Effect of shade treatment on biosynthesis of catechins in tea plants

Transcription

1 Plant & Cell Phynol. 21(6): (1980) Effect of shade treatment on biosynthesis of catechins in tea plants Ryoyasu Saijo National Research Institute of Tea, Kanaya-cho, Haibara-gun, Shizuoka 428, Japan (Received March 17, 1980) When tea plants were shaded with black lawn cloth for several days in the field, the accumulations of ( )-epicatechin, ( )-epicatechin-3-gallate, ( )-epigallocatechin and ( )-epigallocatechin-3-gallate decreased in newly developing tea shoots. Radioactive tracer studies showed that the conversions of glucose-u- 14 C, shikimic acid-g- 14 C and phenylalanine-u- 14 G into ( )-epicatechin and ( )-epigallocatechin moieties were depressed by the shade treatment for tea plants but the incorporation of franj-cinnamic acid-3-14 C was not affected. The treatment was found to have no significant effect on the activities of phospho-2-keto-3-deoxy-heptonate. aldolase (EC ), 3-dehydroquinate synthase (EC ), 3-dehydroquinate dehydratase (EC ), shikimate dehydrogenase (EC ) and fratu-cinnamate 4-monooxygenase (EC ) in the shoots, whereas the activity of phenylalanine ammonia-lyase (EC ) clearly decreased. Key words: Camellia sinensis Catechin biosynthesis Phenylalanine ammonialyase Shade treatment Shikimate pathway Tea plant. The tea industry in Japan has developed an artificial shade culture for preparing tea leaves as raw materials of Gyokuro, fine green tea, and Matcha, ceremony tea; tea fields are shaded with reed blinds for about two weeks and then with rice straw for about a week before the young tea shoots are harvested. This procedure makes the teas comparatively less astringent and provides them with a characteristic "sun-shade" flavor. Changes in the amounts of tea components by the treatment have been demonstrated by a few workers. The contents of free amino acids which contribute to the taste increased and the contents of catechins which contribute to astringency decreased (/, 3). Effects of light and darkness on polyphenol distribution in tea plants have also been investigated (2). Light intensity is one of the most important environmental factors controlling flavonoid biosynthesis in many higher plants, as reviewed in literature (5, 10, 14). This investigation was carried out with radioactive tracer experiments and enzyme studies to obtain information on the regulatory mechanisms involved in catechin biosynthesis in tea plants under reduced light intensity. Materials and methods Materials Newly developing tea shoots, Camellia sinensis L., were obtained from the field of 989

2 990 R. Saijo the National Research Institute of Tea from April to August. Names of the cultivars are given in the tables and figures. Shade treatment of tea plants in the field In order to reduce light intensity, each tea plant was shaded with four layers of black lawn cloth beginning with the opening of the second new leaves. The treatment reduced light intensity to about 1 % of full sunlight. The treatment period is shown in each table and figure. In all cases naturally grown tea plants were used as controls for chemical analyses, radioactive tracer experiments and enzyme studies. Quantitative analysis of four major catechins The method of Nakagawa and Torii was used (6): ( )-epicatechin, ( )- epicatechin-3-gallate, ( )-epigallocatechin and ( )-epigallocatechin-3-gallate were extracted from dried, powdered materials with 80% acetone, separated by twodimensional paper chromatography, and spectrophotometrically determined after the catechins had reacted with diazotized sulfanilic acid reagent. Administration of radioactive precursors and fractionation of the metabolites Two tea shoots consisting of the apical bud and two leaves, 1.2 to 1.4 g, were obtained from the tea plants. Water solutions of glucose-u- 14 C (10/iCi), shikimic acid-g- 14 C (1.5,aCi), phenylalanine-u- 14 C (1.5 pd) and Jraar-cinnamic acid-3-14 C (1.5 fid) were separately administered through the cut stems of the shoots at 25 G for 1 to 2 hr under light of approximately 40,000 lux. The incubation was conducted in a ca. 6-liter glass chamber. Air, at 5 liters per hour, was passed through the chamber and then into an outside trap of aqueous potassium hydroxide (5%) to absorb respiratory carbon dioxide. At the end of the incubation, the shoots were extracted with hot 80% ethanol totaling ca. 100 ml. The extracts were evaporated to dryness under reduced pressure, the residue was dissolved in ca. 30 ml of water, and the sample was subjected to extraction with ethyl acetate (30 ml x 4 times). The ethyl acetate-soluble fraction was concentrated to dryness and the residue dissolved in a small amount of 30% acetone. The acetone solution was applied to a Sephadex LH-20 column (1.8x30 cm) (8), which had been equilibrated with the same solvent, then the column was eluted with an acetone-water mixture, utilizing a linear gradient of acetone from 30 to 70%. The effluent was collected in 10-ml fractions and a portion of the effluents was used to measure radioactivity for determining the fractions containing catechins. The mixture of ( )-epicatechin and ( )-epigallocatechin was separated from their gallic acid esters by this procedure. The free catechins were further separated by descending paper chromatography with phenol-water (3 : 1). The gallic acid esters were hydrolyzed with tannase at 30 C for 2 hr into free catechins and gallic acid, which were separated by paper chromatography with the above solvent. Preparation and assay of the enzymes Phospho-2-keto-3-deoxy-heptonate aldolase, 3-dehydroquinate synthase, 3- dehydroquinate dehydratase and shikimate dehydrogenase were prepared and

3 Catechin biosynthesis in tea plant 991 assayed by previously described methods (9). Preparation and assay of phenylalanine ammonia-lyase and /ra/tr-cinnamate 4-monooxygenase were carried out according to the methods described by Iwasa (4) and by Tanaka et al. (//), respectively. Units of the activities of all the enzymes were defined as /jmoles of products formed (the above five enzymes except 3-dehydroquinate synthase) or substrate utilized (3-dehydroquinate synthase) per min under the assay conditions. Measurement of radioactivity Radioactivity was determined with a liquid scintillation spectrometer, Aloka LSC-502. Portions were counted from samples of materials dissolved in the various solvents used; insoluble materials were combusted in a sample oxidizer, Aloka ASC- II 2, and the carbon dioxide collected in an ethanol amine-methanol mixture for counting. The paper chromatograms were cut into strips 1 cm wide, and a small amount of 80% ethanol was added before the measurement. Chemicals Glucose-U- 14 C (specific activity 5 mci/mmole) was purchased from The Radiochemical Centre, shikimic acid-g- 14 C (sp. act mci/mmole) and phenylalanine-u- 14 C (sp. act mci/mmole) from New England Nuclear, fra/u-cinnamic acid-3-14 C (sp. act. 10 mci/mmole) from Commissariat a L'finergie Atomique. The radioactive precursors were used after adjustment to a specific activity of 5 mci/mmole. Tannase (21,000 units/g) was kindly provided by Sanraku Ocean Co., Ltd. ; Results Effect of reduced light intensity on catechin accumulation The analytical data in Table 1 indicate that the contents of ( )-epicatechin, ( )-epicatechin-3-gallate, ( )-epigallocatechin, and ( )-epigallocatechin-3- gallate in the shade-treated shoots were lower than those in the naturally grown Table 1 Catechin contents in naturally grown and shaded tea shoots Naturally grown shoots Shaded shoots Catechin mmoie/ ^ ^ ole/, e/ Ratio to k/ Ratio to looshoots gfr.wt. gdrywt. 100 shoots decontrol ^» fr _ ^ the control \/o) \/o) EC ECG EGC EGCG Total (73) (80) (53) (76) (68) (80) (89) (59) (86) (77) Ratio to the control (%) (85) (93) (62) (89) (80) Abbreviations used: EC, ( )-epicatechin; ECG, ( )-epicatechin-3-gallate; EGC, ( )-epigallocatechin; EGCG, ( )-epigallocatechin-3-gallate. Tea plant, cultivar Yabukita, was shaded with four layers of black lawn cloth for 7 days in May, after which 100 newly developing shoots were plucked and used for the analyses.

4 992 R. Saijo EC ECG EC + ECG O O to 4 e I 3 EGC EC + EGC 1 1 i i I I I I I 1 I I I I I EGCG ECG+EGCG 8 h EGC+EGCG EC+ECG+ EGC+EGCG 0 April May April May April May 2528 I I > Fig. 1. Changes in the amounts of catechins in naturally grown and shaded tea shoots during development. Abbreviations used are explained in Table 1. Newly developing tea shoots of cultivar Yabukita were sampled every three days from April 25. Part of a teafieldwas shaded with black lawn cloth on May 1 and samplings from both experimental plants were continued until May 19. -O-. naturally grown control shoots; - -, shaded shoots.

5 Catechin biosynthesis in tea plant 993 control shoots on all bases including per 100 shoots, per g fresh weight, and per g dry weight. Of the four compounds, ( )-epigallocatechin had decreased the most. In addition ( )-epicatechin in the treated shoots probably was affected more than its gallic acid ester. To obtain more detailed information concerning the effect of the treatment, the catechin accumulations in the newly developing tea shoots were measured during the shoot development. Fig. 1 illustrates changes in the amounts of the four individual catechins and the sums of the catechins. The moles of ( )-epicatechin plus ( )-epicatechin-3- gallate, ( )-epigallocatechin plus ( )-epigallocatechin-3-gallate, ( )-epicatechin plus ( )-epigallocatechin and ( )-epicatechin-3-gallate plus ( )-epigallocatechin-3-gallate represent those of the ( )-epicatechin moiety, the ( )-epigallocatechin moiety, the two free catechins, and their gallic acid moiety, respectively. The average amounts of the accumulated catechins per 100 shoots a day, calculated from Fig. 1, showed that the treatment resulted in decreases from 24//moles to 6 //moles in ( )-epicatechin, 18//moles to 3//moles in ( )-epicatechin-3-gallate, 116//moles to 23//moles in ( )-epigallocatechin, 79 //moles to 57//moles in ( )- epigallocatechin-3-gallate, 42 //moles to 3 //moles in the ( )-epicatechin moiety, 195 //moles to 80//moles in the ( )-epigallocatechin moiety, 140//moles to 17// moles in the two free catechins, 97 //moles to 60 //moles in the gallic acid moiety, and 237 //moles to 77 //moles in the total catechins. As gallic acid and theogallin (3-galloyl-quinic acid) were detected only in small amounts in the samples compared with the catechins, the amounts of the gallic acid moiety in Fig. 1 are likely to be approximately equal to the total gallic acid moiety in the shoots. Therefore, the comparatively slight reduction in the production of the two gallates under reduced light intensity, as reported by others {1-3), is probably attributable to the minimal effect of shading on systems concerned with gallic acid production. Interestingly, the production of the ( )-epigallocatechin moiety was reduced the most by the shade treatment, followed by that of the ( )-epicatechin moiety and the gallic acid moiety. Effect of shade treatment on the conversion of precursors into catechins As effective utilization of shikimic acid in catechin biosynthesis has been well demonstrated in tea plants (4, 12, 13), the incorporation patterns were investigated with the control and treated shoots. Table 2 indicates that shikimic acid-g- 14 G administered to the tea shoots was actively metabolized to the ( )-epicatechin, ( )-epigallocatechin and gallic acid moieties in a short period of incubation under the experimental conditions used. Most of the radioactivity in the ( )-epicatechin and ( )-epigallocatechin moieties was found in the free catechins in both shoots, although the gallic acid esters are the predominant form in natural catechin distribution (7); thus the radioactivity is likely to be trapped in the free catechin pool prior to the esterification. Paper chromatographic separation showed that radioactivity found in the gallic acid moiety was more abundant in ( )-epigallocatechin-3- gallate than in ( )-epicatechin-3-gallate in the two kinds of shoots. Comparison with the incorporation rates between the control and treated shoots showed that those of the total ( )-epicatechin, ( )-epigallocatechin and gallic acid moieties were

6 994 R. Saijo Table 2 Conversion qfshikimic acid-g- ii C to catechins in naturally grown and shaded tea shoots Naturally grown shoots Shaded shoots Fraction Radioactivity Incorporation Radioactivity Incorporation (dpmxlo-3) (dpmxlo-3) % Total radioactivity absorbed * 80% ethanol-soluble fraction Ethyl acetate-soluble fraction EC EC moiety of ECG Total EC moiety EGC EGC moiety of EGCG Total EGC moiety Gallic acid moiety of ECG &EGCG 80% ethanol-insoluble fraction " 100 x radioactivity in each fraction/total radioactivity absorbed. 4 the ethanol soluble fraction plus the insoluble fraction. Abbreviations used are explained in Table 1. Tea plant of cultivar C81 was shaded for 8 days in early May. reduced 0.94, 1.36 and 0.23% by the treatment, respectively. Radioactivity was not detectable in free gallic acid from both experimental shoots. Furthermore, effects of the treatment on incorporation of certain intermediates in the shikimate pathway and the phenylpropanoid metabolism into catechins were investigated using glucose-u- 14 G, shikimic acid-g- 14 C, phenylalanine-u- 14 G and /ra/u-cinnamic acid-3-14 C. The results are illustrated in Table 3, which excludes the radioactivity found in the gallic acid moiety as it may have been synthesized through at least two different pathways in the tea plant (unpublished data). The incorporation rates of glucose-u- 14 C, shikimic acid-g- 14 C and phenylalanine-u- 14 C into the total ( )-epicatechin and ( )-epigallocatechin moieties were obviously depressed by the shade treatment. In contrast, those offra;u-cinnamicacid-3-14 C did not appear to be affected. These observations strongly suggest that aflowof the intermediates is inhibited at stages before frwu-cinnamic acid in the catechin biosynthetic pathway when the tea plant is grown under reduced light intensity Effect of shade treatment on the enzyme activities Several reports describe the stimulatory effect of light on the activities of enzymes involved in flavonoid biosynthesis such as 3-dehydroquinate dehydratase, shikimate dehydrogenase, phenylalanine ammonia-lyase, /ranj-cinnamate 4-monooxygenase and other enzymes of phenylpropanoid biosynthesis in many higher plants (5, 10, 14). As the reverse phenomena might occur by shading tea plants, activities of the six enzymes involved in the shikimate pathway and the phenylpropanoid metabolism were measured in the control and treated shoots as shown in Fig. 2. Of those enzymes, no significant differences of the activities were observed for phospho-2-keto-3-

7 Table 3 Effect of shndc on conversion of various precursors into (-)-epicatechin and (-)-cpigalfocatcchin in tea plant Fraction Glucose-U-14C Shikimic acid-gj4c Naturally grown shoots Shaded shoots Naturally grown shoots Shaded shoots Radioactivity Incorporation Radioactivity Incorporation Radioactivity Incorporation Radioactivity Incorporation (dpm x 10-3) (dpm x 10-3) rate ' (dpm x 10-3) rate ' (dpm x 10-3) rate ' (%) (%) (%I Total radioactivity absorbed % ethanol-soluble fraction Total EC moiety Total EGC moiety Q 80% ethanol-insoluble fraction Fraction Phenylalanine-U-'QC Naturally grown shoots Shaded shoots Radioactivity Incorporation Radioactivity Incorporation (dpm x 10-3) :g; ' (dpm x 10-3) :z Total radioactivity absorbed %' ethanol-soluble fraction Total EC moietyc Total EGC moiety ' (%I trans-cinnamic acid-3-14c 3 6. Naturally grown shoots Shaded shoots I. Radioactivity Incorporation Radioactivity rncorporation 5' (dpm x 10-3) (dpm x 10-3) (%I (%) 3 a_ 80% ethanol-insoluble fraction ' s b As in Table 2. EC+EC moiety of ECG EGC+EGC moiety of EGCG Abbreviations used are explained in Table 1. Tea plant of cultivar C 81 was used. The shade treatment was continued for 13 days. $ E 5 f

8 996 R. Saijo 15X10"" A B 6XI0" 2 I0XI0" 4 4XI0" 2 % H (uni vity 5XI0" 4 I5XI0' 3 IOXIO- 3 5XI0" X10~ 3 h IOXIO" 3-5XI0" C i i i May D i i i May 4 2XIO" 2 0 6XI0" 1 4XI0" 1 2XI0' 1 0 6XI0" 6 4XI0" 6 2XI0" 6 Fig. 2. Effect of shade treatment on the activities of enzymes involved in catechin biosynthesis of tea plants. A, phospho-2-keto-3-deoxy-heptonate aldolase; B, 3-dehydroquinate synthase; C, 3-dehydroquinate dehydratase; D, shikimate dehydrogenase; E, phenylalanine ammonia-lyase; F, frwu-cinnamate 4-monooxygenase. Newly developing tea shoots of cultivar Okumidori were used. -O- and - -, as in Fig deoxy-heptonate aldolase, 3-dehydroquinate synthase, 3-dehydroquinate dehydratase, shikimate dehydrogenase andfcww-cinnamate4-monooxygenase in both shoots. In contrast, the activity of phenylalanine ammonia-lyase decreased to about 45 and 40% of the original activity after shade treatment of three and six days, respectively. Although the activity in the control plants also decreased to about 65% after six days of development, the results indicated that much of the decrease in the shadetreated shoots was due to the effect of the light intensity as mentioned by Iwasa (4).

9 Catechin biosynthesis in tea plant 997 The observation suggests that phenylalanine ammonia-lyase plays an important role in the regulation of catechin biosynthesis in tea plants under shade treatment. Discussion Table 1 demonstrates that shade treatment reduced the production of the individual catechins to a greater extent than weight increases of the newly developing tea shoots. With the shade treatment, carbohydrate supply for flavonoid biosynthesis may have been reduced, resulting in the decreased production of catechins. However, other mechanisms also should be sought as contributors to the lower content of catechins in the shade-treated tea shoots. 14 C-Incorporation studies using B-ring intermediates suggested that a regulatory point (or points) was present before thefra;u-cinnamicacid synthesis on the catechin biosynthetic pathway (Table 3). In addition, the enzyme studies provided evidence that the level of phenylalanine ammonia-lyase was reduced as a result of the treatment (Fig. 2), while the other five enzymes involved in the B-ring formation of catechins were less affected. The present results and other findings (1-4) lead to the conclusion that shade treatment depressed phenylalanine ammonia-lyase activity, which then contributed to the decrease in catechin biosynthesis, and finally reduced the catechin content in the shade-treated shoots. References ( 1) Anan, T. and M. Nakagawa: Effect of light on chemical constituents in tea leaves (in Japanese). Nippon Nogeikagaku Kaishi48: (1974). (2) Forrest, G. I.: Effects of light and darkness on polyphenol distribution in the tea plant {Camellia sinensisl.). Biochem. J. 113: (1969). ( 3) Iwasa, K.: Influence of the shading culture on catechin composition in tea leaves (in Japanese). Study of Tea 36: (1968). (4) Iwasa, K.: Biosynthesis of catechins in tea plant. Bulletin of the National Research Institute of Tea No. 13: (1977). (5) McClure, J. W.: Physiology and functions of flavonoids. In The Flavonoids. Edited by J. B. Harborne, T. J. Mabry and H. Mabry. p Academic Press, Inc. New York, N. Y., (6) Nakagawa, M. and H. Torii: Studies on theflavanolsin tea (Part 1). A method for the quantitative determination of flavanols by paper partition chromatography. Agric. Biol. Chem. 28: (1964). (7) Nakagawa, M. and H. Torii: Studies on the flavanob in tea (Part 3). Varietal difference of flavanolic constituents in tea leaves (in Japanese). Study of Tea 29: (1964). (5) Saijo, R. and T. Takeo: Incorporation of exogeneously supplied JV-ethyl- 14 C-theanine to catechin in tea shoots (in Japanese). ibid. 56: (1978). ( 9) Saijo, R. and T. Takeo: Some porperties of the initial four enzymes involved in shikimic acid biosynthesis in tea plant. Agric. Biol. Chem. 43: (1979). (10) Smith H.: Regulatory mechanisms in the photocontrol of flavonoid biosynthesis. In Biosynthesis and Its Control in Plants. Edited by B. V. Milborrow. p Academic Press, Inc., New York, N. Y., (//) Tanaka, Y., M. Kojima and I. Uritani: Properties, development and cellular-localization of cinnamic acid 4-hydroxylase in cut-injured sweet potato. Plant & Cell Physiol. 15: (1974).

10 998 R. Saijo (12) Zaprometov, M. N. and V. Ya. Bukhlaeva: The effectiveness of the use of various 14 C- precursors for the biosynthesis of flavonoids in the tea plant. Biokhimiya 36: (1971). (13) Zaprometov, M. N. and V. Ya. Bukhlaeva: The possibility of the existence of alternative pathways of flavonoid biosynthesis in tea plants, ibid. 38: (1973). (14) Zucker, M.: Light and enzymes. Arm. Rev. Plant Physiol. 23: (1972).