60708 (183) Biosci. Biotechnol. Biochem., 71, 60708-1–11, 2007 Chemical Profiling and Gene Expression Profiling during the Manufacturing Process of Taiwan Oolong Tea ‘‘Oriental Beauty’’ y Jeong-Yong CHO,1 Masaharu MIZUTANI,1; Bun-ichi SHIMIZU,1 Tomomi KINOSHITA,1 Miharu OGURA,2 Kazuhiko TOKORO,2 Mu-Lien LIN,3 and Kanzo SAKATA1 1Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan 2Central Research Laboratory, Takasago International Corporation, 1-4-11 Nishi-yawata, Hiratsuka-shi, Kanagawa 254-0073, Japan 3Tea Research and Extension Station, 324 Chunghsin Road, Yangmei, Taoyuan 326, Taiwan Received December 18, 2006; Accepted March 15, 2007; Online Publication, June 7, 2007 [doi:10.1271/bbb.60708] Oriental Beauty, which is made from tea leaves in- infested by the tea green leafhopper (J. formosana). The fested by the tea green leafhopper (Jacobiasca formosa- infested leaves (Fig. 1B) are small and yellowish as na) in Taiwan, has a unique aroma like ripe fruits and compared to healthy leaves (Fig. 1A). It is traditionally honey.Advance To determine what occurs in the tea leaves View dur- known that insect attack promotes the tea quality, es- ing the oolong tea manufacturing process, the gene pecially the strength of the unique aroma of the tea.1) expression profiles and the chemical profiles were inves- Oolong tea is generally produced from fresh tea tigated. Tea samples were prepared from Camellia si- leaves via complicated processes of plucking, solar nensis var. sinensis cv. Chin-shin Dah-pang while the tea withering, indoor withering, turn over, panning, rolling, leaves were attacked by the insect. The main volatile and drying, although various processing methods are compounds, such as linalool-oxides, benzyl alcohol, 2- used for other types of oolong tea.3) The typical man- phenylethanol, and 2,6-dimethylocta-3,7-diene-2,6-diol, ufacturing process for Oriental Beauty is illustrated in increased during manufacture. The gene expression Fig. 1C. There are unusual points in the manufacturing profiles during manufacture were analyzed by differ- process for OrientalProofs Beauty as compared to those for the ential screening between fresh leaves and tea leaves of other types of oolong tea. In Oriental Beauty manufac- the first turn over. Many up-regulated transcripts were ture, the steps of solar withering and indoor withering found to encode various proteins homologous to stress are longer, and the additional step, wetting and soften- response proteins. Accordingly, the endogenous contents ing, is done before rolling. Thus the degree of fermen- of abscisic acid and raffinose increased during manu- tation in Oriental Beauty is higher, and that may also be facture. Thus the traditional manufacturing method is a responsible for the promotion of tea quality. unique process that utilizes plant defense responses to Tea aroma is one of the most important factors in elevate the production of volatile compounds and other determining the character and quality of each tea, es- metabolites. pecially oolong tea and black tea. The main aroma con- stituents of oolong tea are linalool, geraniol, benzyl al- Key words: Oriental Beauty (Pom-Fong tea); oolong cohol, 2-phenylethanol, and linalool oxides.2,4,5) Most of tea; tea green leafhoppers; stress response these alcoholic aroma compounds are mainly present gene; volatile compound as disaccharide glycosides, such as -primeveroside, - acuminoside, and -vicianoside, in fresh leaves of tea Oriental Beauty is a famous Formosa oolong tea that cultivars for oolong tea (C. sinensis var. sinensis cv. has a unique aroma like ripe fruit and honey.1,2) Other Shuixian and Maoxue) and for green tea (cv. Yabu- names are Pom-Fong tea, Champagne oolong tea, Chan kita).6,7) These glycosides are hydrolyzed to release Pin oolong tea, and White Tip oolong tea, etc. Oriental alcoholic aroma compounds by the action of endogenous Beauty is mainly produced in counties of Hsinchu, enzymes ( -glucosidase and -primeverosidase) during Miaoli, Taoyuan, and Taipei in the northern part of the tea manufacturing process, suggesting that the hy- Taiwan. In Oriental Beauty manufacture, one of the drolysis of glycosides is one of the key factors in alco- most characteristic factors is the use of tea leaves holic aroma formation during the processing of oolong y To whom correspondence should be addressed. Tel: +81-774-38-3230; Fax: +81-774-38-3229; E-mail: [email protected] Abbreviations: ABA, abscisic acid; diol, 2,6-dimethylocta-3,7-diene-2,6-diol; EST, expressed sequencing tags; NCED, 9-cis-epoxycarotenoid dioxygenase; AMI, 2-O-( -L-arabinopyranosyl)-myo-inositol 60708-2 J.-Y. CHO et al. AB Healthy tea leaves Infested tea leaves (Non-infested leaves) by tea green leafhoppers C (Jacobiasca formosana) Cultivation Turn over Plucking Solar withering with insects /indoor withering (F) (4 h, SW) (3 weeks) (every 2 h, 5 times, T1-T5) Wetting Panning Rolling Drying Product /softening Fig. 1. Tea Leaves (Healthy and Infested) and Sampling during the Manufacture of Oriental Beauty. Sampling of tea leaves was done at each step of the manufacturing process (F, SW, and T1-T5). For chemical profiling of volatile compounds and metabolites, the sample at each step was subjected to the processes of panning, wetting and softening, rolling, and drying. For gene expression profiling, each sample was immediately frozen without panning. teaAdvance6–10) as well as black tea.11,12) Furthermore, Viewthe cells 104 U2 and tissues of tea leaves are still alive before panning U1 (Fig. 1C), and therefore it is likely that many aroma 103 compounds are produced by de novo biosynthesis in tea leaves. It has been reported that emission of volatiles and 102 expression of the genes involved in volatile biosynthesis is induced by stresses such as insect attack and wound- 101 13–15) ing in various plant species. In addition to increased Intensity of Cy5 (T1) production of volatiles, it is well known that abiotic 100 Proofs stresses trigger a wide variety of plant responses, in- 100 101 102 103 104 cluding alteration in gene expression, the accumulation Intensity of fluorescein (F) of phytohormones such as abscisic acid (ABA), jas- monic acid, and ethylene, and enhanced synthesis of Fig. 2. Differential Screening Analysis with Fresh Leaves (F) and specific proteins (e.g., heat shock proteins and enzymes the Tea Leaves of First Turn Over (T1) during the Oolong Tea that function in various metabolite biosynthesis, such as Manufacturing Process. sugars and osmoprotective raffinose-family oligosaccha- U1, highly induced transcripts; U2, moderately induced tran- scripts. rides).16,17) These plant responses lead to acclimation and tolerance to environmental stresses. In the same way, fluctuation of various metabolites in tea leaves must occur in response to stresses such as UV-irra- during the oolong tea manufacturing process should give diation, drought, and wounding during the manufactur- us valuable information about a vast number of induc- ing process. However, little is known about what occurs ible genes, including stress-response genes and biosyn- in tea leaves during processing. thetic genes of aroma compounds. Recently, molecular biological approaches such as In this study, we report gene expression profiles and differential screening, expressed sequencing tags (EST), chemical profiles to understand what occurs in tea leaves and DNA microarray have been used to identify in- during the manufacturing process of Oriental Beauty. ducible genes in response to various stresses from many First, the changes in the chemical constituents of the plant tissues, including nonwoody plants (Arabidopsis, volatile compounds were examined during manufacture. tomato, Brassica)18–20) and woody plants (poplar, grape- We also investigated the gene expression profiling in tea fruit).21,22) In tea leaves (C. sinensis), Chen et al.23) leaves during the earlier stages of manufacture by dif- performed EST analysis of spring tender shoots. Park et ferential screening analysis. Although we could not find al.24) also reported EST analysis of a subtractive cDNA the transcripts to be directly related to aroma compound library between young leaves and mature leaves, and biosynthesis, we identified various stress response genes identified several genes involved in catechin biosyn- and identified the accumulation of ABA and raffinose thesis. In this way, transcript profiling in tea leaves in response to the manufacturing process. Accordingly, Chemical and Gene Profiling in the Manufacture of Taiwan Oolong Tea 60708-3 we also discuss the relation between the oolong tea thesized by T7 DNA polymerase using Cy5-uridine manufacturing method and plant stress response. triphosphate (UTP) and fluorescein UTP. The probed microbeads (4:0 Â 105 beads, a total of 8:0 Â 105 beads) Materials and Methods for the tea samples (F and T1) were mixed and hy- bridized overnight at 50 C in digoxigenin (DIG) Easy Materials. Tea leaves (cv. Chin-shin Dah-pang) in- Hyb (Roche Diagnostics, Basel, Switzerland). The hy- fested by tea green leafhoppers (J. formosana) for about bridized microbeads were washed in 1Â saline sodium 3 weeks were plucked in Hsinchu County of Taiwan citrate (SSC)/1% sodium dodecyl sulfate (SDS), and in June 2004. The manufacture of Oriental Beauty was then in 0:1Â SSC/0.1% SDS at 65 C. The microbeads carried out in the same county according to the tra- were sorted with a MoFlo fluorescence-activated cell ditional method, as shown in Fig. 1C. After the infested sorter (DakoCytomation, Fort Collins, CO). The distri- tea leaves were exposed to sunlight for 4 h as a solar bution of cDNAs up- and down-regulated during oolong withering process, the leaves were indoor-withered at tea manufacture is shown in Fig. 2. The up-regulated room temperature, accompanied by 5 repetitions (every cDNAs were further fractioned into two zones, highly 2 h) of indoor withering, followed by turn over. Then the induced genes (U1 group) and moderately induced genes leaves were parched at 180–190 C for 3–5 min.