细胞系，本文对茶愈伤组织玻璃化法超低温保存技术进行了研究。结果表明：采用预培养4d、60%PVS2冰浴装载20min、100%PVS2冰浴脱水处理60min、40℃复温解冻细胞，可使茶愈伤组织的细胞存活率达到76%。
2.探索了酯型儿茶素的合成代谢途径及其关键酶。
采用体外酶学手段，结合TLC、HPLC、LC-MS、~1H NMR和13C NMR、蛋白质分离纯化技术，研究了酯型儿茶素合成和水解途径，建立了酯型儿茶素合成代谢相关酶的检测体系，分离纯化、鉴定了酯型儿茶素合成底物1-O-没食子酰-β-葡糖苷，纯化了酯型儿茶素合成酶：表儿茶素没食子酰基转移酶。
研究结果首次提出了茶树酯型儿茶素合成及代谢途径，即茶树酯型儿茶素合成途径包含两步反应，首先没食子酸在依赖UDPG的没食子酰葡萄糖基转移酶(UDP-glucose:galloyl~(-1)-O-β-D-glucosyltransferase，UGGT)作用下，形成1-O-没食子酰-β-D-葡糖苷(βG)。1-O-没食子酰-β-D-葡糖苷(βG)作为活化的没食子供体，在表儿茶素没食子酰基转移酶(epicatechin:1-O-galloyl-β-D-glucose O-galloyltransferase，ECGT)作用下，将没食子基团转移到非酯型表儿茶素的C环-3-位点上，形成酯型儿茶素。此外茶树中酯型儿茶素，在酯型儿茶素水解酶(galloylated catechins hydrolase, GCH)催化下，可被水解形成非酯型儿茶素和没食子酸。
为进一步寻找ECGT和UGGT在茶树中存在的证据，本文从茶树中分离、纯化并鉴定了βG；建立了基于粗酶提取液的UGGT、ECGT和GCH酶最适检测体系；利用硫酸铵分级沉淀、疏水柱层析、亲和柱层析和蛋白质的SDS-PAGE电泳分析技术，对ECGT进行了纯化；纯化后的ECGT其活性提高了1420倍。
3.研究了茶树中酚类物质合成与积累的器官组织及发育特异性。
利用LC-TOF/MS、分光光度计法等技术研究了主要酚类物质在茶树不同器官、叶片不同发育时期的积累规律。利用q-RTPCR技术和酶学分析手段，研究了茶树酚类物质主要合成酶和基因在不同器官、叶片不同发育时期的表达规律。利用香草醛盐酸显色技术，研究了儿茶素在茶树体内定位的器官组织特异性。
研究结果显示，不同器官中酚类物质组分及含量差异较大。在鲜叶和茎中的儿茶素、黄酮醇、酚酸组分较多且含量较高；而在根中则相反，只能少量检测到B环双羟基的儿茶素和黄酮醇，缺乏酯型儿茶素。茶树中原花青素主要以儿茶素的二聚和三聚体形式存在，从含量差异上看，根中原花青素含量明显高于鲜叶和茎；从组分差异上看，根中主要是B环双羟基类型，而叶中则是B环一羟基、双羟基和三羟基类型同时存在。从基因表达差异上看，从鲜叶到茎到根，CHI、F3H和F3’5’H表达依次降低，这些差异可能与根中儿茶素和黄酮醇含量低、缺乏B环三羟基化合物有关。酶学实验显示，从鲜叶到茎到根，GCH酶活逐渐降低，UGGT和EGCT酶活在鲜叶和茎中无明显差异，而在根中则检测不到活性，这可能是根中缺乏酯型儿茶素的原因之一。
研究结果还显示，随着鲜叶的发育，儿茶素、黄酮醇、酚酸、花青素和原花青素表现出不同的变化规律。其中儿茶素含量在一叶中最高，其次是芽；随着鲜叶发育程度增加，儿茶素单体GC、C和EC含量以及儿茶素聚合物含量较低且相对稳定，但EGC含量依次上升，而酯型EGCG和ECG的含量依次下降。黄酮醇的含量在一叶和二叶中较高。鲜叶中花青素的含量较低，且随着鲜叶发育依次减少。qRT-PCR结果显示，PAL，F3H，LAR和DFR基因的表达与不同发育时期鲜叶中儿茶素和黄酮醇积累规律相一致。酶学实验数据显示，随着鲜叶的发育，UGGT和ECGT的活性依次降低，相反GCH的活性依次增高，它们的变化与酯型EGCG和ECG的含量依次下降，而EGC含量依次上升趋势相吻合。
香草醛盐酸试剂显色结果表明，茶树不同器官中儿茶素定位具有特异性。茶鲜叶中，除了主脉薄壁细胞外，大多数组织都有积累，其中以维管束和栅栏组织积累较多；在亚细胞水平上，儿茶素主要定位在叶绿体和导管壁上。在幼茎中，除髓部和皮层的薄壁细胞外，绝大部分组织都有明显的儿茶素积累。而根部的儿茶素主要积累在中柱鞘周围。此外，在光照培养和黑暗培养的愈伤组织中儿茶素分布有所不同。
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~(-1)Dw, the latter was0.13mgg~(-1)DW.
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.
1.建立了茶树次生代谢研究的重要平台---茶树愈伤组织培养体系。
开展了愈伤组织诱导、筛选、保存等方面研究，在此基础上，建立了茶树愈伤组织培养体系，为开展茶树儿茶素合成代谢及调控研究建立了良好平台。
研究结果表明，在H、S和B三种诱导培养基中，只有B培养基适合于诱导生长迅速、组织疏松、质地均一的愈伤组织。从外植体上看，种子诱导出的愈伤组织具有较强的植株再生能力；幼茎和叶片诱导出的愈伤组织，其生长速度快、组织疏松，但再生能力弱。
采用目视法和二分法，结合TLC和HPLC分析，对愈伤组织进行了筛选，得到含不同儿茶素含量的细胞系“yunjing63Y”和“yunjing63X”。分析结果显示，细胞系“yunjing63Y”和“yunjing63X”的儿茶素组分虽与鲜叶相似，但儿茶素含量差异较大，前者为7.77mg g~(-1)DW，后者仅为0.13mg g~(-1)DW。
为保存具有稳定代谢能力的优良茶细胞系，本文对茶愈伤组织玻璃化法超低温保存技术进行了研究。结果表明：采用预培养4d、60%PVS2冰浴装载20min、100%PVS2冰浴脱水处理60min、40℃复温解冻细胞，可使茶愈伤组织的细胞存活率达到76%。
2.探索了酯型儿茶素的合成代谢途径及其关键酶。
采用体外酶学手段，结合TLC、HPLC、LC-MS、~1H NMR和13C NMR、蛋白质分离纯化技术，研究了酯型儿茶素合成和水解途径，建立了酯型儿茶素合成代谢相关酶的检测体系，分离纯化、鉴定了酯型儿茶素合成底物1-O-没食子酰-β-葡糖苷，纯化了酯型儿茶素合成酶：表儿茶素没食子酰基转移酶。
研究结果首次提出了茶树酯型儿茶素合成及代谢途径，即茶树酯型儿茶素合成途径包含两步反应，首先没食子酸在依赖UDPG的没食子酰葡萄糖基转移酶(UDP-glucose:galloyl~(-1)-O-β-D-glucosyltransferase，UGGT)作用下，形成1-O-没食子酰-β-D-葡糖苷(βG)。1-O-没食子酰-β-D-葡糖苷(βG)作为活化的没食子供体，在表儿茶素没食子酰基转移酶(epicatechin:1-O-galloyl-β-D-glucose O-galloyltransferase，ECGT)作用下，将没食子基团转移到非酯型表儿茶素的C环-3-位点上，形成酯型儿茶素。此外茶树中酯型儿茶素，在酯型儿茶素水解酶(galloylated catechins hydrolase, GCH)催化下，可被水解形成非酯型儿茶素和没食子酸。
为进一步寻找ECGT和UGGT在茶树中存在的证据，本文从茶树中分离、纯化并鉴定了βG；建立了基于粗酶提取液的UGGT、ECGT和GCH酶最适检测体系；利用硫酸铵分级沉淀、疏水柱层析、亲和柱层析和蛋白质的SDS-PAGE电泳分析技术，对ECGT进行了纯化；纯化后的ECGT其活性提高了1420倍。
3.研究了茶树中酚类物质合成与积累的器官组织及发育特异性。
利用LC-TOF/MS、分光光度计法等技术研究了主要酚类物质在茶树不同器官、叶片不同发育时期的积累规律。利用q-RTPCR技术和酶学分析手段，研究了茶树酚类物质主要合成酶和基因在不同器官、叶片不同发育时期的表达规律。利用香草醛盐酸显色技术，研究了儿茶素在茶树体内定位的器官组织特异性。
研究结果显示，不同器官中酚类物质组分及含量差异较大。在鲜叶和茎中的儿茶素、黄酮醇、酚酸组分较多且含量较高；而在根中则相反，只能少量检测到B环双羟基的儿茶素和黄酮醇，缺乏酯型儿茶素。茶树中原花青素主要以儿茶素的二聚和三聚体形式存在，从含量差异上看，根中原花青素含量明显高于鲜叶和茎；从组分差异上看，根中主要是B环双羟基类型，而叶中则是B环一羟基、双羟基和三羟基类型同时存在。从基因表达差异上看，从鲜叶到茎到根，CHI、F3H和F3’5’H表达依次降低，这些差异可能与根中儿茶素和黄酮醇含量低、缺乏B环三羟基化合物有关。酶学实验显示，从鲜叶到茎到根，GCH酶活逐渐降低，UGGT和EGCT酶活在鲜叶和茎中无明显差异，而在根中则检测不到活性，这可能是根中缺乏酯型儿茶素的原因之一。
研究结果还显示，随着鲜叶的发育，儿茶素、黄酮醇、酚酸、花青素和原花青素表现出不同的变化规律。其中儿茶素含量在一叶中最高，其次是芽；随着鲜叶发育程度增加，儿茶素单体GC、C和EC含量以及儿茶素聚合物含量较低且相对稳定，但EGC含量依次上升，而酯型EGCG和ECG的含量依次下降。黄酮醇的含量在一叶和二叶中较高。鲜叶中花青素的含量较低，且随着鲜叶发育依次减少。qRT-PCR结果显示，PAL，F3H，LAR和DFR基因的表达与不同发育时期鲜叶中儿茶素和黄酮醇积累规律相一致。酶学实验数据显示，随着鲜叶的发育，UGGT和ECGT的活性依次降低，相反GCH的活性依次增高，它们的变化与酯型EGCG和ECG的含量依次下降，而EGC含量依次上升趋势相吻合。
香草醛盐酸试剂显色结果表明，茶树不同器官中儿茶素定位具有特异性。茶鲜叶中，除了主脉薄壁细胞外，大多数组织都有积累，其中以维管束和栅栏组织积累较多；在亚细胞水平上，儿茶素主要定位在叶绿体和导管壁上。在幼茎中，除髓部和皮层的薄壁细胞外，绝大部分组织都有明显的儿茶素积累。而根部的儿茶素主要积累在中柱鞘周围。此外，在光照培养和黑暗培养的愈伤组织中儿茶素分布有所不同。
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~(-1)Dw, the latter was0.13mgg~(-1)DW.
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.
引文
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