Effect of shade treatment on biosynthesis of catechins in tea plants

When tea plants were shaded with black lawn cloth for severaldays in the field, the accumulations of (—)-epicatechin,(—)-epicatechin-3-gallate, (—)-epigallocatechinand (—)-epigallocatechin-3-gallate decreased in newlydeveloping tea shoots. Radioactive tracer studies showed thatthe conversions of glucose-U-¹⁴C, shikimic acid-G-¹⁴C and phenylalanine-U-¹⁴Cinto (—)-epicatechin and (—)-epigallocatechin moietieswere depressed by the shade treatment for tea plants but theincorporation of trans-cinnamic acid-3-¹⁴C was not affected.The treatment was found to have no significant effect on theactivities of phospho-2-keto-3-deoxy-heptonate. aldolase (EC.4.1.2.15 [EC] ), 3-dehydroquinate synthase (EC. 4.6.1.3 [EC] ), 3-dehydroquinatedehydratase (EC. 4.2.1.10 [EC] ), shikimate dehydrogenase (EC. 1.1.1.25 [EC] )and trans-cinnamate 4-monooxygenase (EC. 1.14.13.11 [EC] ) in theshoots, whereas the activity of phenylalanine ammonia-lyase(EC. 4.3.1.5 [EC] ) clearly decreased. (Received March 17, 1980; )     

Fine control of caffeine biosynthesis in tissue cultures of Camellia sinensis

To determine whether caffeine biosynthesis is controlled by the availability of purine precursors and/or methyl-donors, we examined the effect of some purine compounds on purine alkaloid accumulation, using tea callus cultures. No stimulation of caffeine biosynthesis was observed when the calli were cultured with 0.5 mM adenosine, guanosine or hypoxanthine for 3 weeks. However, 0.5 mM paraxanthine doubled the caffeine level relative to controls. Adenosine stimulated the growth of callus and reduced the caffeine concentration 3 months after inoculation. These results indicate that methylation of xanthosine by 7-methylxanthosine synthase is the most plausible rate-limiting step of caffeine biosynthesis; the supply of non-methylated purine precursors or availability of S-adenosyl-l-methionine are not the principal controlling factors of caffeine biosynthesis. Adenosine salvage to adenine nucleotide synthesis may contribute to the growth of tea calli, but not to caffeine biosynthesis.     

Profiles of purine metabolism in leaves and roots of Camellia sinensis seedlings

To determine the metabolic profiles of purine nucleotides and related compounds in leaves and roots of tea (Camellia sinensis), we studied the in situ metabolic fate of 10 different (14)C-labeled precursors in segments from tea seedlings. The activities of key enzymes in tea leaf extracts were also investigated. The rates of uptake of purine precursors were greater in leaf segments than in root segments. Adenine and adenosine were taken up more rapidly than other purine bases and nucleosides. Xanthosine was slowest. Some adenosine, guanosine and inosine was converted to nucleotides by adenosine kinase and inosine/guanosine kinase, but these compounds were easily hydrolyzed, and adenine, guanine and hypoxanthine were generated. These purine bases were salvaged by adenine phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase. Salvage activity of adenine and adenosine was high, and they were converted exclusively to nucleotides. Inosine and hypoxanthine were salvaged to a lesser extent. In situ (14)C-tracer experiments revealed that xanthosine and xanthine were not salvaged, although xanthine phosphoribosyltransferase activity was found in tea extracts. Only some deoxyadenosine and deoxyguanosine was salvaged and utilized for DNA synthesis. However, most of these deoxynucleosides were hydrolyzed to adenine and guanine and then utilized for RNA synthesis. Purine alkaloid biosynthesis in leaves is much greater than in roots. In situ experiments indicate that adenosine, adenine, guanosine, guanine and inosine are better precursors than xanthosine, which is a direct precursor of a major pathway of caffeine biosynthesis. Based on these results, possible routes of purine metabolism are discussed.     

大规模茶叶细胞悬浮培养茶氨酸合成工艺优化研究

以婺源绿茶为材料进行茶叶愈伤组织悬浮培养,采用正交实验设计进行了大规模茶叶细胞悬浮培养合成茶氨酸工艺条件优化研究。结果显示,NH4+/NO-30.0/60.0mmol/L、K+100.0mmol/L、Mg++3.0mmol/L、H2PO-43.0mmol/L、蔗糖30.0g/L、水解酪蛋白2.0g/L条件下,茶叶细胞生长量(速率)和茶氨酸积累量均达到最高值;提高培养基中蔗糖和水解酪蛋白浓度可延长细胞对数生长期和稳定生长期,从而有利于茶氨酸积累;H2PO-4浓度主要影响细胞生长速率和茶氨酸积累速率的同步性,低H2PO4-浓度环境中茶氨酸积累速率峰值滞后于细胞增长速率峰值,高H2PO4-浓度环境中早于细胞生长速率峰值出现时间;K+和Mg++对细胞生长的影响不明显,但影响茶氨酸合成酶活性,维持适量的K+和Mg++有利于茶氨酸积累。先加入一定量盐酸乙胺再每天进行少量补充,茶氨酸合成量比一次性加入的效果要好。从生产效率考虑,培养周期以19～22d为宜。     

Distribution and biosynthesis of theanine in Theaceae plants

The theanine content of the leaves of 27 species or varieties of Theaceae plants was investigated. Theanine was present in 21 species or varieties, but in much lower amounts (<0.2 μmol/g fresh weight) than the quantity detected in Camellia sinensis var. sinensis. The major free amino acids in leaves of four species belonging to the genera Schima and Eurya, were glutamic acid, aspartic acid, glutamine, asparagine, alanine and proline and content of these amino acids is similar to or higher than theanine. Accumulation of free amino acids in these plants was generally lower than in C. sinensis var. sinensis. The biosynthetic activity of theanine, assessed by the incorporation of radioactivity from [¹⁴C]ethylamine, was detected in seedlings of two species of Schima. The theanine biosynthetic activity in roots was higher than that of leaves.     

l-alanine decarboxylase in Camellia sinensis

l-Alanine decarboxylase, extracted from the cotyledon and root of tea seedling, had a pH optimum of 6.25. Pyridoxal-5-phosphate did not activate the enzyme. The enzyme was partially stabilized by l-alanine. Enzyme activity in green seedlings was higher than in etiolated seedlings.     

茶类胡萝卜素研究进展

类胡萝卜素是茶(Camellia sinensis (L.) O. Ktze.)中一类重要的光合色素,具有光保护、抗氧化等众多生理功能,同时也是脱落酸、独脚金内酯和胡萝卜内脂等植物激素的合成前体,在茶生长发育过程中起极其重要的作用。类胡萝卜素还是构成茶叶外形、叶底色泽的重要成分,也是茶叶重要致香物质的前体物,其种类、含量对茶叶品质起着至关重要的作用。本文对茶中类胡萝卜素种类、代谢途径及其对制茶品质的影响等方面的研究进展进行了综述,同时对茶类胡萝卜素下一步的研究方向进行了展望。     

Metabolism of methionine and biosynthesis of caffeine in the tea plant (Camellia sinensis L.).

1. Caffeine biosynthesis was studied by following the incorporation of 14C into the products of L-[Me-14C]methionine metabolism in tea shoot tips. 2. After administration of a 'pulse' of L-[Me-14C]methionine, almost all of the L-[Me-14C]methionine supplied disappeared within 1 h, and 14C-labelled caffeine synthesis increased throughout the experimental periods, whereas the radioactivities of an unknown compound and theobromine were highest at 3 h after the uptake of L-[Me-14C]methionine, followed by a steady decrease. There was also slight incorporation of the label into 7-methylxanthine, serine, glutamate and aspartate, disappearing by 36 h after the absorption of L-[Me-14C]methionine. 3. The radioactivities of nucleic acids derived from L-[Me-14C]methionine increased rapidly during the first 12 h incubation period and then decreased steadily. Sedimentation analysis of nucleic acids by sucrose-gradient centrifugation showed that methylation of nucleic acids in tea shoot tips occurred mainly in the tRNA fraction. The main product among the methylated bases in tea shoot tips was identified as 1-methyladenine. 4. The results indicated that the purine ring in caffeine is derived from the purine nucleotides in the nucleotide pool rather than in nucleic acids. A metabolic scheme to show the production of caffeine and related methylxanthines from the nucleotides in tea plants is discussed.     

Molecular regulation of catechins biosynthesis in tea [Camellia sinensis (L.) O. Kuntze

Catechins are bioprospecting molecules present in tea and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. These are synthesized through the activities of phenylpropanoid and flavonoid pathways. Expression regulation of various genes of these pathways namely phenylalanine ammonia-lyase (CsPAL), cinnamate 4-hydroxylase (CsC4H), p-coumarate:CoA ligase (Cs4CL), flavanone 3-hydroxylase (CsF3H), dihydroflavonol 4-reductase (CsDFR) and anthocyanidin reductase (CsANR) was accomplished previously. In depth analyses of the remaining genes namely, chalcone synthase (CsCHS), chalcone isomerase (CsCHI), flavonoid 3'5'-hydroxylase (CsF3'5'H) and anthocyanidin synthase (CsANS) were lacking. The objective of the work was to clone and analyze these genes so as to generate a comprehensive knowledge on the critical genes of catechins biosynthesis pathway. Gene expression analysis was carried out in response to leaf age and external cues (drought stress, abscisic acid, gibberellic acid treatments and wounding). A holistic analysis suggested that CsCHI, CsF3H, CsDFR, CsANS and CsANR were amongst the critical regulatory genes in regulating catechins content.     

The Effect of Phosphorus and Potassium Deficiencies on Transpiration in Tea (Camellia sinensis)

The effect of phosphorus and potassium deficiencies on transpiration in tea (Camellia sinensis L. Clone DT 1) was studied. The plants were grown in sand culture, and measurements were made after the plants showed phosphorus or potassium deficiency symptoms. The overall growth of plants was reduced by potassium deficiency but not by phosphorus deficiency. Both deficiencies reduced stomatal aperture and increased leaf water potential. Stomatal density decreased in phosphorus deficient leaves and it increased in potassium deficient leaves. The transpiration of whole plants was reduced by both deficiencies. The relative sensitivity of transpiration to water stress was increased by potassium deficiency but not by phosphorus deficiency.     

Effect of kinetin on early stages of chlorophyll biosynthesis in streptomycin-treated barley seedlings

Treatment of barley seeds (Hordeum vulgare L.) with streptomycin, an inhibitor of plastid protein synthesis, resulted in growth of the albino phenotype seedlings with ribosome-deficient undifferentiated plastids and chlorophyll (Chl) level as low as 0.1% of that in control plant leaves. A major effect of the antibiotic was almost complete suppression of the ability of plants to synthesize 5-aminolevulinic acid (ALA) intended for Chl biosynthesis. The activity of synthesis of ALA intended for heme porphyrin biosynthesis in etiolated and greening seedlings and in light-grown albinophenotype plants was insensitive to light and cytokinins. In the upper parts of leaves of streptomycin-treated plants, exhibiting 60% Chl deficit, the cells with three types of chloroplasts could be observed: normally developed chloroplasts, chloroplasts composed of single thylakoids and grana, and completely undifferentiated plastids. In this Chl-deficient tissue, ALA synthesis was found to be stimulated by kinetin but much less than in leaves of the control plants. The endogenous cytokinin content in etiolated and greening seedlings treated with streptomycin was almost the same as it was in untreated control seedlings. The cytokinin level in the white tissue of plants grown in the light was on average twice as high as that in green leaves of the control plants. The capability of kinetin to stimulate the synthesis of ALA used for Chl biosynthesis was found to correlate with the Chl content and organization of the chloroplast internal structure. This correlation confirms the hypothesis that the normally developed internal structure of plastids is essential for the adequate phytohormone response in plants.     

The Effect of Potassium Deficiency on Stomatal and Cuticular Resistance in Tea (Camellia sinensis)

The effect of potassium deficiency on cuticular resistance and diurnal variation in stomatal diffusive resistance was studied in tea (Camellia sinensis). The plants were grown in sand and potassium deficiency induced by withholding the supply of potassium. The results showed that during the day potassium-deficient leaves had a higher stomatal diffusive resistance than control leaves. However when solar radiation was reduced by clouds the stomatal diffusive resistance in both control and potassium-deficient leaves was not significantly different. Night opening of stomata was observed in both control and potassium-deficient leaves, but noticeably lower in the latter. Potassium-deficient leaves had a lower cuticular resistance than control leaves.     

Flavonoid biosynthesis in the tea plant Camellia sinensis: properties of enzymes of the prominent epicatechin and catechin pathways

Leaves of tea (Camellia sinensis L.) contain extraordinary large amounts of (-)-epigallocatechin, (-)-epicatechin, (+)-gallocatechin, and (+)-catechin and derivatives of these compounds that show positive effects on human health. The health-promoting effects of flavan 3-ols, especially those of green tea, are of scientific and public interest. Furthermore, they play a crucial role in defense against pathogens of tea. Therefore, biosynthesis of these flavonoid compounds was investigated. The anthocyanidin reductase enzyme recently described from Arabidopsis and Medicago was shown to be present in tea with very high activity and produces epicatechin as well as epigallocatechin from the respective anthocyanidins, thus explaining the very high contents of these compounds. A strong combined dihydroflavonol 4-reductase/leucoanthocyanidin 4-reductase activity was demonstrated and catalyzes the key steps in catechin and gallocatechin formation. Together with the enzyme activities and substrate specificities of the preceding enzymatic reactions, the biosynthesis of the most prominent flavonoids of tea is elucidated.     

外源物质对茶树耐寒及蔗糖代谢关键基因表达的影响

冬春季节低温伤害是影响茶树(Camellia sinensis)生产的重要因素。以茶树盆栽苗为试验材料,通过喷施不同浓度的γ-氨基丁酸(GABA)、绿藻粉和竹醋液溶液,研究低温胁迫下3种外源物质对茶树耐寒能力的影响,并对蔗糖代谢关键基因SPS、SUS4、INV4和INV5表达量及耐寒相关生理指标进行分析,解析外源物质影响茶树耐寒性的生理与分子机制。结果表明,低温胁迫下,用不同浓度GABA、绿藻粉和竹醋液喷施处理茶树盆栽苗叶片,其冻害指数和相对电导率显著低于对照;可溶性糖含量显著高于对照,以10 mmol·L^–1 GABA、0.22 mg·mL^–1绿藻粉及2.5 mg·mL^–1竹醋液处理效果最佳。低温胁迫下,与清水处理相比,分别用10 mmol·L^–1 GABA、0.22 mg·mL^–1绿藻粉或2.5 mg·mL^–1竹醋液处理茶树盆栽苗后,茶树苗丙二醛含量显著降低,抗氧化酶活性显著提高;叶片叶绿素、可溶性糖和脯氨酸含量显著增加,蔗糖含量在处理72小时后分别增加了15.24%、11.39%和5.97%;SPS、SUS4、INV4和INV5基因的表达量显著升高。实验结果表明,GABA、绿藻粉和竹醋液能显著增强茶树的耐寒性。研究结果可为茶树抗寒剂的筛选提供理论依据。