Molecular Characterization of WRKY Transcription Factors that Act as

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Agricultural and Environmental Chemistry

Molecular Characterization of WRKY Transcription Factors that Act as Negative Regulators of O-methylated Catechins Biosynthesis in Tea Plant (Camellia Sinensis L.) Yong Luo, Shuangshuang Yu, Juan Li, Qin Li, Kunbo Wang, Jianan Huang, and Zhonghua Liu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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Journal of Agricultural and Food Chemistry

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Molecular Characterization of WRKY Transcription Factors that Act as

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Negative Regulators of O-methylated Catechins Biosynthesis in Tea Plant

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(Camellia Sinensis L.)

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Authors: Yong Luo1, Shuangshuang Yu1, Juan Li1,2, Qin Li1,2, Kunbo Wang1,2*, Jianan Huang1,2,*, Zhonghua

5

Liu1,2,*

6 7

1

8

China

9

2

Key Laboratory of Tea Science of Ministry of Education, Hunan Agricultural University, Changsha, 410128, P. R.

National Research Center of Engineering and Technology for Utilization of Botanical Functional Ingredients &

10

Hunan Co-innovation Center for Utilization of Botanical Functional Ingredients, Hunan Agricultural University,

11

Changsha, 410128, P. R. China

12 13 14 15 16 17 18 19 20 21 22 23 1

ACS Paragon Plus Environment


24 25

ABSTRACT: Tea O-methylated catechins especially (-)-epigallocatechin-3-O-(3-O-methyl)-gallate

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(EGCG3"Me) have been attracted much attention due to their positive health effects. The

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transcription regulators of O-methylated catechins biosynthesis remains elusive. In this study,

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expression pattern of genes related to O-methylated catechins biosynthesis including CsLAR, CsANS,

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CsDFR, CsANR and CCoAOMT in three tea cultivars with different content of EGCG3"Me was

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investigated. Two WRKY transcription factors (TFs), designated as CsWRKY31 and CsWRKY48,

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belonging to group IIc and IIb of the WRKY family respectively, were further identified.

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CsWRKY31 and CsWRKY48 were nuclear-localized proteins, and possessed transcriptional

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repression ability. Furthermore, expression of CsWRKY31 and CsWRKY48 showed negative

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correlation with CsLAR, CsDFR, and CCoAOMT, during EGCG3"Me accumulation in tea leaves.

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More importantly, W-box (C/T)TGAC(T/C) elements were located in the promoter of CsLAR,

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CsDFR, and CCoAOMT, and further assays revealed that CsWRKY31 and CsWRKY48 were

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capable of repressing the transcription of CsLAR, CsDFR, and CCoAOMT, via the attachment of

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their promoters to the W-box elements. Collectively, our findings identify two novel negative

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regulators of O-methylated catechins biosynthesis in tea plant, which might provide a potential

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strategy to breed high quality tea cultivar.

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KEYWORDS: Camellia sinensis, Catechins biosynthesis, EGCG3"Me, WRKY, Transcriptional regulator

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INTRODUCTION

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The tea plant (Camellia Sinensis L.) is one of the most important commercial plants. It has been cultivated

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worldwide for at least 2000 years due to it stimulating and soothing effects as well as positive effects on human

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health. Numerous studies showed that tea can prevent diseases including anti-cancer, anti-cardiovascular,

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anti-neurodegenerative, and oxidative stress1-3. In general, the bioactive properties of tea are catechins. A new

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catechin, (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3"Me) that has been reported recently to have

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stronger anti-allergic and anti-hypertensive effects than epigallocatechin-3-O-gallate (EGCG)4-6. Another

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biologically important characteristic of EGCG3"Me is absorbed more easily than EGCG4-6. The quality and taste of

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tea is predominantly determined by flavonoids and catechins. Previously, we have identified two tea cultivars

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‘Jinmudan’ and ‘Jinguanyin’ with high EGCG3"Me content7, but the molecular mechanism(s) concerning

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EGCG3"Me biosynthesis in these two cultivars remain unclear. Therefore, exploring the regulatory mechanisms of

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catechins biosynthesis by diverse factors may contribute to greater understanding of mechanisms for increasing

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catechins accumulation in tea plants by effective strategies such as environmental regulation and genetic breeding.

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In general, catechins and EGCG3"Me are thought to be biosynthesized through the flavonoid pathways5, 8-9. As

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shown in Fig. 1a, the biosynthesis of catechins and EGCG3"Me is regulated by the dynamic balance of the

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expression of biosynthesis-related genes leucocyanidin reductase (LAR), anthocyanidin synthase (ANS),

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dihydroflavonol-4-reductase(DFR)

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3-O-methyltransferase (CCoAOMT) has been suggested to be directly associated with EGCG3"Me accumulation5.

and

anthocyanidin

reductase

(ANR).

Moreover,

caffeoyl-CoA

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Recently, many transcription regulators including MYB, bHLH, MADS and WD40 were found to be associated

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with catechins metabolic regulation by controlling the expression of important enzyme genes participate in 3



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catechins biosynthetic pathway10-11. As a major transcription factor(TF) familys in plants, WRKYs are key

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regulators of plant development, and stress defenses12-14. The WRKY family proteins have one or two WRKY

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domains consisting of a 60-amino-acid region that possesses a highly conserved WRKYGQK in their N-termini,

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and C2HC zinc finger in C-termini12. WRKY transcription factors can regulate downstream transcription by

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specifically binding to the W-box (C/T)TGAC(T/C)15. Upon the completion of genome sequencing, WRKY TFs

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have been isolated and identified in many plants, including Arabidopsis, Glycine max, Oryza sativa and Hordeum

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vulgare16-18. WRKY TFs are also reported to play regulatory roles in plant metabolite biosynthesis, including

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phenolic, phenylpropanoids, terpenes and alkaloids by regulating the downstream genes19. For example, a opium

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poppy (Papaver somniferum) WRKY was found to affect the biosynthesis of alkaloids20. Similarly, OsWRKY76

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was reported to be regulating the production of terpenes and phenylpropanoids21. WRKYs were reported to

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up-regulate the biosynthesis of phenylpropanoids, such as flavonols, but few down-regulated regulators was

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reported from various plants22-23. Knowledge regarding the involvement of WRKY TFs in EGCG3"Me

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biosynthesis is far from complete.

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In this study, two tea cultivars Jinmudan and Jinguanyin with high levels of EGCG3"Me were used. The

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expression of EGCG3"Me biosynthesis-related genes including CsLAR, CsDFR and CCoAOMT were performed.

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Moreover, two potential WRKY TFs termed CsWRKY31 and CsWRKY48 that negatively associated with

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EGCG3"Me biosynthesis were identified and characterized. Further experiments revealed that CsWRKY31 and

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CsWRKY48 directly bound to the promoter of CsLAR, CsDFR and CCoAOMT, and repressed their expressions.

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Our findings demonstrate that CsWRKY31 and CsWRKY48 might negatively participate in catechins biosynthesis

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by down-regulating CsLAR, CsDFR and CCoAOMT expressions. Our study disseminate novel knowledge about the

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transcriptional regulatory of plant catechins biosynthesis and clues for the breeding of tea cultivar resources with 4


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high EGCG3"Me levels.

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MATERIALS AND METHODS

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Plant materials and treatments.

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Three different tea cultivars (Camellia sinensis (L.) O. Kuntze.) including Fudingdabai, Jinmudan and

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Jinguanyin, were cultivated in the Gao qiao tea farm of the Tea Research Institute of Hunan Agricultural Sciences,

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Changsha, China. For each cultivar, leaves at standard stage with “one bud and two leaves” from ten different tea

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plants were collected (Figure 1b), then frozen in liquid nitrogen and stored at −80 °C until later use for

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physiological and molecular analysis.

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Quantification of EGCG and EGCG3"Me.

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The EGCG3"Me contents in tea leaves in the different three cultivars were detected by LC-10ATVP HPLC

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system (Shimadzu, Tokyo, Japan). The tea samples were separated using a reversed-phase column (Welchorm C18

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200×4.6, 5mm). Standards of EGCG and EGCG3"Me(cas:83104-874) were purchased from Sigma-Aldrich (St.

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Louis, MO, USA).

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Gene Isolation, Sequence and Expression Analysis.

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Each tea leaf sample was used for the extraction of total RNA using the RNeasy Mini kit (Qiagen, Hilden,

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Germany) according to the manufacturer’s instructions. RNA quality and quantity were monitored by gel

103

electrophoresis and spectrophotometry. The DNA-free total RNA was reversely transcribed into the first strand of

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cDNAs using a Prime ScriptRT Reagent Kit (Takara, Dalian, China). Three genes including CsLAR, CsDFR and

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CCoAOMT related to catechins and EGCG3"Me biosynthesis and two WRKY genes CsWRKY31 and CsWRKY48

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were identified and selected from the Camellia sinensis genome (http://www.plantkingdomgdb.com/tea_tree/) and

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our RNA-seq database by using leaves from different cultivars (unpublished data). The full-length sequences of 5



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CsWRKY31 and CsWRKY48 were amplified with specific primers (Primers are shown in Table S1), and blasted in

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the National Center for Biotechnology Information (NCBI). The sequence alignment and phylogenetic tree were

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constructed using ClustalW and MEGA5, respectively.

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Gene sequences we used in this article were downloaded from NCBI, including DFR (GenBank Accession

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No. AY648027.1), ANS (GenBank Accession No. AY830416.1), ANR (GenBank Accession No. GU944768.1),

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LAR(GenBank Accession No. EF205148.1), CCoAOMT(GenBank Accession No. KF268598.1). Gene expression

114

level was performed using Go Taq qPCR Master Mix Kit (Promega, Madison, WI, USA) on a Bio-Rad CFX96

115

real-time PCR system. The PCR conditions started with an pre-denaturation step at 94°C for 5 min, then followed

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by 40 cycles of 94°C for 10s, 60°C for 30s, and 72°C for 30s, in a 20-mL reaction. Primers are shown in Table S1.

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Subcellular Localization Analysis.

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The coding region of CsWRKY31 or CsWRKY48 was cloned into the pEAQ-GFP vector (Primers used are shown

119

in Table S1)and was verified by further sequencing. About 4 to 6 weeks-old N. benthamiana leaves were transiently

120

transformed with the well constructed vectors through Agrobacterium tumefaciens strain GV3101 as described

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previously24. GFP fluorescence was recorded by a fluorescence microscope after 2-3 days of infiltration.

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Transcriptional Activation Assay.

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CsWRKY31 and CsWRKY48 were inserted into the pGBKT7 vector (Clontech, USA). Then the fusion construct

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pGBKT7-CsWRKY31 or -CsWRKY48, positive control (p53+T-antigen) and negative control (pGBKT7 vector)

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were transformed separately into yeast cells. The transcriptional activation ability of CsWRKY31 and CsWRKY48

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was evaluated basing on the growth status and α-galactosidase activity of yeast cells that grow on SD medium

127

(SD/-Trp), or (SD/-Trp-His-Ade).

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Promoter Isolation and Analysis. 6


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The promoter sequences of CsLAR, CsDFR and CCoAOMT were amplified by PCR (primers are shown in Table

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S1) using genomic DNA of tea leaves as the template according to the DNeasy Plant Mini Kit (Qiagen). Conserved

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W-box motifs presented in the promoters were predicted and identified through the Plant-CARE online software

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(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

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Protein Expression and Electrophoretic Mobility Shift Assay (EMSA).

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The sequence of CsWRKY31 N-terminus (from 475 to 753 bp including the WRKY domain) and the coding

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region of CsWRKY48 were cloned into the pGEX-4T-1 (Amersham Biosciences) vector and transformed into

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Escherichia coli strain BM Rosetta (DE3). The induced GST-CsWRKY31-N and GST-CsWRKY48 protein was

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induced and purified using a Glutathione-Superflow Resin (Clontech) and stored at −80 °C. The 5’ ends of

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synthesized oligonucleotide probes containing W-box in the promoters of CsLAR, CsDFR and CCoAOMT were

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labeled with biotin. The Light Shift Chemiluminescent EMSA Kit (Thermo Scientific) was used for EMSA

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experiment as previously described25. Briefly, GST-CsWRKY31-N and GST-CsWRKY48 protein and

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biotin-labeled probes were incubated together, then the assay mixtures were analyzed by 6% native polyacrylamide

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gel electrophoresis, after transferred onto nylon membrane, the protein-DNA complexes was detected using a

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ChemiDoc™ MP Imaging System (Bio-Rad, USA). GST protein alone as well as the unlabeled and mutated probes

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were used as negative control.

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Dual Luciferase Reporter Assay.

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To investigate the transcriptional activity of CsWRKY31 and CsWRKY48, their coding sequences were

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cloned into pBD vector. The double-reporter vector included a GAL4-LUC and an internal control REN as

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previously described25. To assess the binding activity of CsWRKY31 and CsWRKY48 to the promoters of CsLAR,

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CsDFR and CCoAOMT. CsWRKY31 and CsWRKY48 were cloned into pEAQ vector as effectors. The promoters 7



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of CsLAR, CsDFR and CCoAOMT were recombined to pGreenII 0800-LUC vector as reporters. The constructed

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effectors and reporters plasmids with different combinations were co-transformed into the Nicotiana benthamiana

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leaves .

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Firefly luciferase and Renilla luciferase were quantified after 2 d of infiltration with the dual luciferase assay

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kit (Promega, USA). The transactivation ability of CsWRKY31 and CsWRKY48 was assessed by the LUC to REN

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ratio. At least six independent experiments were included for each combination.

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Statistical Analyses.

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All data are presented as means ± standard errors (S.E.) of three or six independent biological replicates. To

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compare the statistical difference and determine the significance of experimental means, the data were evaluated by

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student's t-test at P value < 0.05 or 0.01.

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RESULTS AND DISCUSSION

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The

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Biosynthesis-Related Genes among Cultivars

Variation

of

EGCG

and

EGCG3"Me

Contents,

and

the

Expression

of

EGCG3"Me

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As a bioactive constituent, EGCG3"Me was recently reported to occur naturally in tea leaves, but the quantities

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of EGCG3"Me was extremely limited and detected in few tea cultivars5. In this study, EGCG and EGCG3"Me

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levels in tea leaves of three cultivars including ‘Fudingdabai’, ‘Jinmudan’ and ‘Jinguanyin’ at the standard stage

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with “one bud and two young leaves” were measured by HPLC. As shown in Figure 2a, EGCG3"Me content in the

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leaves of ‘Jinguanyin’ and ‘Jinmudan’ was higher than that in ‘Fudingdabai’. EGCG3"Me level in ‘Jinguanyin’ and

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‘Jinmudan’ was 6.12 ± 0.35 mg/g and 7.81 ± 0.30 mg/g respectively, whereas was almost undetectable in

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‘Fudingdabai’. The contents of EGCG in leaves of ‘Jinguanyin’ and ‘Jinmudan’ was also higher than in

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‘Fudingdabai’ (Figure 2b). Lv et al. (2014)26 reported that different tea cultivars contained different EGCG3"Me 8


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content and only 4 tea cultivars were found to be rich in EGCG3"Me.

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LAR, ANS, DFR, ANR and CCoAOMT are important enzymes in catechins and EGCG3"Me biosynthesis

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(Figure 1a). To further investigate the association of LAR, ANS, DFR, ANR and CCoAOMT with EGCG3"Me

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biosynthesis, their gene expression levels in leaves of ‘Fudingdabai’, ‘Jinmudan’ and ‘Jinguanyin’ were compared.

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Some significant correlations were noted. As shown in Figure 2c, transcript levels of CsLAR, CsDFR and

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CCoAOMT were obviously higher in ‘Jinguanyin’ and ‘Jinmudan’ than in ‘Fudingdabai’, while no obvious

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difference among these three cultivars was observed in CsANR and CsANS expression.

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Identification and Bioinformatics Analysis of CsWRKY31 and CsWRKY48.

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To date, ~72, 109, 197 and 45 WRKY members have been identified in Arabidopsis, Oryza sativa, Glycine max

180

and Hordeum vulgare respectively16, 18-19, 27. About 50 WRKY genes have been identified in tea plants28, and two

181

WRKY genes attracted our attention as their expression was obviously down-regulated in ‘Jinguanyin’ and

182

‘Jinmudan’ according to our RNA-seq database. Therefore, the full-length of these two genes were cloned and

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designated as CsWRKY31 and CsWRKY48 since the sequence of these two WRKY genes had high degree of

184

homology with AtWRKY31 (NP_567644.1) (58%) and AtWRKY48 (NP_199763.1) (54%) respectively.

185

CsWRKY31 and CsWRKY48 cDNA contains an Open Reading Frame (ORF) of 1260 and 486 bp in length,

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encoding 419 and 161 amino acids that resulted in calculated molecular mass of 45.13 and 17.87 kDa, respectively.

187

WRKY proteins can be categorized into three major groups (I-III) according to the number of WRKY DNA

188

binding domain and Zinc-finger motif. And group II can be further divided into IIa, IIb, IIc, IId, and IIe12, 29.

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Multiple sequence alignment showed that CsWRKY31 and CsWRKY48 consisted of a highly conserved WRKY

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DNA binding domain (DBD) and a zinc finger (C2H2 motif) (Figure 3a), which shared a high degree of homology

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with the group II WRKY family. Phylogenetic analysis further indicated that CsWRKY31 and CsWRKY48 was 9



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clustered with AtWRKY31 and AtWRKY48 into the group IIb and group IIc WRKY family, respectively (Figure

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3b). The group IIb Solanum lycopersicum SlWRKY73 and group IIc AtWRKY23 have been reported to regulate

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plant-specialized metabolism, such as the biosynthesis of important pharmaceutical, aromatherapy, biofuel, and

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industrial components30-31. Similarly, pitaya HpWRKY44 could be clustered with the Arabidopsis thaliana Group I

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WRKYs including AtWRKY44 and AtWRKY33. Notably, the Arabidopsis thaliana WRKY44 and WRKY33 were

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reported to regulate the biosynthesis of indole alkaloid and phenylpropanoid. Accordingly, HpWRKY44 was

198

reported to be involved in the biosynthesis of secondary metabolites in pitaya24. Thus, it could be speculated that

199

CsWRKY31 and CsWRKY48 might be associated with the biosynthesis of EGCG3"Me in tea plants.

200

Expression and Molecular Characterization of CsWRKY31 and CsWRKY48.

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Similar to the RNA-seq data, RT-qPCR analysis exhibited that CsWRKY31 and CsWRKY48 transcript levels

202

were remarkably lower in ‘Jinguanyin’ and ‘Jinmudan’ than in ‘Fudingdabai’ (Figure 4a), showing the opposite

203

pattern with CsLAR, CsDFR and CCoAOMT expression, and EGCG3"Me accumulation, implied that CsWRKY31

204

and CsWRKY48 might be negatively involved in EGCG3"Me biosynthesis in the tea plant. WRKYs are usually

205

nuclear proteins27,

206

GFP-empty proteins with these two WRKY proteins transiently expressed into tobacco leaves. As shown in Figure

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4b, the 35S:GFP-empty signal was found in the nucleus and cytoplasm, whereas the 35S:CsWRKY31-GFP and

208

35S:CsWRKY48-GFP fusion proteins were predominately observed in the nucleus, indicating that CsWRKY31

209

and CsWRKY48 are nucleus-localized proteins.

32-33.

To find out the subcellular location of CsWRKY31 and CsWRKY48, we fused the

210

Transcriptional ability of TFs has a key role in regulating downstream genes34-35. The transcriptional activities

211

of CsWRKY31 and CsWRKY48 were further investigated using a GAL4-responsive reporter system in yeast cells.

212

The positive control (pGBKT7-53 + pGADT7-T) had normal growth in the SD plates without tryptophan, histidine, 10


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and adenine and showed α-galactosidase activity, whereas the yeasts transformed with the pGBKT7-empty vector,

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pGBKT7-CsWRKY31 and pGBKT7-CsWRKY48 did not grow and lacked the α-galactosidase activity (Figure 5a),

215

indicating that CsWRKY31 and CsWRKY48 did not possess any transcriptional activation activity in the yeast

216

cells, and they might be transcriptional repressors. The transcriptional repression activities of CsWRKY31 and

217

CsWRKY48 were further verified by dual-luciferase assays (Figure 5b and 5c). Compared with the pBD-empty

218

control, both CsWRKY31 and CsWRKY48 significantly repressed the LUC reporter activities (Figure 5c),

219

suggesting that CsWRKY31 and CsWRKY48 are likely act as transcriptional repressors.

220

CsWRKY31 and CsWRKY48 Target CsLAR, CsDFR and CCoAOMT Promoters via the W-box Element.

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It is well-known that WRKYs regulate their target genes expressions by binding to consensus sequence

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(C/T)TGAC(T/C), known as W-box presented in the promoters15,

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CsLAR, CsDFR and CCoAOMT promoters (Text S1). EMSA using the purified recombinant GST-CsWRKY31-N

224

and GST-CsWRKY48 protein (Figure 6a and 6c) was performed to confirm whether CsWRKY31 and CsWRKY48

225

could target CsLAR, CsDFR and CCoAOMT promoters via the W-box. As expected, both GST-CsWRKY31-N and

226

CsWRKY48 fusion proteins could directly bind to labeled CsLAR, CsDFR and CCoAOMT fragment containing

227

W-box and caused mobility shifts. The mobility shift was effectively abolished due to unlabeled fragment being

228

used as a cold probe in a dose-dependent manner, but not by the mutated probes (Figure 6b and 6d). In addition,

229

when the probes was incubated with GST alone, the mobility shift was not observed. These data reveal that

230

CsWRKY31 and CsWRKY48 target the W-box motif in the CsLAR, CsDFR and CCoAOMT promoters.

231

Trans-repression of CsWRKY31 and CsWRKY48 on CsLAR, CsDFR and CCoAOMT Promoters

19.

Indeed, the W-box motifs were found in

232

To further investigate whether the promoter activity of CsLAR, CsDFR and CCoAOMT could be repressed by

233

CsWRKY31 or CsWRKY48, the well-established transient dual luciferase assay was employed, using a double 11



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reporter plasmid containing the LUC driven by CsLAR, CsDFR and CCoAOMT promoters and the REN driven by

235

the CaMV35S promoter, together with an effector plasmid expressing CsWRKY31 or CsWRKY48 (Figure 7a).

236

The results showed that LUC/REN ratio was remarkably decreased when the CsLAR, CsDFR and CCoAOMT

237

pro-LUC reporter construct was co-transfected with CsWRKY31 or CsWRKY48. Compared with the empty

238

control (Figure 7b), suggesting that CsWRKY31 and CsWRKY48 repressed the promoter activity of CsLAR,

239

CsDFR and CCoAOMT. Taken together, our data support the notion that CsWRKY31 and CsWRKY48 act as

240

transcriptional repressors of CsLAR, CsDFR and CCoAOMT through direct binding to their promoters. In tea plant,

241

the R2R3-MYB, bHLH and WD40 were reported to be involved in flavonoid biosynthesis10,36. For instance,

242

MYB4a, a R2R3-MYB TF, was reported negatively regulate the phenylpropanoid and shikimate pathways37.

243

WRKY TFs were mostly reported in plant responses to stress. For example, Arabidopsis WRKY52 was able to

244

confer resistance against bacterial pathogen Ralstonia solanacearum38. In tea plants, WRKY2 is shown to function

245

in cold and drought stress responses39. Nevertheless, several studies suggest that WRKY TFs regulate the

246

production of several secondary metabolites by regulating the genes within the metabolite pathway. For instance,

247

Arabidopsis WRKY12, WRKY23 and WRKY44 played an important role in regulating the production of lignin,

248

flavonol and tannin, respectively19, 30, 40-41. While Rice (Oryza sativa) OsWRKY45 up-regulated the accumulation

249

of oryzalexin, phytocassane and momilactone, by priming the target gene expression22. Overexpression of

250

OsWRKY13 up-regulates phenylpropanoid pathway related-genes23. Moreover, it has been well documented that

251

regulatory proteins such as TFs, seldom act alone. Numerous studies reveal that WRKY TFs physically interact

252

with a wide range of proteins including themselves, as well as VQ and MAPKs42-45. Therefore, it would be

253

meaningful to focus on determining whether CsWRKY31 and CsWRKY48 can interact with each other or other

254

proteins to co-regulate EGCG3"Me biosynthesis-related genes in the near future studies. 12


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255

In summary, two tea cultivars Jinmudan and Jinguanyin with high contents EGCG3"Me were used to explore the

256

underlying mechanism of EGCG3"Me biosynthesis. The expression levels of CsLAR, CsDFR and CCoAOMT were

257

obviously higher in Jinmudan and Jinguanyin than Fudingdabai. Moreover, we characterized two transcriptional

258

repressors CsWRKY31 and CsWRKY48, and revealed that CsWRKY31 and CsWRKY48 repressed CsLAR,

259

CsDFR and CCoAOMT transcription via directly targeting their promoters. We propose that CsWRKY31 and

260

CsWRKY48 might be acting as negatively regulators of EGCG3"Me biosynthesis in tea plants through directly

261

repressing catechins biosynthetic genes. These findings help us better understanding the transcriptional regulation

262

of EGCG3"Me biosynthesis involving WRKY TFs.

263

ABBREVIATIONS USED

264

EGCG, (-)-epigallocatechin-3-gallate; EGCG3"Me, Epigallocatechin-3-O-(3-O-methyl) gallate ; HPLC, high

265

performance liquid chromatography; DFR, dihydroflavonol; 4-reductase; LAR, leucoanthocyanidinase; reductase;

266

ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; CCoAOMT, Caffeoyl-CoA 3-O-methyltransferase;

267

bHLH, basic Helix-Loop-Helix; TF, transcription factor; ORF, Open Reading Frame; NCBI, National Center for

268

Biotechnology Information; qRT-PCR, Quantitative real-time PCR; GFP,

269

RT-PCR, Reverse transcriptionpolymerase chain reaction.

270

ASSOCIATED CONTENT

271

Supporting Information

272

Primers used in this study (Table S1), and nucleotide sequences of CCoAOMT, CsLAR and CsDFR promoters

273

(Text S1)

274

Corresponding Author

275

* Telephone: +86-73184635304. Fax: +86-73184635304, email: [email protected] 13


Green fluorescence protein;


276

* Telephone: +86-73184635306. Fax: +86-73184635304, email: [email protected]

277

* Telephone: +86-73184635306. Fax: +86-73184635304, email: [email protected]

Page 14 of 26

278 279

Author Contributions

280

Yong Luo, Shuangshuang Yu, Kunbo Wang, Jianan Huang and Zhonghua Liu designed and performed the

281

experiments, analyzed the data, and co-wrote the manuscript. Juan Li and Qin Li helped with collecting materials,

282

extracting total RNA, and performing qRT-PCR.

283

Funding

284

The work was financially supported by the National Natural Science Foundation of China (31470692, 31670691

285

and 31500567).

286

Notes

287

The authors declare no competing financial interest.

288 289

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398 399 400 401 402 403 404

Figure legends

405 406 407

Figure 1. The biosynthetic pathways of EGCG3"Me, and the plant materials of tea leaves.(a)Possible biosynthetic pathways of EGCG3"Me. (b) Plant materials of tea leaves (a terminal bud and two young leaves).

408 409 410 411 412 413 414 415 416 417 418

Figure 2. The contents of EGCG3"Me and EGCG, and the expression pattern of EGCG3"Me biosynthesis related-genes. (a)The content of EGCG3"Me in different tea cultivars by HPLC in dry weight of leaf sample, “n.d.”indicates not detected. (b)The content of EGCG in different tea cultivars. (c)Relative expression analyzed by real time quantitative reverse transcription-PCR (qRT-PCR) of EGCG3"Me biosynthesis related-genes, β-actin was used as an internal control. Each value represents the mean ± SE of three replicates. *P ≤ 0.05, **P ≤ 0.01, compared with the Fudingdabai, respectively. “NS” indicate no differences by Student’s t test. (Note: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase;4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3b-hydroxylase; F3’H, flavonoid 30-hydroxylase; F3’5H, flavonoid 3’5’-hy-droxylase; DFR, dihydroflavonol reductase; LAR, leuacoanthocyanidin reductase; ANS, anthocyanin synthase; ANR, anthocyanidin reductase; CCoAOMT, caffeoyl-CoA 3-O-methyltransferase.)

419 420 421 422 423 424 425 426 427

Figure 3. Identification and amino acid analysis of CsWRKY31 and CsWRKY48 involved in EGCG3"Me biosynthesis in tea tree (Camellia sinensis). (a)Comparative sequence analysis and dendrogram of CsWRKY31 and CsWRKY48 protein with the other WRKY proteins. The conserved WRKY domain and the C2H2 zinc-finger motifs are indicated by red letters. (b) Phylogenetic tree analysis of CsWRKY31 and CsWRKY48 protein comparison with amino acid sequences of the WRKY family isolated from Arabidopsis. The full-length amino acid sequences were downloaded from the institute for Genomic Research (http://www.tigr.org) and the National Center for Biotechnology information (http://www.ncbi.nlm.nih.gov). The amino acid sequences were aligned with ClustalW and the phylogenetic tree was constructed by MEGA 5.0 using the neighbor-joining method .

428 429 430 431

Figure 4. Expression pattern of CsWRKY31 and CsWRKY48 in different tea cultivars and their subcellular localizations. (a) Relative expressions analyzed by real time quantitative reverse transcription-PCR (qRT-PCR). **Significant differences in values (P < 0.01) by Student's t-test. (b)Subcellular localizations of CsWRKY31 and CsWRKY48 in 17



432 433 434

Page 18 of 26

tobacco leaves. CsWRKY31 and CsWRKY48 fused with the GFP or GFP control were infiltrated into tobacco leaves via Agrobacterium tumefaciens strain GV3101. After 48 h of the infiltration, GFP fluorescence was visualized using a fluorescence microscope, Scale bars=50µm.

435 436 437 438 439 440 441 442 443 444 445 446 447 448

Figure 5. Molecular Characterization of CsWRKY31 and CsWRKY48. (a)The transcriptional activity analysis of CsWRKY31 and CsWRKY48 in yeast cell assays. Both the full length of the two WRKYs are fused with pGBKT7, and transformed yeasts were selected on SD/-Trp or SD/-Trp/-His/-Ade/ X-α-gal media for 3-5 d at 30°C. Transcription activation was monitored by detecting yeast growth and the expression of a-galactosidase (a-Gal) activity. (b ＆ c)Transcriptional activation ability of CsWRKY31 and CsWRKY48 in tobacco leaves. The double-reporter plasmids contain 5×GAL4 and mini CaMV35S fused to firefly luciferase (LUC) and renilla luciferase (REN) driven by CaMV35S. The effector plasmids contain the CsWRKY31 and CsWRKY48 genes fused to GAL4BD driven by the CaMV35S. The dual REN/LUC reporter and effectors were co-transformed into tobacco leaves by Agrobacterium tumefaciensstrain GV3101. After 48-72h of the infiltration, LUC and REN luciferase activities were assayed, and the transcription activation ability of CsWRKY31 and CsWRKY48 is indicated by the ratio of LUC to REN. Each value represents the means of six biological replicates, and vertical bars represent the S.E. **Significant differences in values (P < 0.01) by Student's t-test, compared with pBD-empty.

449 450 451 452 453 454 455 456 457

Figure 6. Electrophoretic mobility shiſt assay (EMSA) showing both CsWRKY31 and CsWRKY48 can specifically bind to the promoter region of EGCG3"Me biosynthetic genes including CCoAOMT, CsLAR, and CsDFR. (a＆c) SDS–PAGE gel stained with Coomassie blue demonstrating affinity purification of the recombinant GST-CsWRKY31-N and GST-CsWRKY48 protein used for the EMSA. (b＆d) Recombinant GST-CsWRKY31-N and GST-CsWRKY48 protein bind directly to the promoters of CCoAOMT, CsLAR, and CsDFR containing W-box(T/CTGACT/C) element, respectively. Biotin-labeled DNA probe from the promoters or mutant probe was incubated with the two recombinant protein, and the DNA-protein complexes were separated on 6% native polyacrylamide gels. +and++indicate increasing amounts unlabeled probes for competition.

458 459 460 461 462 463 464

Figure 7. CsWRKY31 and CsWRKY48 repressed the promoter activity of genes involved in the EGCG3"Me biosynthesis pathways.(a)Constructs used in the transient transactivation assays. (b)The LUC/REN ratio was calculated as the final transcriptional activity. Empty vector was used as the effector in the control assay. The promoters of CCoAOMT, CsLAR, and CsDFR genes were used in dual luciferase assays, and each value represents the means of six biological replicates. Vertical bars represent the S.E.. Significant differences in values (*P < 0.05, **P < 0.01) by Student's t-test.

465 466 467 468 469 18


Page 19 of 26


Figure 1. The biosynthetic pathways of EGCG3"Me, and the plant materials of tea leaves.(a)Possible biosynthetic pathways of EGCG3"Me. (b) Plant materials of tea leaves (a terminal bud and two young leaves). 419x217mm (300 x 300 DPI)



Figure 2. The contents of EGCG3"Me and EGCG, and the expression pattern of EGCG3"Me biosynthesis related-genes. (a)The content of EGCG3"Me in different tea cultivars by HPLC in dry weight of leaf sample, “n.d.”indicates not detected. (b)The content of EGCG in different tea cultivars. (c)Relative expression analyzed by real time quantitative reverse transcription-PCR (qRT-PCR) of EGCG3"Me biosynthesis relatedgenes, β-actin was used as an internal control. Each value represents the mean ± SE of three replicates. *P ≤ 0.05, **P ≤ 0.01, compared with the Fudingdabai, respectively. “NS” indicate no differences by Student’s t test. (Note: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase;4CL, 4-coumarate:CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3b-hydroxylase; F3’H, flavonoid 30-hydroxylase; F3’5H, flavonoid 3’5’-hy-droxylase; DFR, dihydroflavonol reductase; LAR, leuacoanthocyanidin reductase; ANS, anthocyanin synthase; ANR, anthocyanidin reductase; CCoAOMT, caffeoyl-CoA 3-O-methyltransferase.) 422x130mm (300 x 300 DPI)


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Figure 3. Identification and amino acid analysis of CsWRKY31 and CsWRKY48 involved in EGCG3"Me biosynthesis in tea tree (Camellia sinensis). (a)Comparative sequence analysis and dendrogram of CsWRKY31 and CsWRKY48 protein with the other WRKY proteins. The conserved WRKY domain and the C2H2 zinc-finger motifs are indicated by red letters. (b) Phylogenetic tree analysis of CsWRKY31 and CsWRKY48 protein comparison with amino acid sequences of the WRKY family isolated from Arabidopsis. The full-length amino acid sequences were downloaded from the institute for Genomic Research (http://www.tigr.org) and the National Center for Biotechnology information (http://www.ncbi.nlm.nih.gov). The amino acid sequences were aligned with ClustalW and the phylogenetic tree was constructed by MEGA 5.0 using the neighbor-joining method . 170x203mm (300 x 300 DPI)



Figure 4. Expression pattern of CsWRKY31 and CsWRKY48 in different tea cultivars and their subcellular localizations. (a) Relative expressions analyzed by real time quantitative reverse transcription-PCR (qRTPCR). **Significant differences in values (P < 0.01) by Student's t-test. (b)Subcellular localizations of CsWRKY31 and CsWRKY48 in tobacco leaves. CsWRKY31 and CsWRKY48 fused with the GFP or GFP control were infiltrated into tobacco leaves via Agrobacterium tumefaciens strain GV3101. After 48 h of the infiltration, GFP fluorescence was visualized using a fluorescence microscope, Scale bars=50µm. 170x59mm (300 x 300 DPI)


Page 22 of 26

Page 23 of 26


Figure 5. Molecular Characterization of CsWRKY31 and CsWRKY48. (a)The transcriptional activity analysis of CsWRKY31 and CsWRKY48 in yeast cell assays. Both the full length of the two WRKYs are fused with pGBKT7, and transformed yeasts were selected on SD/-Trp or SD/-Trp/-His/-Ade/ X-α-gal media for 3-5 d at 30°C. Transcription activation was monitored by detecting yeast growth and the expression of agalactosidase (a-Gal) activity. (b ＆ c)Transcriptional activation ability of CsWRKY31 and CsWRKY48 in tobacco leaves. The double-reporter plasmids contain 5×GAL4 and mini CaMV35S fused to firefly luciferase (LUC) and renilla luciferase (REN) driven by CaMV35S. The effector plasmids contain the CsWRKY31 and CsWRKY48 genes fused to GAL4BD driven by the CaMV35S. The dual REN/LUC reporter and effectors were co-transformed into tobacco leaves by Agrobacterium tumefaciensstrain GV3101. After 48-72h of the infiltration, LUC and REN luciferase activities were assayed, and the transcription activation ability of CsWRKY31 and CsWRKY48 is indicated by the ratio of LUC to REN. Each value represents the means of six biological replicates, and vertical bars represent the S.E. **Significant differences in values (P < 0.01) by Student's t-test, compared with pBD-empty. 170x109mm (300 x 300 DPI)



Figure 6. Electrophoretic mobility shiſt assay (EMSA) showing both CsWRKY31 and CsWRKY48 can specifically bind to the promoter region of EGCG3"Me biosynthetic genes including CCoAOMT, CsLAR, and CsDFR. (a＆c) SDS–PAGE gel stained with Coomassie blue demonstrating affinity purification of the recombinant GST-CsWRKY31-N and GST-CsWRKY48 protein used for the EMSA. (b＆d) Recombinant GSTCsWRKY31-N and GST-CsWRKY48 protein bind directly to the promoters of CCoAOMT, CsLAR, and CsDFR containing W-box(T/CTGACT/C) element, respectively. Biotin-labeled DNA probe from the promoters or mutant probe was incubated with the two recombinant protein, and the DNA-protein complexes were separated on 6% native polyacrylamide gels. +and++indicate increasing amounts unlabeled probes for competition. 135x181mm (300 x 300 DPI)


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Figure 7. CsWRKY31 and CsWRKY48 repressed the promoter activity of genes involved in the EGCG3"Me biosynthesis pathways.(a)Constructs used in the transient transactivation assays. (b)The LUC/REN ratio was calculated as the final transcriptional activity. Empty vector was used as the effector in the control assay. The promoters of CCoAOMT, CsLAR, and CsDFR genes were used in dual luciferase assays, and each value represents the means of six biological replicates. Vertical bars represent the S.E.. Significant differences in values (*P < 0.05, **P < 0.01) by Student's t-test. 256x323mm (300 x 300 DPI)



TOC 84x47mm (300 x 300 DPI)


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Molecular Characterization of WRKY Transcription Factors that Act as

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