Light-induced expression of genes involved in phenylpropanoid biosynthetic pathways in callus of tea (Camellia sinensis (L.) O. Kuntze)

Light-induced expression of genes involved in phenylpropanoid biosynthetic pathways in callus of tea (Camellia sinensis (L.) O. Kuntze)

Scientia Horticulturae 133 (2012) 72–83 Contents lists available at SciVerse ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com...

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Scientia Horticulturae 133 (2012) 72–83

Contents lists available at SciVerse ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Light-induced expression of genes involved in phenylpropanoid biosynthetic pathways in callus of tea (Camellia sinensis (L.) O. Kuntze) YunSheng Wang a,b,1 , LiPing Gao b,1 , ZhengRong Wang b , YaJun Liu a , MeiLian Sun a , DongQing Yang a , ChaoLing Wei a , Yu Shan a , Tao Xia a,∗ a b

Key Laboratory of Tea Biochemistry & Biotechnology, Ministry of Education in China, Anhui Agricultural University, China School of Life Science, Anhui Agricultural University, China

a r t i c l e

i n f o

Article history: Received 6 August 2011 Received in revised form 12 October 2011 Accepted 16 October 2011 Keywords: Tea Light inducement Phenylpropanoid biosynthesis Gene expression SSH cDNA library

a b s t r a c t Tea (Camellia sinensis (L.) O. Kuntze) is a commercially important crop that is valued for its secondary metabolites. Light is an important environmental parameter that regulates plant growth and development and influences the phenylpropanoid metabolism in plants. To investigate the molecular mechanism by which light regulates phenylpropanoid metabolism, we established light-induced suppression subtractive hybridization (SSH) cDNA libraries of tea calli. A total of 265 clones from the library were selected, sequenced, and analyzed in this study. Nine diverse ESTs involved in phenylpropanoid biosynthesis were detected in the library. A new CsDFR gene (CsDFR2), higher increment of the expression activated by light than the previously reported CsDFR gene (CsDFR1), was cloned. The key phenylpropanoid compounds and representative genes expression analysis implied that light could be effective for activation of the biosynthesis of phenylpropanoids. Compared to the darkness control, levels of lignins, catechins, and PAs were increased 3.46, 3.00, and 1.21-fold, in light-induced calli, respectively. And lignin biosynthesis genes, involved in CCoAOMT, HCT and CCR, were identified in the light-induced SSH library. Therefore it was assumed that lignins might be the main phenylpropanoid metabolites activated by light in tea calli. In addition, our researches found that catechins, as the main secondary metabolites, significantly decreased in the tea calli compared to those in tea mature leaves, While PAs (polymer of catechins) in calli did not decrease compared to mature leaves. The data suggest that polymerization reaction might be the main pathway of flavonoid metabolism in tea callus. The SSH library established in this study represents a valuable resource for better understanding the mechanisms of light-induced secondary metabolism in tea plants. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Tea (Camellia sinensis (L.) O. Kuntze) is an important commercial crop grown in over 30 countries and consumed worldwide

Abbreviations: ABC protein, ATP-binding cassette type membrane protein; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; C, catechin; CAD, cinnamoyl alcohol dehydrogenase; CCH, coumaroyl-CoA 3-hydroxylase; CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; CCR, cinnamoyl-CoA reductase; C3H, coumarate 3-hydroxylase; C4H, cinnamate 4-hydroxylase; CHS, chalcone synthase; CHI, chalcone isomerase; 4CL, 4-coumaroyl-CoA ligase; COMT, caffeic acid-3-O-methyltransferase; DFR, dihydroflavonol 4-reductase; EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3gallate; FST, flavonol 4 -sulfotransferase; GC, gallocatechin; GST, glutathione S-transferase; HCT, hydroxycinnamoyl transferase; LAR, leucoanthocyanidin reductase; PAL, phenylalanine ammonialyase; SA, salicylic acid; SSH, suppression subtractive hybridization; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase. ∗ Corresponding author at: 130 West Changjiang Rd., Hefei, Anhui 230036, China. Tel.: +86 551 5786003; fax: +86 551 5785729. E-mail address: [email protected] (T. Xia). 1 These authors contributed equally to this work. 0304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2011.10.017

primarily as a beverage made from the processed leaves. As the main secondary metabolites, flavan 3-ols (catechins), along with other phenylpropanoids including flavonols, proanthocyanins (PAs), anthocyanins and lignins, are derived from multiple branches of the phenylpropanoid biosynthetic pathways (Fig. 1), one of the most-characterized secondary metabolic routes in plant systems. The metabolic genes related to catechins biosynthesis (including catechin (C), gallocatechin (GC), epicatechin (EC), and epigallocatechin (EGC)) comprise flavanone 3 -hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS), and anthocyanidin reductase (ANR) (Tanner et al., 2003; Xie et al., 2003). The ester catechins (including epicatechin-3-gallate (ECG) and epigallocatechin-3gallate (EGCG)) were synthesized by esterification reaction of gallic acid or proanthocyanidins (PAs) were synthesized by polymerization of the monomer catechins. In addition to phenylalanine ammonialyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumaroyl-CoA ligase (4CL), and chalcone synthase (CHS), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) and hydroxycinnamoyl transferase (HCT) are key genes in biotechnological

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Fig. 1. Possible phenylpropanoid and flavonoid pathways in Camellia sinensis (L.) O. Kuntze (Punyasiri et al., 2004, Rogers and Campbell, 2004). (A) basic phenylpropanoid biosynthetic pathway; (B) early flavonoid biosynthetic pathway; (C) late flavonoid biosynthetic pathway; (D) lignin biosynthetic pathway. PAL, phenylalanine ammonialyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3 -hydroxylase; F3 H, flavonoid 3 -hydroxylase; F3 5 H, flavonoid 3 5 -hydroxylase; FLS, flavonol synthase; FST, flavonol 4 -sulfo-transferase; DFR, dihydroflavonol 4-reductase; LAR, leucoanthocyanidin reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; UFGT, UDP-glucose:flavonoid 3-O-glucosyltransferase; C3H, coumarate 3-hydroxylase; HCT, hydroxycinnamoyl transferase; COMT, caffeic acid-3-O-methyltransferase; CCoAOMT: caffeoyl-CoA 3-O-methyltransferase; CCH, coumaroyl-CoA 3-hydroxylase; CCR, cinnamoyl-CoA reductase; CAD, cinnamoyl alcohol dehydrogenase; F5H, ferulate 5-hydroxylase; CoA, coenzyme A.

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alteration of lignin biosynthesis to improve wood properties (Meyermans et al., 2000; Zhong et al., 2000). However, because of competition for the substrate and multiplex branches in the biosynthetic pathways, the relationships among phenylpropanoids in plants are still unclear. Compared with certain model plants such as Arabidopsis thaliana, Vitis vinifera, Petunia hybrida and Populus bonatii, the biosynthesis of phenylpropanoids in tea plants is poorly understood. Light is an important environmental parameter that drives photosynthesis and regulates plant growth and development. The physiological and molecular basis of light-induced processes have been well discussed (for review, see Ferenc et al., 2001; Castillon, 2007). Plants are capable of undergoing physiological adjustments in response to light stimuli. They possess a substantial number of inducible defense mechanisms that protect the plants against the stimulation. These defense mechanisms might include activation of phenylpropanoid biosynthesis (Cominelli et al., 2008). For example, flavonoids serve as protectants against UV irradiation (Winkel-Shirley, 2002). And the modification of cell wall structure by lignin deposition is one of the defense mechanisms induced (Rogers et al., 2005). However, no major systematic or global analysis has focused on the molecular mechanisms of phenylpropanoid biosynthesis induced by light in the tea plant. Plant cell culture offers important advantages by minimizing environmental fluctuation. Plant callus and suspension cell lines are suitable for examining the effects of a variety of factors on plant secondary metabolism. In our present study, comparisons were made between light and full darkness treatment of tea calli from morphological, flavonoid (catechin and proanthocyanin) and lignin contents, and gene expression perspectives. To further elucidate the molecular mechanisms by which light influences the phenylpropanoid biosynthetic pathways, a particular focus of the study was to analyze differences in genes expression between lightand dark-treated tea calli by suppression subtractive hybridization (SSH). Most of the light-induced ESTs were identified so as to get a comprehensive picture of the light response in the plant at the level of gene, which might be useful in elucidating the molecular mechanism underlying of light response in tea plant.

2. Materials and methods 2.1. Materials and culture conditions Shoot tip explants excised from tea (C. sinensis (L.) O. Kuntze) variety, ‘Yunnandaye’, was sterilized. The explants were cultured in solid B5 medium (pH 5.5) containing 0.5 mg L−1 2,4dichlorophenoxyacetic acid, 0.1 mg L−1 kinetin, 30 mg L−1 sucrose and 0.7% (w/v) agar. The explants were grown at 25.0 ± 2.0 ◦ C and 70% humidity in the dark in a growth chamber. Callus was subcultured every 20 days by transferring about 5 g (fresh weight) of callus to fresh medium. The cell line Yunjing 63Y was acquired by subculture. For light treatment and the full darkness control, 3 g Yunjing 63Y callus generated from suspension cultures were plated on B5 solid media containing 0.3% phytagel. The plates were cultured in total darkness (the control; culture dishes were covered with aluminium foil) or 50 ± 5 ␮mol m−2 s−1 light (Cool white, 55 W, Philips, Netherlands) for 20 days. Images of the morphology of tea calli were captured with a Fujifilm 9500 camera (Fujifilm, Japan). Six culture dishes were selected from the light treatment and full darkness control, and the average fresh weight was recorded. The water content was calculated after the cultures were oven-dried at 105 ◦ C. The relative growth rate (RGR) was estimated using the method of Schnute (1981).

2.2. Paraffin section Tea calli were infused with fresh ethanol:acetic acid (3:1, v/v) solution and fixed for 7 days. Tissue samples were dehydrated and embedded in paraffin. Serial sections 15 ␮m thick were cut from the paraffin blocks and mounted on glass slides. The sections were deparaffinized and stained with safranin and Fast Green (Schichnes et al., 2001). The sections were inspected with a Nikon Eclipse E600 microscope (Nikon, Japan).

2.3. Analysis and identification of catechins Catechins were extracted from the tea callus and analyzed according to the procedure described by Liu et al. (2009). Three grams of callus was crushed in liquid N2 and macerated with 10 mL 95% ethanol. The ethanol extract was centrifuged at 4000 × g for 15 min to separate the supernatant and precipitate. The supernatant was evaporated then dissolved in hot water. The water solution was extracted three times with ethyl acetate. The organic phase was evaporated and dissolved in 1 mL methanol. Catechins in the methanolic solution were analyzed by high-performance liquid chromatography (HPLC). All samples were filtered through a 0.22 ␮m membrane then separated on a Phenomenex Synergi 4u Fusion-RP80 column (250 mm × 4.6 mm) with detection at 280 nm using a HPLC–UV detector (Waters 2478, Waters Instruments). The binary solvent system consisted of 1% (v/v) acetic acid (A) and 100% acetonitrile (B). Following injection of 5 ␮L of sample, a linear gradient was initiated at a flow rate of 1.2 mL min−1 : B from 10% to 13% over 20 min, then from 13% to 30% between 20 and 40 min, and from 30% to 10% between 40 and 41 min. Peaks were identified by retention time compared with catechin standards. The retention times of GC, EGC, C, EC, EGCG, and ECG are 5.67 min, 10.08 min, 12.58 min, 19.09 min, 23.06 min, and 34.51 min, respectively.

2.4. Measurement of proanthocyanin and lignin contents Total soluble and insoluble PAs were measured according to the simplified method of Pang et al. (2009). Samples (0.5 g) were extracted with 5 mL of 70% acetone–0.5% acetic acid (extraction solution). Following centrifugation at 2500 × g for 10 min, the residues were extracted twice with 5 mL extraction solution. Butanol–HCl reagent (3 mL) was added to the supernatants (containing soluble PAs) and residues (containing insoluble PAs), and the mixtures were pyrolyzed at 100 ◦ C for 1 h. The absorption of the supernatants was measured at 550 nm. Absorbance values were converted into PA equivalents using a standard curve for delphinidin chloride (Sigma, USA). Total lignin content was determined with a modified Klason method, where extracted ground stem tissue (0.2 g) was treated with 3 mL 72% H2 SO4 following Coleman et al. (2006). The lignin content was calculated as the mean percentage of the dry weight of extract-free stem material. Six replicates were analyzed.

2.5. Construction and analysis of SSH cDNA libraries 2.5.1. RNA isolation and cDNA preparation Total RNA was isolated from tea calli or leaves with the RNAiso Plus and RNAiso-mate for Plant Tissue kits (Takara, China). Doublestranded cDNA was prepared by reverse transcription of 4 ␮g purified mRNA in a 20 ␮L reaction solution using the Super SMART PCR cDNA Synthesis Kit (Clontech, Palo Alto, USA) following the manufacturer’s instructions.

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2.5.2. Construction of SSH cDNA Libraries The suppression subtractive hybridization (SSH) cDNA libraries included forward (the callus in light) and reverse (in full darkness) libraries. Double-stranded cDNA prepared from dark-cultured Yunjing 63Y calli was used as the driver and double-stranded cDNA prepared from the light-treated calli was used as the tester. The cDNA was digested with RsaI for about 3–4 h and then ligated to adapters 1 and 2R provided in the kit. Two rounds of hybridization and PCR amplification were performed to normalize and enrich differentially expressed cDNA. The subtractive products were inserted into the pMD18-T vector with a blue-white selection marker (Takara, China) and transformed into Escherichia coli DH5␣ competent cells. The transformed bacteria for both the forward and reverse SSH cDNA libraries were plated separately on four LB agar plates (15 ␮L SSH cDNA per plate), incubated at 37 ◦ C for 24 h, and white colonies were picked-up. Approximately 384 colonies from both the forward and the reverse SSH libraries were picked-up. These colonies were grown individually in liquid LB medium at 37 ◦ C overnight at 250 rpm in 96-well plates. Inocula of the individual cultures were then grown in 2 mL of the same medium (supplemented with 100 g mL−1 ampicillin) at 37 ◦ C and 250 rpm overnight. The plasmids were isolated using the Qiaprep Spin Mini-Prep Kit (Qiagen, Germany) following the manufacturer’s protocol. 2.5.3. Screening and authentication of the SSH libraries The plasmid samples of each library were spotted (approximately 50 ng plasmid DNA per spot) separately on an 8 in. × 10 in. nylon membrane (N+, Amersham Hybond) in duplicate. The secondary PCR products of the forward and the reverse SSH cDNA libraries were labeled separately with ␣-32 P dATP by random primer labeling using the PCR-select Screening Kit (Clontech) following the manufacturer’s instructions. The plasmid-spotted membranes were incubated separately for 30 min in 30 mL prehybridization buffer (7% SDS and 10 mM Na-EDTA in 0.5 M sodium phosphate buffer, pH 7.2) at 65 ◦ C. The desired denatured probe was added to the individual reaction buffers and hybridization was allowed to continue overnight at 65 ◦ C. After the hybridization reaction, the membranes were washed with 30 mL wash buffer I (1× SSC, pH 7.0, containing 150 mM NaCl, 15 mM trisodium citrate and 0.1% SDS) for 30 min at 65 ◦ C followed by washing with 30 mL wash buffer II (0.5× SSC and 0.1% SDS) at 65 ◦ C for 15 min. The membranes were air-dried and exposed to X-ray film at −70 ◦ C overnight and then the film was developed. 2.5.4. Sequencing and analysis of the cloned ESTs Positive clones from the SSH cDNA libraries were sequenced. The sequences of expressed sequence tags (ESTs) were grouped into singletons and contigs using the TIGR assembler (http://nbc11.biologie.uni-kl.de/framed/ Left/menu/auto/rightigr assembler) and were termed unigenes. The unigene sequences were subjected to a BLASTX homology search against the NCBI database and the unigenes were categorized into proteins with known functions, proteins with unknown functions and proteins with no match in the database. The unigenes were then grouped into functional categories using the MIPS (Munich Information for Protein Sequences) function catalogue (http://mips.gsf.de/projects/function). 2.6. Expression validation by quantitative real time RT-PCR In order to verify further the light-induced gene expression, real time quantitative RT-PCR (qRT-PCR) was quantify the transcript levels of 14 representative genes, which were selected for gene expression analysis from the SSH library and GenBank, comprising PAL, CHS, CHI, DFR, ANS, ANR, flavonol 4 -sulfotransferase

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(FST; GW316722), CCoAOMT, HCT, SA-binding protein, ACC oxidase, Ca-binding protein, and serine/threonine-PK. The housekeeping gene, GAPDH, was employed as the reference gene. Primers were designed with the Primer 5.0 software. The primer sequences for each gene are listed in Table 1. The qRT-PCR assays were conducted using the QuantiFast SYBR Green RT-PCR Kit (Qiagen, USA) on an Opticon-2 qRT-PCR machine (MJ Research, Bio-Rad). The PCR reaction conditions were set according to the RT-PCR kit manufacturer’s manual. After completion of the reactions, the threshold cycle (CT ) value for each reaction was recorded and the fold difference in transcript level between the dark and light-treated samples was calculated using the method of Pfaffl (2001). 2.7. Cloning of CsDFR2 One EST sequence, homologous with the reported CsDFR1, was selected to design primers for rapid amplification of cDNA ends (RACE) to clone the full-length cDNA of CsDFR2. The 3 end and 5 end of the cDNA sequence was isolated using a 3 -Full RACE Core Set Ver. 2.0 (Takara, China) and 5 -Full RACE Kit (Takara, China). The outer and inner specific primers used for 3 RACE were 5 ACTTGCCCAATGCTACCACA-3 and 5 -CCACTCCCATGAATTTCGTT3 , respectively, and the outer and inner specific primers for 5 RACE were 5 -GTGTACGGCATGTCCTTGGGTCAGC-3 and 5 CAGAAATCCACATCGGTCC-3 , respectively. The reaction products were purified, cloned and sequenced. The sequences of reaction products were analyzed with the BLAST programs against the NCBI databases. Protein sequences were aligned using Clustal W (http://www.ebi.ac.uk/clustalw). The pI and molecular weight were calculated using the pI/molecular weight calculation tools at http://www.expasy.org. Secondary structure of the deduced CsDFR protein was predicted by the SOPMA program (http://npsapbil.ibcp.fr/). 2.8. Statistical analyses Data were expressed as the mean ± SD. The statistical significance of differences between groups was determined with Student’s t-test using SPSS software (Chicago, IL, USA). Values of P < 0.05 were considered statistically significant. 3. Results 3.1. Morphogenetic changes Light irradiation remarkably affected morphogenesis of tea calli. The Yunjing 63Y callus cultured in complete darkness maintained a white-yellow color, while the cells exposed to light became smaller and distinctly bright yellowish (Fig. 2a), and failed to become green even with irradiation for 80 days (Fig. S1). The cell size of lighttreated callus was also noticeably smaller than that of the dark control (Fig. 2b). As shown in Table 2, light treatment significantly inhibited the relative growth rate of tea callus, which was 1.72-fold lower compared to the dark control. 3.2. Changes in flavonoid and lignin contents To investigate whether the phenylpropanoid biosynthetic pathways were enhanced by light treatment, catechins, PAs and lignins were extracted and quantified. As shown in Table 2, compared to those in tea mature leaves, the contents of phenylpropanoids significantly decreased in the calli. The content of catechins in calli is 100 fold less than in leaves, and the content of lignins is 10 fold less than in leaves. However, the content of soluble and insoluble PAs of tea callus in complete darkness did not differ from tea mature leaves (4.60 ± 0.81, 5.25 ± 0.79, P > 0.05).

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Table 1 Sequences of primers used to amplify genes involved in phenylpropanoid and flavonoid biosynthesis of Camellia sinensis. Gene name

GenBank accession number

Primer sequence

Length of PCR product (bp)

GADPHa

XM 002263109

213

PALa

D26596

CHSa

AY169403

F5 -TTGGCATCGTTGAGGGTCT-3 R5 -CAGTGGGAACACGGAAAGC-3 F5 -CCAAGTTTTCGGGAAGTAAATG-3 R5 -GTGATTAGGCTTGGTGGGAA-3 F5 -GGCAATCAAAGAATGGGG-3 R5 -ATGGGCGAAGACCGAGT-3 F5 -CTACTCAAGATGGCCCGACAA-3 R5 -ACAACACCTCCAGCAACTTGC-3 F5 -ATTGGCAGAGAAAGCAGCAT-3 R5 -GTGATTAGGCTTGGTGGGAA-3 F5 -TTGCCCAATGCTACCACA-3 R5 -TGCCTTCCACAAGCTGAG-3 F5 -TTTAAAAGCCTGCGCGAAAG-3 R5 -ATGACAAGCCCCGTTCCAT-3 F5 -GCGAAGTTGATCCTCTCGTC-3 R5 -AACCACATCGTCAAGTGAACA-3 F5 -GGCCACAAGTGCCTACAATTG-3 R5 -CCCATGATTCACCAAATGCA-3 F5 -GGGTCGTTTGATTTCATTTTCG-3 R5 -AGAGCCGTTCCAGAGGGTGT-3 F5 -GGAGCCCATACTTTTAGGT-3 R5 -CATCTTTGGTTGGACTTGG-3 F5 -CTAATAGAGCCAGGAAGTGC-3 R5 -GAAAACCCATTGCTCACC-3 F5 -TTGATGGAGGTCATGGAA-3 R5 -ACGAAGACAGCAGTGGAG-3 F5 -GAGGTTTAGGGAGCAGAGG-3 R5 -TGTTGGATTGAGGGAGT-3 F5 -GGGAGTGGTGATAATAGACG-3 R5 -TCCAATCGAGACAAAACG-3 F5 -TGGAGCAGCAAGGGGTTT-3 R5 -CTTTAGCATTCAAGCGTTCA-3

F3Ha

AY641730 a

DFR1

AB018685

DFR 2b

HO703569

ANR1a

AY169404

a

ANR2

AY641729

ANSa

AY830416

CCoAOMTb

GW316714

HCTb

GT087980

b

FST

GW316722

SA-binding proteinb

GW316715

ACC oxidaseb

GW316719 b

Ca binding protein

GW316723

Serine/threonine-PKb

GW316718

a b

101 122 101 120 95 102 198 128 126 160 155 128 125 129 115

Genes from GenBank. ESTs from the light-induced SSH cDNA library.

Light irradiation had notably positive effects on both flavonoid (including catechins and PAs) and lignin accumulation (Fig. 2c and Table 2), but had no significant effect on anthocyanin accumulation (data not shown). Compared with the dark control, the total lignin, catechin, and PA contents of light-treated tea callus increased 3.46, 3.00, and 1.21-fold, respectively. These results indicate that light irradiation is effective for the activation of the phenylpropanoid biosynthesis pathways. In addition, compared with the HPLC spectral profiles, the EC, ECG, and EGCG peaks were notably enhanced in light-treated tea calli (Fig. 2c).

3.3. SSH cDNA library construction Approximately 384 colonies from both the forward and the reverse SSH libraries were obtained. The lengths of the inserted cDNA fragments ranged from 100 to 750 bp for both forward and reverse SSH libraries and the average length was about 500 bp (Fig. S2).

To eliminate false positive clones and quantify the relative expression level of the cloned cDNAs more accurately, we performed further cDNA differential screening. The cDNA clones showing differential expression were identified by successive screenings with the subtracted tester and driver as probes (Fig. S3). We identified 265 positive clones whose tester expression levels were significantly higher (≥2.0-fold) than these from the driver, comprising 191 positive clones from the forward SSH library and 74 positive clones from the reverse. The EST sequencing and cluster analysis of the forward SSH cDNA library (191 clones identified by differential screening) indicated that these sequences represented 136 unique ESTs. The unique ESTs were submitted to the EST database of GenBank (http://www.ncbi.nlm.nih.gov/dbEST). These 136 unique ESTs comprised nine contigs and 127 singletons. Based on a homology search with BLASTX, among the 127 singletons, 104 clones (81.9%) were homologous to known genes, and 23 clones (18.1%) to the genes with unknown function or without matches in the database. The subtractive ESTs were further grouped into 12 different

Table 2 Influence of light on growth, water content and phenylpropanoids accumulation in tea callus. Light callus, the callus was grown for 20 days under light treatment; dark callus, the callus was grown for 20 days under full darkness; tea mature leaves, the 3rd leaf of tea plant was grown in full illumination. Following letters (A–C) indicate significant differences (P < 0.05). Treatment

Water content (%)

Relative growth rate (mg g−1 DW day−1 )

Total catechins content (mg g−1 DW)

Soluble proanthocyanidins content (mg g−1 DW)

Insoluble proanthocyanidins content (mg g−1 DW)

Total lignins content (mg g−1 DW)

Light callus Dark callus Tea mature leaves

92.44 ± 1.65A 94.40 ± 1.03A 71.31 ± 0.26B

4.28 ± 0.29B 7.36 ± 0.47A –

1.24 ± 0.21B 0.43 ± 0.14C 159.97 ± 13.61A

7.02 ± 1.47A 4.13 ± 0.69B 4.87 ± 0.76B

0.97 ± 0.16A 0.47 ± 0.12B 0.38 ± 0.06B

84.67 ± 0.99B 24.49 ± 1.54C 538.10 ± 52.33A

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Fig. 2. Influence of light on callus morphology and catechins accumulation in tea callus. (a) Tea callus grown for 20 days under light (light) and full darkness (dark); (b) micrographs of tea callus grown for 20 days under light (light) and full darkness (dark) (Bar = 250 ␮m); (c) HPLC spectral profiles of catechins from tea callus grown for 20 days under light (light) and full darkness (dark) ((1) epicatechin, (2) epigallocatechin-3-gallate, and (3) epicatechin-3-gallate).

categories based on their putative cellular functions (Fig. 3), charactering from Arabidopsis (Arabidopsis Genome Initiative, 2000) and our research interest: metabolism (15.75%), phenylpropanoid and secondary metabolism (7.09%), energy (2.36%), transcription (11.81%), protein synthesis (4.72%), transport (8.66%), cell structure (2.36%), cell growth and division (4.72%), signal transduction (11.02%), stress defense (13.39%), unclear classification (12.60%), and unclassified (5.51%). Phenylpropanoid biosynthetic pathways are the mostcharacterized secondary metabolic routes in plant systems,

and might be activated during the course of plant photomorphogenesis and/or stress-related responses. Many studies have shown that light quality and intensity affect phenylpropanoid biosynthetic pathways in plants. Light irradiation had notable positive effects on both flavonoid (catechins and proanthocyanins) and lignins accumulation (Table 2). In the light-induced SSH library, nine diverse ESTs involved in phenylpropanoid biosynthesis were detected, such as DFR (GenBank accession no. HO703569), CCoAOMT (GW316714), HCT (GT087980), and CCR (GW316717). In addition, several ESTs involved in flavonoid transport, such

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Fig. 3. Functional categories of ESTs in the forward (light-induced) SSH cDNA library of tea callus.

as the ATP-binding cassette type membrane protein (ABC protein; GT088071), glutathione S-transferase (GST, GT087944), and flavonol 4 -sulfotransferase (FST, GW316722), were identified in this library. Light is an important exogenic signal for plants to drive photosynthesis, including the effects of light on callus growth, differentiation and development. A few ESTs in the library appeared to be associated with enzymes localized in plastids, including plastid alpha-amylase (GenBank accession no. GT088062), a chloroplasttargeted copper chaperone (GT087925) and rubisco subunit binding-protein beta subunit (GT088004). Although ESTs involved in phytochromes and additional photoreceptors were not identified, the transcriptional regulators mediated by phytochromes or other signal pathways appeared in the SSH cDNA library, such as a GH3 family protein (GenBank accession no. GT087965), and translationally controlled tumor protein (GT087960). The GH3 family proteins act as phytohormone-amino acid synthetases and play a role in stress responses, the auxin signal pathway, and photo signal pathway, by modulating endogenous levels of active auxin through a negative feedback regulation (Park et al., 2007). The translationally controlled tumor protein, as an important regulator of growth, is expressed throughout plant tissues and developmental stages with increased expression in meristematic and expanding cells in Arabidopsis (Berkowitza et al., 2008).

Additionally, some ESTs in our library were related to stressrelated chaperone proteins, such as the chloroplast-targeted copper chaperone, heat-shock protein 70 (GenBank accession no. GT088011), and DNA photolyase (GT088049). Strong increases of many defense-related transcripts were also observed in the library, such as chitinase (GT088016), endo-1,4-betaglucanase (GT087954), GST, GRAS protein (GT088050), glycine-rich RNA-binding protein (GRP; GT087909) and ABC proteins. In addition, a few sulfation genes, including a metallothioneinlike protein (GenBank accession no. GT087972) and flavonol 4 -sulfotransferase (GW316722), were identified in the library. Sulfonate conjugation not only facilitates transport and excretion of hydrophobic molecules by increasing their water solubility, but also modulates the biological activity of hormones (Rouleau et al., 1999). The above results suggested that photooxidation stress might be generated in the light-treated tea callus. Many diverse sequences involved in signal transduction were also detected in the light-induced SSH library. The plant hormone metabolism ESTs included indole-3-acetic acid-amido synthetase (GenBank accession no. GW316713), cystathionine gamma synthase (GW316716), ACC oxidase (GW316719), and flavonol 4 sulfotransferase. The membrane signal transduction ESTs included a pheromone receptor (GT088015), ADP-ribosylation factor-like

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Fig. 4. Expression of representative phenylpropanoid and flavonoid biosynthetic genes as analyzed by real time quantitative RT-PCR (qRT-PCR). Total RNA isolated from the calli and leaves was separately applied to qRT-PCR using GAPDH gene as an internal control. The relative expression values were calculated by the 2−Ct method. Values were normalized against the expression level of GAPDH, and relative to the darkness control callus as a calibrator. CT = CTtarget − CTGAPDH , −CT = −(CTtarget − CTthe dark control calli ), where CTtarget and CTGAPDH are the threshold cycles of target and GAPDH, respectively. The values presented are the mean ± standard deviation of three independent analyses.

protein (GT088065), and SA-binding protein (GW316715). The secondary messenger ESTs included a small GTP-binding protein (GW316710), adenylyl cyclase-associated protein (GT087983), and calcium ion-binding protein (GW316723). The ESTs associated with protein phosphatic reactions included protein kinase (GT088051), PKN/PRK1 (GT088026), serine/threonine–protein kinase (GW316718), and UDP-glucosyltransferase (GT087918 and GT087929). The EST sequencing and cluster analysis of the reverse SSH cDNA library (74 clones identified by differential screening) indicated that these sequences represented 64 unique ESTs. The unique ESTs were also submitted to the EST database of GenBank. The result of the classification of the ESTs is shown in Fig. S4. In contrast with light-treated calli, highly expressed genes in dark-cultured callus cells (the reverse library) were related to plant development and cell wall modification, such as pectin methylesterase (GenBank accession no. GW316724), pectinesterase precursor (GW316725), arabinogalactan protein (GT088081), 3-hydroxy-3methylglutaryl-CoA synthase (GW316726), NAC domain protein (GW316727), auxin-induced putative CP12 domain-containing protein (GT088114), and small chloroplast protein (GT088078).

3.4. Expression of genes involved in phenylpropanoid biosynthetic pathways To further evaluate the relationship of catechins and other phenylpropanoid biosynthetic end-products, the expression of structural genes of the phenylpropanoid biosynthetic pathways was analyzed by qRT-PCR. In the present study, the 14 representative genes, comprising basic phenylpropanoid biosynthetic genes (PAL), early flavonoid biosynthetic genes (CHS), late flavonoid biosynthetic genes (DFR, ANS, and ANR), and lignin and flavonol biosynthetic genes (HCT, CCoAOMT and FST), and the signal transduction genes (comprising ACC oxidase, SA-binding protein,

Ca-binding protein, and serine/threonine PK) were selected for gene expression analysis. The GADPH cDNA, expected to show a constitutive expression pattern, was used as the control. Every other selected gene, except PAL and ANR2, showed higher transcription in the lighttreated callus than in the dark control (Fig. 4). The expression of SA-binding protein in light-treated calli was the highest among the genes selected for qRT-PCR, increasing 3.27-fold compared with the dark control. And the other ESTs from the light-induced SSH library, including CCoAOMT, HCT, FST, ACC oxidase, SA-binding protein, Ca-binding protein, and serine/threonine PK, were also overexpressed (>2.0-fold) in the light-treated callus. These findings were also reflected in the slot-blot hybridization. It was noteworthy that the two dfr genes, as the key genes in catechin biosynthesis, showed different expression levels induced by light. The expression of CsDFR2 increased 2.57-fold higher in the light-exposed calli compared to the darkness control calli, while CsDFR1 expression increased 2.14-fold. As shown in Fig. 4, some difference was found between the calli and leaves: the expression of the lignin biosynthesis genes, comprising CCoAOMT and HCT, were notably higher in calli than in the leaves, whereas the flavonoid biosynthesis genes, including CsDFR1 (from GenBank, AB018685), CsDFR2 (from the SSH library), CHS, ANS, ANR1, and ANR2, in calli were significantly lower than in the leaves. 3.5. Cloning of CsDFR2 Among the analyzed ESTs, a 228 bp fragment (accession no. HO703569) exhibited significant homology with previously reported DFRs. Based on the sequence, a full-length CsDFR2 was cloned by 5 and 3 RACE. The CsDFR2 comprised a 1392 bp fulllength cDNA with an ORF of 996 bp, starting from 1 to 996 encoding a protein with 332 amino acid residues. Sequence analysis revealed that CsDFR2 had 51.5–60.9% homology with other reported DFR

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Fig. 5. Comparison of amino acid sequences encoded by CsDFRs with other reported DFR proteins. The GenBank accession numbers for the sequences are: Camellia sinensis DFR1 AAT66505, Vitis vinifera 3 XP 002281250, Vitis vinifera 4 XP 002281758, Arabidopsis thaliana NP 199094, and Daucus carota AAD56579. Gaps are represented as dashes; asterisks, colons and dots indicate identical amino acid residues, conserved substitutions, and semi-conserved substitutions, respectively, in all sequences used in the alignment. The boxed region is a putative NADPH-binding region. The region predicted to be related to substrate specificity is underlined.

proteins (Fig. 5). Phylogenetic analysis indicated the protein sequence of CsDFR2 showed higher identity with Daucus carota DFR (GenBank accession no. AAD56579), and lower identity with CsDFR1 (AAT66505) (Fig. 6). SMART domain analysis indicated amino acids at positions 6–210 had a domain resembling that of the NADPH-dependent epimerase/dehydratase family (Kashmir et al., 2009), which utilize nucleotide sugar substrates for a variety of chemical reactions involving NADPH as a cofactor. The CsDFR2

theoretical molecular weight and isoelectric point were 37.38 kDa and 6.00, respectively. Its secondary structure predicted by SOPMA indicated that the molecule contained 134 ␣-helices, 48 ␤-turns, 23 extended strands and 127 random coils (Fig. 7), whereas CsDFR1 has 140 ␣-helices, 47 ␤-turns, 22 extended strands and 138 random coils (Combet et al., 2000). The similarity in secondary structures of CsDFR2 and CsDFR1 indicated that CsDFR2 was a functional gene.

Y. Wang et al. / Scientia Horticulturae 133 (2012) 72–83

Fig. 6. Phylogenetic tree derived from amino acid sequences of genes encoding DFRs in plants. The GenBank accession numbers for the sequences are: Cs DFR1 AAT66505, M truncatula AAR27015, P communis AAO39818, M X domestica AAD26204, P trichocarpa EEE94663, D carota AAD56579, C sinensis AAS00611, G hirsutum ACV72642, P tremuloides AN63056, A thaliana 1 NP 199094, A thaliana 2 NP 182064, A thaliana 3 NP 195268, A thaliana 4 NP 194455, V vinifera 1 CAA72420, V vinifera 2 XP 002281858, V vinifera 3 XP 002281250, V vinifera 4 XP 002281758, O sativa 1 BAA36183, O sativa 2 AAD24584, O sativa 3 BAD09991. The phylogenetic analysis was performed using the maximum parsimony method. Numbers on branches correspond to bootstrap estimates for 100 replicate analyses using 500× stepwise addition of taxa; values less than 50% are not indicated.

4. Discussion Light induces many adaptive responses in plants. Morphological comparison in this study showed that, during exposure to light, the cells of tea callus became smaller and cell growth was significantly inhibited. Several genes involved in plastid development were identified in the light-induced SSH library. In addition, A few genes involved in lignins biosynthesis, namely CCoAOMT, HCT and CCR, were identified in the light-induced SSH library. As key enzymes in lignin biosynthesis, CCoAOMT and HCT were overexpressed in the light-treated callus, increasing 2.68 and 2.39-fold, respectively. The lignin content of light-treated tea callus increased 3.46-fold compared with the dark control. It is suggested that with cell differentiation and the development of plastids or chloroplasts, light irradiation could enhance lignin biosynthesis and accumulation in tea cells.

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Catechins, as the main secondary metabolites in tea plant, are derived from multiple branches of the phenylpropanoid and flavonoid biosynthetic pathways. PAs in green tea are dimmers or oligomers of catechin and epicatechin, and are responsible for bitterness and astringency in tea beverages (Dixon et al., 2005). Phenylpropanoid compounds and gene expression analyses showed that the biosynthesis of catechins in the tea calli significantly decreased compared to those in tea mature leaves (Table 2, and Fig. 4). While the content of soluble and insoluble PAs in calli did not decrease (in darkness control), or increase (in light treatment) compared to those in tea mature leaves. The data suggest that PAs synthesized by polymerization of catechins might be the main pathway of flavonoid metabolism in tea calli. Our previous study indicated that, in green calli cultured under light, catechins were observed in chloroplasts and the cytoplasm as well as in vacuoles (Liu et al., 2009). Therefore, it was assumed that PAs might be the storage state of catechins in cell organelles. Recent researches have revealed that the expression of PAL, CHS, C4H, F3H, and DFR are in accordance with the catechin contents (Singh et al., 2008; Eungwanichayapant and Popluechai, 2009). Unexpectedly, the qRT-PCR analyses in this study showed that expression of PAL and ANR2 were reduced in the light-treated calli. Therefore, further study is required to better understand the relationship between the relative gene expression and flavonoid accumulation induced by light in tea plant. DFR is an important enzyme that catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key step in controlling metabolic flux into biosynthetic pathway branches leading to anthocyanins and proanthocyanidins (Xie et al., 2004). A number of DFR genes have been isolated from a variety of higher plants (Sparvoli et al., 1994; Shimada et al., 2005; Piero et al., 2006). In this paper, a novel CsDFR gene (CsDFR2), exhibiting strong homology with previously reported DFRs, was cloned using its EST sequence information. The qRT-PCR analyses showed that the two CsDFRs were overexpressed in the light-treated calli, while the expression increment of CsDFR2 with light treatment was higher than the increment of CsDFR1. Further research is required to determine the exact functional difference of the two CsDFRs. It is known that photooxidation stress may be generated when dark-cultured tea calli were exposed to light for a long period. The expression of genes encoding enzymes involved in chaperone proteins, antioxidant proteins, and detoxification enzymes (Table 3) in the SSH library indicated the presence of photooxidative stress in the callus. Accordingly, endogenous plant hormones, stimulated by photooxidative stress, may activate intracellular signal transduction mediated by pheromone receptors. A few ESTs involved in phytohormone signal pathways were detected, including ACC oxidase, indole-3-acetic acid-amido synthetase, and SA-binding protein. The qRT-PCR analyses confirmed that a SA, ethylene, and Ca2+ messenger might be involved in the regulation of lightinduced phenylpropanoid accumulation in the tea plant.

Fig. 7. Secondary structure of the deduced amino acid sequence of the two DFRs predicted by SOPMA. ␣-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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Table 3 Related ESTs (partial) in the SSH library. Molecular function

EST accession number

ESTs involved in cell growth and division GT087925 Chloroplast-targeted copper chaperone Rubisco subunit binding-protein beta subunit GT088004 GT088062 Plastid alpha-amylase GT087965 GH3 family protein GT087960 Translationally controlled tumor protein GT087984 Cell division control protein 50 ESTs involved in stress defense GT087920 Chaperone protein dnaJ GT088011 Heat shock protein 70 GT088049 DNA photolyase GT088016 Chitinase GT087954 Endo-1,4-beta-glucanase GT088042 Glutathione transferase GT087944 Glutathione S-transferase (GST)a GT088050 GRAS protein GT087909 Glycine-rich RNA-binding protein (GRP) ATP-binding cassette type membrane protein GT088071 (ABC protein)a GT087972 Metallothionein-like protein GW316722 Flavonol 4 -sulfotransferase (FST)a GT087907 Cysteine protease Cp6 Alcohol dehydrogenase GT087955 GT088017 Dopamine beta-monooxygenase Hypersensitive-induced response protein GT088072 GT088064 Selenium binding ESTs involved in signal transduction GW316713 Indole-3-acetic acid-amido synthetase GW316716 Cystathionine gamma synthase (CGS) ACC oxidase GW316719 GT088015 Pheromone receptor Small GTP-binding protein GW316710 GT088065 ADP-ribosylation factor-like protein GT087983 Adenylyl cyclase-associated protein GW316715 SA-binding protein GT088051 Protein kinase PKN/PRK1 GT088026 Serine/threonine-PK GW316718 UDP-glucosyltransferase GT087918 GT087929 UDP-glucosyltransferase GW316723 Cabinding protein ESTs involved in phenylpropanoid biosynthetic pathways GW316712 Cytochrome P450, family 81 Cytochrome P450 GW316721 GW316714 Caffeoyl-CoA-O-methyltransferase (CCoAOMT) GW316711 Epoxide hydrolase GT087981 IMP dehydrogenase GT087980 Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) Cinnamoyl-CoA reductase family (CCR) GW316717 HO703569n 2 -Hydroxydihydrodaidzein reductase (DFR) GT087914 Beta-glucosidase ESTs down-regulated with light treatment GT088081 Arabinogalactan protein (AGP) GW316726 3-Hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA) NAC domain protein GW316727 GT088114 Auxin-induced putative CP12 domain-containing protein GT088078 Small chloroplast protein GW316725 Pectinesterase precursor GW316724 Pectin methylesterase a

Organism

BlastX

Number of ESTs

Score

E-value

Ricinus communis Ricinus communis Actinidia chinensis Populus trichocarpa Ricinus communis Zea mays

83.5 409.0 442.0 150.0 182.0 215.0

7.00E−11 2.00E−118 2.00E−122 3.00E−35 1.00E−44 1.00E−54

1 1 1 1 2 1

Ricinus communis Camellia sinensis Ricinus communis Persea americana Ricinus communis Glycine max Glycine max Capsicum annuum Vitis vinifera Ricinus communis

63.9 408.0 199.0 422.0 195.0 128.0 155.0 103.0 149.0 164.0

5.30E−11 3.00E−112 2.00E−49 1.00E−116 1.00E−48 2.00E−28 2.00E−36 4.00E−21 7.00E−35 2.00E−44

2 1 1 1 1 1 3 1 1 2

Camellia sinensis Ricinus communis Actinidia deliciosa Ricinus communis Ricinus communis Oryza sativa Arabidopsis thaliana

74.7 118.0 370.0 75.5 146.0 187.0 118.0

3.00E−12 2.00E−25 5.00E−101 2.00E−12 7.00E−34 4.00E−46 3.00E−25

1 1 4 1 1 1 1

Ricinus communis Solanum lycopersicum Camellia sinensis Mus musculus Nicotiana tabacum Musa acuminata AAA Group Gossypium arboreum Nicotiana tabacum Medicago truncatula Vitis vinifera Ricinus communis Ricinus communis Stevia rebaudiana Ricinus communis

263.0 192.0 185.0 65.8 330.0 233.0 60.8 176.0 155.0 293.0 360.0 232.0 171.0 152.0

4.00E−69 1.00E−47 1.00E−45 1.40E−11 1.00E−88 3.00E−60 4.40E−11 7.00E−43 3.00E−36 2.00E−77 1.00E−97 9.00E−60 2.00E−41 1.00E−35

2 1 1 1 1 1 1 1 1 1 1 1 1 1

Arabidopsis thaliana Populus trichocarpa Broussonetia papyrifera Arabidopsis thaliana Toxoplasma gondii GT1 Coffea canephora

107.0 156.0 397.0 293.0 73.9 171.0

5.00E−22 7.00E−37 5.00E−109 1.00E−77 5.30E−11 4.00E−47

1 1 1 1 1 1

Arabidopsis thaliana Glycine max Zea mays

148.0 97.8 104.0

1.00E−34 3.00E−19 1.00E−22

3 1 1

Daucus carota Camptotheca acuminata

78.6 45.6

2.00E−13 1.00E−03

1 1

Populus trichocarpa Arachis hypogaea

53.9 80.1

1.00E−05 8.00E−14

1 2

96.3 166 79.0

1.00E−18 7.00E−40 1.00E−13

2 1

Galdieria sulphuraria Ricinus communis Eucalyptus globulus

ESTs representing proteins involved in flavonoid transport.

5. Conclusions In conclusion, our results indicate that expression of the key genes involved in phenylpropanoid metabolism is notably enhanced, and phenylpropanoids are accumulated in response to light in tea callus. The key phenylpropanoid compounds and

representative genes expression analysis implied that light could be effective for activation of the biosynthesis of phenylpropanoids, and lignins might be the main phenylpropanoid metabolites activated by light in tea calli. In addition, our researches suggest that PAs synthesized by polymerization of catechins might be the main pathway of flavonoid metabolism in tea callus. A new

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CsDFR gene, actively responsive to light treatment, was cloned in this study. Further expression and functional analyses of fulllength forms corresponding to target ESTs of the SSH library will accelerate discovery and characterization of light-induced genes. These genes will constitute potential targets for genetic engineering of secondary metabolites to enhance the functional secondary metabolite composition in tea plants. Acknowledgments The authors thank Dr. Shu Wei for his critical review of the manuscript. This work was supported by the National Natural Science Foundation of China (nos. 30771755, 30972401, and 31000314), the Natural Science Foundation of Anhui Province China (no. 090411006), the Natural Science Foundation of Anhui Department of Education (no. KJ2010A117), and the President Youthful Foundation of Anhui Agricultural University (no. 2009zd09). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.scienta.2011.10.017. References Arabidopsis Genome Initiative, 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815. Berkowitza, O., Josta, R., Pollmannb, S., Maslea, J., 2008. Characterization of TCTP, the translationally controlled tumor protein, from Arabidopsis thaliana. Plant Cell 20, 3430–3447. Castillon, A., Shen, H., Huq, E., 2007. Phytochrome interacting factors: Central players in phytochrome-mediated light signaling networks. Trends Plant Sci. 12, 514–521. Coleman, H.D., Ellis, D.D., Gilbert, M., Mansfield, S.D., 2006. Up-regulation of sucrose synthase and UDP-glucose pyrophosphorylase impacts plant growth and metabolism. Plant Biotechnol. J. 4, 87–101. Combet, C., Blanchet, C., Geourjon, C., Deleˇıage, G., 2000. NPS@: network protein sequence analysis. Trends Biochem. Sci. 25, 147–150. Cominelli, E., Gusmaroli, G., Allegra, D., Galbiati, M., Wade, H.K., Jenkins, G.I., Tonelli, C., 2008. Expression analysis of anthocyanin regulatory genes in response to different light qualities in Arabidopsis thaliana. J. Plant Physiol. 165, 886–894. Dixon, R.A., Sharma, S.B., Xie, D., 2005. Proanthocyanidins – a final frontier in flavonoid research? New Phytol. 165, 9–28. Eungwanichayapant, P.D., Popluechai, S., 2009. Accumulation of catechins in tea in relation to accumulation of mRNA from genes involved in catechin biosynthesis. Plant Physiol. Biochem. 47, 94–97. Ferenc, N., Stefan, K., Eberhard, S., 2001. Intracellular trafficking of photoreceptors during lightinduced signal transduction in plants. J. Cell Sci. 114, 475–480. Kashmir, S., Sanjay, K., Sudesh, K.Y., Paramvir, S.A., 2009. Characterization of dihydroflavonol 4-reductase cDNA in tea [Camellia sinensis (L.) O. Kuntze]. Plant Biotechnol. Rep. 3, 95–101. Liu, Y., Gao, L., Xia, T., Zhao, L., 2009. Investigation of the site-specific accumulation of catechins in the tea plant (Camellia sinensis (L.) O. Kuntze) via vanillin–HCl staining. J. Agric. Food Chem. 57, 10371–10376.

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