Tree Physiology 29, 837846 doi:10.

1093/treephys/tpp022

Dierential display mediated cloning of anthocyanidin reductase gene from tea (Camellia sinensis) and its relationship with the concentration of epicatechins
KASHMIR SINGH,1,* ARTI RANI,1,** ASOSII PAUL,1 SOM DUTT,1 ROBIN JOSHI,3 ASHU GULATI,3 PARAMVIR SINGH AHUJA1 and SANJAY KUMAR1,2
1 2 3

Biotechnology Division, Institute of Himalayan Bioresource Technology (CSIR), P.O. Box No. 6, Palampur 176061, India Corresponding author (sanjayplp@redimail.com, sanjaykumar@ihbt.res.in) Hill Area Tea Science Division, Institute of Himalayan Bioresource Technology (CSIR), P.O. Box No. 6, Palampur 176061, India

Received November 27, 2008; accepted March 16, 2009; published online April 20, 2009

Summary Tea [Camellia sinensis (L.) O. Kuntze] leaves are a major source of epicatechin (EC) and its gallolyl derivatives epicatechin gallate, epigallocatechin and epigallocatechin gallate, collectively known as epicatechins (ECs). Epicatechins are important factors determining tea quality, and they also possess many medicinal properties. To gain further information about the regulation of the biosynthesis of ECs, we cloned the gene encoding anthocyanidin reductase from tea (CsANR) by rst quantifying changes in the concentrations of ECs in response to drought, gibberellic acid (GA3), abscisic acid (ABA) and wounding treatments, followed by dierential display of mRNAs and analysis of those bands exhibiting a change in expression paralleling the treatment-induced changes observed in the EC data. Analysis of 133 bands yielded a partial cDNA of CsANR that was later cloned to the full length by rapid amplication of the cDNA ends. The full-length CsANR (Accession No. AY641729) comprised 1233 bp with an ORF of 1014 bp (from 79 to 1092 bp) encoding a polypeptide of 337 amino acids. Expression of CsANR in an Escherichia coli expression vector yielded a functional protein that catalyzed the conversion of cyanidin to EC in the presence of NADPH. Analysis of ECs and gene expression in leaves at dierent developmental stages and across ve tea clones exhibiting variable concentrations of ECs revealed a positive correlation between concentration of ECs and CsANR expression. Expression of CsANR was down-regulated in response to drought, ABA and GA3 treatments and upregulated in response to wounding. Keywords: catechin, avonoid.

Introduction
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Present address: Department of Biotechnology, Punjab University, Chandigarh 160014, India. ** Present address: Vittal Mallya Scientic Research Foundation, Bangalore, India.

Tea [Camellia sinensis (L.) O. Kuntze] is grown in more than 30 countries around the world (Jain 1999). Tea leaves are a major source of catechins that constitute 2530% of the dry mass of young tea leaves (Lin et al. 1996, Singh et al. 1999). Catechins are water-soluble, polyhydroxylated avonoids that impart the characteristic astringency and bitterness to black tea (Chen 2002). In the animal system, catechins and their derivatives are reported to prevent cancer, cardiovascular, neurodegenerative and other oxidativestress-related diseases (Bordoni et al. 2002, Levites et al. 2002, Nie et al. 2002), possibly by inhibiting the activities of certain enzymes such as ornithine decarboxylase, urokinase and protein kinase C (Agarwal et al. 1992, Yoshizawa et al. 1992, Jankun et al. 1997). Catechins is the general term used for epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG), catechin and catechin gallate. Epicatechin and its gallolyl derivatives EGC, ECG, EGCG [collectively termed epicatechins (ECs)] constitute about 90% of the total catechins in tea leaves (Zhen et al. 2002). Epicatechins are synthesized through the avonoid (FL) pathway (Figure 1) and 4-coumaroyl CoA, the substrate for the rst committed step of the FL pathway, is supplied by the phenylpropanoid (PP) pathway (Rani et al. 2008). Anthocyanidins are synthesized through a series of reactions, involving the enzymes chalcone synthase (CHS), chalcone isomerase, dihydroavonol reductase and anthocyanidin synthase (Punyasiri et al. 2004, Singh et al. 2008c). Anthocyanidin reductase (ANR) uses anthocyanidins such as cyanidin to synthesize EC in the presence of NADH or NADPH (Xie et al. 2003). The rst evidence for the role of ANR in EC biosynthesis was provided by the analysis of the BANYULS genes of Arabidopsis thaliana (L.) Heynh. and Medicago truncatula Gaertn. (Xie et al. 2003). The biochemical properties of

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sea level) of the Institute of Himalayan Bioresource Technology was selected for all the experiments except for the reverse northern analysis. Experiments were performed on the apical bud (AB; youngest), and on leaves taken at nodal positions 1, 2, 3 and 4 on the shoot (position with reference to the AB); hereafter referred to as AB, 1st, 2nd, 3rd and 4th leaf. Leaves were collected between 09:00 and 10:00 h during the period of active growth (July; mean temperature 22.5 C), immediately frozen in liquid nitrogen and stored at 80 C until being analyzed. All experiments included three separate biological samples. For reverse northern analysis, clones TS-449, RYDAK-1, AV-2 and HV-39 were selected. RYDAK-1 and AV-2 are Chinary-type tea clones and were obtained from the Makaibari Tea Estate, Darjeeling (India). TS-449, which is an Assamica-type tea clone, and HV-39 (Happy Valley-39), which is a ChinaAssam hybrid tea clone, were obtained from Tocklai Experimental Station, Jorhat (India). These tea clones were maintained at the Banuri tea experimental farm of the Institute of Himalayan Bioresource Technology.
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Imposition of external treatments

Figure 1. Schematic diagram of the biosynthesis of ECs (adapted from Xie et al. 2003, Punyasiri et al. 2004, Singh et al. 2008c). Abbreviations: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, avanone-3-hydroxylase; DFR, dihydroavonol 4-reductase; ANS, anthocyanidin synthase; LAR, leucoanthocyanidin reductase and ANR, anthocyanidin reductase. If the anthocyanidin is cyanidin, EC will be synthesized, whereas if delphinidin is the anthocyanidin, EGC will be synthesized.

ANR from M. truncatula (MtANR) and Arabidopsis (AtANR) dier (Xie et al. 2004), with MtANR using both NADPH and NADH as reductant with a slight preference for NADPH, whereas AtANR uses only NADPH and exhibits positive cooperativity for this cosubstrate. In addition, MtANR shows a preference for cyanidin, whereas AtANR exhibits a preference for delphinidin. Although tea has been shown to possess high ANR activity (Punyasiri et al. 2004), neither the gene nor its regulation has been characterized in this species. The knowledge is critical to (1) understand the role of the gene encoding ANR in EC biosynthesis and (2) exploit it in metabolic engineering. We cloned the full-length ANR gene of tea (CsANR) through dierential display (DD) of mRNA followed by rapid amplication of cDNA ends (RACE), validated the function of the expressed protein in Escherichia coli and obtained further evidence of its role in EC biosynthesis. Materials and methods Plant materials and growth conditions UPASI-10, a Chinary-type tea clone (Balasaravanan et al. 2003), growing at the Banuri tea experimental farm (3206 0 3200 N and 7633 0 4300 E, altitude 1300 m above mean

Two-year-old tea plants of UPASI-10 were raised in plastic pots in a plant growth chamber (temperature, 25 1 C; irradiance, 200 lmol m2 s1; RH, 7080%; Saveer Biotech, India). Drought stress (DS) was imposed by withholding water during the entire 8-day treatment period. Abscisic acid (ABA; 100 lM) and gibberellic acid (GA3; 50 lM) were sprayed on the plants and also applied to the soil (Sharma and Kumar 2005, Singh et al. 2008a). After the 8-day treatment period, the AB and 1st leaf of each plant were harvested and combined for subsequent analyses. For the wounding experiment, because the AB, 1st and 2nd leaves were too small to apply the wounding treatment, the margins of the 3rd leaf of each plant were wounded with the aid of a haemostat. Twelve hours after wounding, each wounded leaf was harvested for analyses. Control samples were harvested at the corresponding times from untreated control plants that were kept well watered. Quantication of ECs Epicatechins were quantied as described by Sharma et al. (2005). Apical bud and leaf samples were ground in liquid nitrogen and extracted with aqueous methanol (70%) with intermittent shaking followed by centrifugation at 1400g for 10 min. The extraction steps were repeated resulting in a nal extract volume of 10 ml. The extracts were ltered through a 0.5-lm Millipore lter before being injected on an analytical semi-preparative Merck-Hitachi HPLC system tted with a C-18 Lichrocart column (250 4.0 mm; 5 lm), along with suitable guard column. Samples were eluted with 0.1% orthophosphoric acid in water (solvent A) and acetonitrile (solvent B) as the mobile phase. The HPLC elution program consisted of ratios of solvents A:B of 90:10 (010 min), 90:1080:20 (1012 min),

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80:2065:35 (1215 min), 65:3564:36 (1518 min), 64:36 (1820 min), 64:3670:30 (2024 min), 70:3080:20 (24 28 min) and 80: 2090:10 (2830 min). The ow rate was 1 ml min1 and the injection volume was 20 ll. Various ECs were detected at 280 nm. For identication and quantication of EC, ECG, EGC and EGCG, the area and retention time of each analyte peak were matched with the corresponding reference standard chromatographed under similar conditions. Standard curves were prepared by plotting concentration versus area for each reference standard. Cloning of CsANR by DD of mRNA Dierential display was performed with total RNA isolated from the shoots of control plants and plants subjected to 8 days of DS, GA3 treatment and 12 h of leaf wounding. The ABA-treated samples were omitted from this experiment because the DS and ABA treatments likely modulate similar genes (Shinozaki and Yamaguchi-Shinozaki 1997). Isolation of total RNA, removal of contaminating DNA, cDNA synthesis, radioactive PCR (polymerase chain reaction), separation of PCR products on a denaturating polyacrylamide gel, elution of cDNA bands and cloning of the PCR products were performed as described previously (Sharma and Kumar 2005, Singh et al. 2008a), except that we used kits supplied by GenHunter Corporation (TN). These kits included MessageClean to remove contaminating DNA, RNAimage for performing DD, PCR-TRAP vector to clone PCR products after eluting and amplifying from the polyacrylamide gel and AidSeq primer set C for sequencing. Taq polymerase was purchased from Qiagen (Germany) and 33P-dATP (specic activity, 2500 Ci mmol1, 10 mCi ml1) was purchased from Bhabha Atomic Research Centre, Bombay, India. We selected and analyzed the cDNA bands that exhibited down-regulation in response to DS and GA3, and up-regulation in response to wounding compared with the control. These cDNA bands were subjected to reverse northern analysis (Singh et al. 2008b) to eliminate PCR artefacts using RNA isolated from UPASI-10. We followed the method for reverse northern analysis described below, except that 140 ESTs of putative dierentially expressed cDNAs were blotted onto the membrane and RNA of UPASI-10 (AB and the 1st leaf combined) was used for probe preparation. The gene fragment showing homology with the reported ANRs was selected to design primers for rapid amplication of the cDNA ends (RACE) to clone the full-length cDNA of CsANR. The primers used for 3 0 RACE were GSP1 5 0 -GTGTCAGTCCAGTGACTCTCATCC AT-3 0 and NGSP1 5 0 -GTGTCAGTCCAGTGACTCTCATGGAT-3 0 , and the primers used for 5 0 RACE were GSP2 5 0 -CAC CTTCTAGCACTAAAGGGTTCAGG-3 0 and NGSP2 5 0 -TGAACAGAGCTTT GACACCCCTGTAG-3 0 . Plasmids were isolated using the Qiagen plasmid mini isolation kit and sequencing was performed using BigDye
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terminator v 3.1 cycle sequencing mix (Applied Biosystems, CA) and an automated DNA sequencer (ABI Prism 310, Genetic Analyzer, Applied Biosystems). Homology searches of sequences in databases were conducted using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/). The deduced amino acid sequence was used to analyze protein families and domains using various tools available at PROSITE database at ExPASy proteomics server (http:// ca.expasy.org/) and SMART (http://smart.embl-heidel berg.de/). Secondary structures of the deduced CsANR protein were predicted by SOPMA (http://npsa-pbil.ibcp.fr/). Recombinant protein expression The open reading frame (ORF) of CsANR was cloned into expression vector pQE-30 U/A and transformed into M15 pREP-4 competent cells (The QIAexpressionist, Qiagen, Germany). pQE-30 U/A allows in-frame cloning of PCR products resulting in a 6 His tag attached to the N-terminal end of the recombinant protein. In-frame cloning of the ORF of the gene with the bacterial promoter was conrmed by sequencing. Expression of the recombinant protein was induced by 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) dissolved in sterile water and added to the liquid culture. Cells were harvested at dierent times and expression of the recombinant protein was checked on an SDSpolyacrylamide gel stained with Coomassie Brilliant Blue (Hames and Rickwood 1990). Since maximum induction was observed 6 h after IPTG addition, cells were harvested after a 6-h induction period and assayed for ANR activity. Assay of CsANR We assayed ANR activity as described by Xie et al. (2004) and Punyasiri et al. (2004) with slight modications. The reaction mixture (500 ll) contained 50 mM TrisHCl (pH 7.0), 4 mM NADPH, 100 ll of 0.1% aqueous cyanidin chloride (Roth, Germany) and 50 lg crude protein from recombinant E. coli harbouring CsANR. Crude extract from a similar but uninduced E. coli culture was used as a control. After incubation of the assay mixture for 1 h at 25 C, the reaction products were extracted twice with 500 ll of ethyl acetate each time. The organic phase was pooled, evaporated under vacuum and the residue dissolved in 100 ll of methanol for estimation of EC as described previously. Semi-quantitative expression analysis by RT-PCR To study gene expression, cDNA was synthesized using 2 lg of DNA-free RNA (RNA was digested with 2 U amplication grade DNase I from Invitrogen, CA) in the presence of 400 U reverse transcriptase Superscript II (Invitrogen) and 1.0 lg oligo (dT) 1218 (Singh et al. 2004). The resultant cDNA was used as template for PCR using forward primer 5 0 -CAC CTT CTA GCA CTA AAG GGT TCA GG-3 0 , and reverse primer

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5 0 -GTG TCA GTC CAG TGA CTC TCA TCC AT-3 0 . The thermal cycler (GeneAmp 9700 ABI, Applied Biosystems) was programmed at 94 C for 3 min for 1 cycle, then at 94 C for 30 s, 61 C for 30 s, 72 C for 30 s for 28 cycles and nally at 72 C for 7 min. Expression of 26S rRNA was used as a control to normalize the amount of cDNA in the various reactions (Singh et al. 2004). Gels were scanned with a gel documentation system (Alpha DigiDocTM, Alpha Innotech, CA) and integrated density values (IDV) were calculated with AD-1000 software to calculate the changes in gene expression. Reverse northern analysis of CsANR in tea clones Reverse northern analysis was performed in ve clones of tea with varying concentrations of ECs. These were RYDAK-1 and AV-2, Chinary type; TS-449, Assamica type; and HV39, ChinaAssam hybrid type. Since these clones have dierent genetic background and there exists enormous polymorphism in tea (Balasaravanan et al. 2003), reverse northern analysis was selected rather than RT-PCR analysis (Singh et al. 2008b). The RNA from AB and the 1st leaf combined was isolated from each of the ve clones and separate cDNA probes were prepared and puried as specied by the manufacturer of the ReversePrime cDNA labelling kit and QIAquick nucleotide removal kit (Qiagen, Germany). We amplied CsANR from UPASI-10 using the forward and reverse primers as 5 0 -GTCATTTTAACATCATCAG CAG-3 0 and 5 0 -AGCTTTCTCAGCTAGTGTTTTC-3 0 , respectively. The PCR product was denatured and blotted onto a nylon membrane (Hybond-XL, Amersham, Little
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Chalfont, UK) essentially as described by the manufacturer (ReversePrime cDNA labeling kit from GenHunter Corporation). Prehybridization, hybridization and washing followed standard protocols (Sambrook et al. 1989). For hybridization, we used equal amounts of probe as quantied by radioactivity. To check that equal quantities of RNA were used, the PCR-amplied product of 26S rRNA (Singh et al. 2004) was blotted and hybridized to serve as a control.
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Results and discussion Cloning of functional CsANR Dierential display is a powerful method for identifying and cloning novel genes and dierent members of a gene family (Joshi et al. 1995, Sharma and Kumar 2005, Singh et al. 2008b). We used DD to clone CsANR by rst imposing treatments that modulated the concentrations of ECs in tea leaves followed by DD reactions and analysis of those bands that exhibited expression paralleling the ECs data. The use of degenerate primers would have been another way to clone the gene, but we were unable to obtain functional primers. Other methods, such as analysis of subtraction suppressive libraries (Diatchenko et al. 1996), require screening of large numbers of clones and simultaneous comparison of more than two mRNA populations poses logistic problems of handling and operation. For these reasons, we chose DD that allows comparison of several treatments simultaneously.

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Figure 2. Concentrations of ECs (total ECs, EC, ECG, EGC and EGCG) in response to (A) DS, (B) ABA treatment, (C) GA3 treatment and (D) wounding (WS) in AB and 1st leaf combined in tea clone UPASI-10. The eects of WS on concentrations of ECs were determined only at 0, 12, 24 and 48 h after wounding because wounded leaves did not permit the experiments beyond 48 h. Each value represents the mean standard error of three separate biological replicates. A color version of this gure is available as Supplementary Data at Tree Physiology Online.

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We found that the DS, ABA and GA3 treatments decreased ECs in the AB and 1st leaf combined, starting from Day 2 and onward, whereas wounding enhanced the concentration of ECs compared with control values

(Figure 2). The cDNA bands that showed down-regulation in response to DS and GA3, and up-regulation in response to wounding were selected for analysis (Figure 1 in Supplementary Data). This approach, which minimized the num-

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Figure 3. Multiple amino acid sequence alignment of CsANR of tea clone UPASI-10 using ClustalW program with the previously reported sequences from V. vinifera (AAZ82409), P. communis (ABB77695), G. hirsutum (ABM64802), M. truncatula (AAN77735) and A. thaliana (NP176365). Gaps are represented as - and *, : and . indicate identical amino acid residues, conserved substitutions and semi-conserved substitutions in all sequences used in the alignment, respectively.

Figure 4. Predicted secondary structure of CsANR of tea clone UPASI-10 (A) by SOPMA (http://npsa-pbil.ibcp.fr/) and comparison with that of AtANR (B) and MtANR (C). Helices, sheets, turns and coils are indicated by the longest, the second longest, the second shortest and the shortest vertical lines, respectively.

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ber of clones selected for analysis, led to the identication of 140 bands that were subjected to reverse northern analysis using RNA isolated from the AB and 1st leaf combined of UPASI-10 to eliminate PCR artefacts (Figure 2 in Supplementary Data). Of these 140 bands, 133 bands were submitted to the NCBI EST database (Gene bank Accession Nos. DN976085 to DN976213, DR397420, AY694187, AY694189 and AY694190; Table 1 in Supplementary Data). Based on the BLAST analysis, these genes were putatively grouped into those involved in (1) primary metabolism (9.7%), (2) cell cycle (3.7%), (3) secondary metabolism (8.2%), (4) cell signalling (9.0%), (5) stress response (11.2%), (6) transcription regulators (4.2%), (7) growth and development (12.0%), (8) high cellular activities (4.5%), (9) defense (6%) and (10) those showing nonsignicant homology were considered novel (31.5%). Of the analyzed cDNAs, a 350-bp band (dbEST Accession No. DN976194) exhibited signicant homology with reported ANRs. Based on the sequence data, a full-length CsANR was cloned by 5 0 and 3 0 RACE using SMART RACE cDNA Amplication Kit (BD, Biosciences, CA) following the recommended protocol. CsANR (Accession No. AY641729) comprised a 1233 bp full-length cDNA with an ORF of 1014 bp, extending from 79 to 1092 bp. CsANR showed 6382% similarity at the amino acid level and 4575% similarity at the nucleotide level with ANR sequences reported from other plants. Phylogenetic analysis of the deduced amino acid sequence of CsANR with ve sequences (Vitis vinifera L., BAD89742; Pyrus communis L., ABB77695; Gossypium hirsutum L., ABM64802; M. truncatula, AAN77735; A. thaliana, NP176365) (Figure 3) in the NCBI database revealed that CsANR was closely related to Gossypium species and distantly to P. communis. The deduced amino acid sequence revealed the presence of characteristic domains of the dehydrogenase superfamily. This is a very large family of enzymes, most of which are known to be NAD- or NADP-dependent oxidoreductases. Most members of this family are proteins of about 250300 aa residues. Most dehydrogenases possess at least two domains; the rst binds the co-substrate, often NAD, and the second binds the substrate such as cyanidin. This latter domain determines the substrate specicity and contains the amino acids involved in the catalysis. It has, therefore, been suggested that the structure of dehydrogenases has arisen through gene fusion of a common ancestral coenzyme nucleotide sequence with various substrate-specic domains (Benyajati et al. 1981). The theoretical molecular mass and isoelectric point of the deduced amino sequence of CsANR were 36.218 and 6.54 kDa, respectively. The prediction of secondary structure by SOPMA (Combet et al. 2000) indicated that the deduced CsANR contained 131 aa constituting a-helices, 26 aa b-turns, 61 aa extended strands and 119 aa random coils (Figure 4). Both AtANR and MtANR also have 141 and 132 aa as a-helices, 28 and 22 aa in b-turns, 68 and 54 aa in extended strands and 105 and 130 aa as random coils,
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respectively. The similarity in the secondary structure of CsANR with functional AtANR and MtANR indicated that CsANR encodes a functional protein (Figure 4). Functional characterization of recombinant CsANR To conrm that the cDNA yielded a functional protein, the entire coding region of CsANR was amplied and cloned into pQE-30 U/A. The recombinant plasmid was transformed into M15 pREP-4 competent cells and induced with 1 mM IPTG. The recombinant CsANR was assayed for enzymatic activity. Analysis of the reaction product by HPLC indicated that cyanidin was converted to EC (Figure 5) conrming that the protein encoded by CsANR is a functional enzyme. Concentration of ECs and expression of CsANR in response to leaf age and tea clone Because the most actively growing tissue of the tea plant, that is, the AB and the associated leaves up to the second node, commonly referred to as two and a bud, are used to manufacture high-quality tea (Jain 1999), we examined the eects of leaf age on the concentrations of ECs and CsANR expression. Concentrations of ECs did not dier signicantly from ABs to the 3rd leaf, but were signicantly decreased in the 4th leaf. Similarly, CsANR expression was high in AB, and 1st, 2nd and 3rd leaves, but very low expression was observed in the 4th leaf (Figure 6). High ANR expression in actively growing tissues has also been reported in V. vinifera (Bogs et al. 2005). Such results are in agreement with earlier reports of a positive relationship between the accumulation of the FLs and the activities of FL biosynthetic enzymes and genes (Koes et al. 1994, Jaakola et al. 2002). To obtain further evidence for this relationship in tea, the concentrations of ECs and CsANR expression were studied in tea clones TS-449, RYDAK-1, AV-2, UPASI 10 and HV-39 having dry-mass-based concentration of ECs of 8.3, 8.4, 24.0, 24.2 and 24.9%, respectively. Clones with higher concentrations of ECs had higher expression of CsANR than clones with lower concentrations of ECs (Figure 7). Concentrations of ECs and expression of CsANR in response to external cues In a separate experiment, potted plants of UPASI-10 were subjected to DS, ABA, GA3 and wounding treatments and the concentrations of ECs were measured. The DS, ABA and GA3 treatments decreased the concentrations of ECs in ABs and the 1st leaf combined, starting from Day 2 and onward. On Day 8 of the DS, ABA and GA3 treatments, the concentrations of ECs had decreased by 23, 21 and 15%, respectively, compared with values at Day 0 (Figures 2AC). Wounding, however, enhanced the concentrations of ECs by 17% and 21% after 12 and

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8-day treatment period (Figure 8E). Correlation coecient analysis of the relationship between the extent of CsANR expression and concentration of ECs in plants subjected to the ABA, GA3, DS and wounding treatments indicated a strong positive relationship between the concentration of ECs and IDVs, with Pearson correlation coecients of 0.96, 0.79, 0.096 and 0.69 in plants subjected to the DS, ABA, GA3 and wounding treatments, respectively.

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Figure 5. Formation of EC from cyanidin by recombinant CsANR by the induced cultures of E. coli harbouring CsANR (A). Formation of EC was not detected in the uninduced cultures of E. coli harbouring CsANR (B); and (C) standard -(-) EC. The experiment was repeated at least four times.

Figure 6. Eect of leaf age on the expression of CsANR in AB, and the 1st, 2nd, 3rd and 4th leaf (in relation to AB) of tea clone UPASI-10. Concentrations of ECs (% dry mass) in dierent leaf positions are indicated below each panel. Expression of 26S rRNA served as an internal control. Bar diagram shows IDVs of the amplied bands measured with Alpha Digidoc 1000 software. Each value represents the mean standard error of three separate biological replicates.

24 h of treatment, respectively, compared with control values (Figure 2D). The concentration of ECs at 48 h after wounding was 21% higher than the control value. The physical condition of the wounded leaves did not permit further measurements after 48 h. Compared with the control, CsANR expression decreased in response to the DS, ABA and GA3 treatments by 94, 66 and 52%, respectively, at Day 8 of the treatments (Figures 8AC). In contrast, CsANR expression was upregulated by 23% in response to wounding after 12 h and up-regulated by 10% and 20% at 24 and 48 h after wounding, respectively (Figure 8D). No change in CsANR expression was observed in untreated control plants during the

Figure 7. Reverse northern analysis for tea clones diering in concentration of ECs. Name of the clone and concentration of ECs (% dry mass) is indicated above each lane. Epicatechins values are mean standard error of three separate biological replicates. To check that an equal quantity of RNA was used for each clone, the PCR-amplied product of 26S rRNA (Singh et al. 2004), which was blotted and hybridized as described in the Materials and methods section, served as a control.

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Figure 8. Eects of DS (A), ABA treatment (100 lM) (B), GA3 treatment (50 lM) (C) and wounding on CsANR expression in tea clone UPASI-10 at dierent times (D). Control plants were kept well watered (E). Expression of 26S rRNA served as an internal control. Bar diagram shows IDVs of the amplied bands measured with Alpha Digidoc 1000 software. Each value represents the mean standard error of three separate biological replicates.

Drought is known to down-regulate the expression of phenylalanine ammonia-lyase (PAL) in tea (Jeyaramraja et al. 2003). A decrease in CsANR expression in response to DS or ABA treatment could be part of the general response of tea to stress. In soybean, susceptibility to ABA is expressed by decreases in the accumulation of isoavonoid and PAL mRNAs (Ward et al. 1989). Abscisic acid induces susceptibility by suppressing PAL activity and consequently the production of isoavonoid, glyceollin and other PP during development of the hostpathogen interaction (Ward et al. 1989, Graham and Graham 1996). The hormone GA3 probably controls plant growth and development through DELLA proteins (Thomas and Sun 2004,

Alvey and Harberd 2005), and it also modulates FL biosynthesis indirectly by inducing the production of one or more regulatory proteins that later modulates the pathway directly (Weiss et al. 1995). The hormone GA3 has been shown to inhibit the FL pathway in growing and non-growing cultures of two species of Spirodela (Furuya and Thimann 1964) and in pea (Russel and Galston 1969), at the level of CHS in carrot (Daucus carota L.) cell cultures (Hinderer et al. 1983) and at the level of PAL in bayberry (Myrica rubra Bieb) (Li et al. 2003). Decreases in the expression of PAL, C4H and F3H have been observed in tea in response to DS, ABA and GA3 treatments that paralleled decreases in the concentrations of ECs (Singh et al. 2008b, 2008c). Similarly, we found

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that DS, ABA and GA3 treatments led to down-regulation of the concentrations of ECs and CsANR transcripts, suggesting down-regulation of the FL pathway. Wounding aects the FL pathway by inducing PAL (Logemann et al. 1995, Singh et al. 2008c), C4H (Chapple 1998, Singh et al. 2008c), 4CL (Douglas et al. 1991, Rani et al. 2008), CHS (Brignolas et al. 1995, Richard et al. 2000) and F3H expression (Singh et al. 2008b). The concentrations of ECs increased in response to wounding in Picea abies (L.) Karst. (Brignolas et al. 1995). Similarly, we found that the concentrations of ECs and CsANR transcripts increased in response to wounding at 12 h after treatment and remained high for 24 h. Brignolas et al. (1995) concluded that wounding-induced enhancement of the concentrations of ECs and other FL is an adaptive response to protect against the tissue damage. In conclusion, we isolated a functional CsANR gene and demonstrated a strong correlation between the concentrations of ECs and CsANR expression, indicating a regulatory role of the enzyme in the biosynthesis of ECs. Modulation of CsANR expression, therefore, oers opportunities for regulating the biosynthesis of ECs. Supplementary Data Supplementary Data for this article are available at Tree Physiology Online.

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