Functional characterization of salicylic acid carboxyl

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Functional characterization of salicylic acid carboxyl methyltransferase from Camellia sinensis, providing the aroma compound of methyl salicylate during withering process of white tea Wei-Wei Deng, Rongxiu Wang, Tianyuan Yang, Li'na Jiang, and Zhengzhu Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04575 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017

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Journal of Agricultural and Food Chemistry

Functional characterization of salicylic acid carboxyl methyltransferase from Camellia sinensis, providing the aroma compound of methyl salicylate during withering process of white tea Wei-Wei Deng#, Rongxiu Wang#, Tianyuan Yang, Li’na Jiang, Zheng-Zhu Zhang*

State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130 Changjiang West Road, Hefei, Anhui 230036, China

#

These authors contributed equally to this work.

W.-W. Deng and Z.-Z. Zhang designed the research; W.-W. Deng and R. Wang performed the experiments and wrote the manuscript; T. Yang revised the figures and L. Jiang helped to do some experiments for this research.

*

Corresponding author; Tel/fax: +86 551 65785471;

Email address: [email protected] (Z-Z. Zhang)

1

ACS Paragon Plus Environment


1

Abstract

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Methyl salicylate (MeSA) is one of the volatile organic compounds (VOCs) that

3

releases floral scent, and plays an important role in tea sweet flowery aroma. During

4

the withering process for white tea producing, MeSA was generated by salicylic acid

5

carboxyl methyltransferase (SAMT) with salicylic acid (SA), and the specific floral

6

scent was formed. In this study, we firstly cloned a CsSAMT from tea leaves

7

(GenBank accession No. MG459470) and used Escherichia coli and Saccharomyces

8

cerevisiae to express the recombinant CsSAMT. The enzyme activity in prokaryotic

9

and eukaryotic expression systems was identified; and the protein purification,

10

substrate specificity, pH and temperature optima were investigated. It was shown that

11

CsSAMT located in the chloroplast, and the gene expression profiles were quite

12

different in tea organs. The obtained results might give a new understanding for tea

13

aroma formation, optimization and regulation, and have great significance for

14

improving the specific quality of white tea.

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Keywords: CsSAMT, characterization, white tea, withering, MeSA

16 17 18 19 20 21 22 2


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Introduction

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Methyl salicylate (MeSA) is synthetically used as a specific fragrance, in foods,

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beverages and liniments nowadays, which is one of the organic esters, naturally

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produced by many species of plants, especially wintergreens. Together with methyl

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benzoate (MeBA), MeSA is one of the volatile organic compounds (VOCs) that

28

releases the floral scent, which plays an important role in plant defense mechanism 1, 2.

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Tea is the most popular beverage in the world, and processed from the leaves of tea

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plants (Camellia sinensis). There are six types of teas (green tea, black tea, yellow tea,

31

dark tea, oolong tea and white tea) in China, based on their specific processing

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methods. The aroma of different teas is quite discriminative because they could be

33

defined by the tea plant variety itself, withering duration and rolling phase. In tea,

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even though the VOCs are existed in a very limited amount (i.e. 0.01% of the total dry

35

weight), they have a very high impact on tea aroma 3. Among the VOGs in tea, MeSA

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is one of the important compounds who give the high quality level of sweet flowery

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and floral aroma to tea.

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After comparing the content of MeSA in the six types of teas made of the same tea

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leaves, the highest content of MeSA was shown in white tea sample (white tea > black

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tea > oolong tea > yellow tea > green tea > dark tea, unpublished data). The traditional

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manufacturing process of white tea is only two stages of withering and drying. During

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these two stages, many compounds are generated to express the specific characters of

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smell and taste for white tea. The prolonged withering stage of white tea causes

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complex internal physical and chemical changes in leaves: the loss of water results in 3



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an increase of volatile compounds in leaf cells and also changes the permeability of

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cell membranes 4; hydrolases are activated and catalyzed the formation of amino acids

47

in leaves, and unsaturated fatty acids such as linoleic acid and linolenic acid are

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enzymatically degraded to small molecule compounds of alcohol, aldehydes, ketones,

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and acids 4. MeSA, as an important and specific floral scent, can be preserved and

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enhanced in the final tea product, especially in white tea with the long time withering.

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However, the biosynthesis of MeSA from salicylic acid (SA) in the withering

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process of white tea was still not reported. SA, a substance of plant hormone, is a one

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of the necessary endogenous signal molecules. Due to the published reports, salicylic

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acid carboxyl methyltransferase (SAMT) catalyzes the production of MeSA from SA

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(Figure

56

S-adenosyl-L-methionine-dependent methyltransferase is a ubiquitous reaction that

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occurs in plants 8. Recently, SAMTs in plants have been studied gradually, but SAMTs

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in tea plants and MeSA formation in white tea have not been well studied. To

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elucidate the enzymatic function of SAMT from C. sinensis (CsSAMT) in generating

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floral aroma, the variation of MeSA in tea leaves during the withering for white tea

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processing was investigated in the present study. A CsSAMT was isolated from tea

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leaves and the encoded protein was characterized using Escherichia coli and

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Saccharomyces cerevisiae individually. This study provides the first evidence of

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CsSAMT in tea leaves and the amount of MeSA in white tea during the withering

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process, and advances the better understanding and regulation of aroma formation in

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tea and especially in white tea.

1)

5-7

.

The

methylation

reaction

4


catalyzed

by

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Materials and Methods

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Materials

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Different tea organs (buds, first leaf, second leaf, old leaf, stem, and root) were

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collected from C. sinensis Shuchazao in the Tea Garden of Anhui Agricultural

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University, China. These organs were frozen in liquid nitrogen and stored at −80 °C

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until use. One bud with two tea leaves was also collected and was used in indoor

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withering of white tea.

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pET-32a(+), pESC-His, pDONR207, and pK7GWF2.0 vectors were obtained from

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the State Key Laboratory of Tea Plant Biology and Utilization in Anhui Agricultural

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University. A BD GenomeWalker™ Kit was obtained from Clontech (Mountain View,

78

CA, USA). Benzoic acid (BA) and methyl benzoate (MeBA) were purchased from

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Aladin (Shanghai, China). Cinnamic acid, vanillic acid, caffeic acid, and jasmonic

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acid were obtained from Yuanye Bio-Technology (Shanghai, China). Salicylic acid

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(SA) and methyl salicylate (MeSA) were purchased from Dr. Ehrenstorfer GmbH

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(Augsburg, Germany).

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MeSA content in white tea after indoor withering

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Two tea leaves with a bud were withered in a wilting sifter and then dried

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immediately at 80 °C for 30 min after 6, 12, 24, 36, and 72 h of withering. As a

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control, fresh leaves were directly steamed for 2–3 min (to deactivate the enzymes)

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and then dried at 80 °C for 30 min. MeSA was extracted from dry tea leaves (20 g)

88

with 30 mL anhydrous diethyl ether by using simultaneous distillation–extraction, and 5



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the anhydrous diethyl ether was concentrated to 1 mL by nitrogen blow controller and

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was analyzed using gas chromatography–mass spectrometry (GC–MS)

91

retention index was calculated using a mixture of n-paraffin C7–C25 as standards.

9

. The

92

The sample was injected into an Agilent 7890A/5975C GC–MS instrument. The

93

injection temperature and injected volume were set at 250 °C and 1 µL, respectively.

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The split ratio was 5:1, and the column (HP-5MS, 30 m×0.25 mm×0.25 µm,

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Agilent, USA) temperature ranged from 50 to 250 °C. MS was performed in the

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selected-ion monitoring mode after electron ionization (70 eV). The following

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GC–MS temperature program was applied: the initial column temperature was set at

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50 °C for 5 min, increased to 180 °C at a rate of 2 °C/min and maintained for 1 min,

99

and then increased to 250 °C at a rate of 10 °C/min and maintained for 2 min 10.

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Cloning of CsSAMT from tea leaves

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The annotated sequence of CsSAMT was screened out from a tea leaves

102

transcriptome database (unpublished data). Total RNA was isolated from the tea

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leaves of C. sinensis Shuchazao by using an RNA prep pure plant kit (Axygen,

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Tewksbury, MA, USA), and the first-strand cDNA was synthesized using a

105

PrimeScript RT Reagent Kit (Takara Bio, Dalian, China). The polymerase chain

106

reaction (PCR)-specific primers TYF and TYR with BamH I and Sac I restriction

107

enzyme sites used for cloning were designed using Primer Premier 5.0 software after

108

bioinformatics analysis (primer sequences were shown in Supplemental table 1); the

109

primers were then synthesized by Sangon (Shanghai, China). The PCR product (the

110

open reading frame, ORF) was purified from agarose gels by using an AxyPrep DNA 6


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Gel Extraction Kit (Axygen) and was ligated into the pEASY-Blunt vector (TransGen

112

Biotech, China) after the gene sequence was confirmed through sequencing (Sangon).

113

Construction of CsSAMT expression vector, prokaryotic expression and enzyme

114

assay

115

The plasmid containing recombinant CsSAMT and the pET-32a(+) vector were

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digested using the respective restriction enzymes (BamH I and Sac I), and the ORF of

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CsSAMT

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pET-32a(+)–CsSAMT expression vector. After the ligation of the cloned fragment

119

was confirmed, the recombinant expression vector was transformed into Escherichia

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coli (BL21) (TransGen Biotech) for inducible His-tagged protein expression. For

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comparison, the pET-32a(+) vector served as a control. Transformed cells were

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cultured overnight at 37 °C in LB media (Lysogeny broth) containing ampicillin (100

123

µg/mL) 11.

was

ligated

into

pET-32a(+)

to

obtain

the

recombinant

124

The supernatants from the disrupted cells were used to measure the activity of

125

recombinant pET-32a(+)–CsSAMT. The reaction mixture for the enzyme activity

126

determination was prepared using the following components: MgCl2 (200 mM), 20

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µL; SAM (S-adenosyl-L-methionine, 5 mM), 50 µL; adenosine triphosphate (1 mM),

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10 µL; dithiothreitol (400 mM), 10 µL; one of substrates (SA, benzoic acid,

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cinnamic acid, vanillic acid, caffeic acid, and jasmonic acid), 100 mM, 100 µL; and

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crude proteins, 810 µL. The mixture was adjusted to a final volume of 1.0 mL and

131

then incubated at 30 °C for 1 h. The activity of recombinant pET–32a(+)-CsSAMT

132

was assayed and analyzed using GC–MS. 7



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The extract was injected into an Agilent 7890A/5975C GC–MS instrument. When

134

SA and benzoic acid were used as substrates, the following GC–MS temperature

135

program was applied: the initial column temperature was set at 50 °C for 5 min,

136

increased to 180 °C at a rate of 2 °C/min and maintained for 1 min, and then

137

increased to 250 °C at a rate of 10 °C/min and maintained for 2 min

138

cinnamic acid, vanillic acid, caffeic acid, and jasmonic acid were used as substrates,

139

the initial column temperature was set at 60 °C for 3 min.

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Purification of the recombinant protein and activity assay

141

10

. When

The protein was purified using the method reported previously 8. The recombinant TM

142

histidine-tagged protein was purified using the HisTrap

HP (GE Healthcare Life

143

Sciences, Marlborough, MA, USA) according to the manufacturer’s instructions. The

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purified protein was used for further enzymatic assays. For the later optimum pH and

145

temperature determination, reactions were carried out according to previous report 8.

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For the pH optima, assays were performed at 30 °C for 30 min using 100 mM PBS

147

buffer with pH levels ranging from 6.0 to 8.0. For the temperature optima, reactions

148

were performed at pH 7.5 for 30 min, with the temperature ranging from 10 to 60 °C.

149

The enzyme assay was performed according to the same procedure in the former

150

section.

151

Construction of CsSAMT expression vector, eukaryotic expression and enzyme

152

assay

153

The PCR specific primers TZF and TZR with EcoR I and Spe I restriction enzyme

154

sites were designed based on the sequence of CsSAMT by using Primer Premier 5.0. 8


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One pair of primers GALI-F and GALI-R was used to identify the positive clones of

156

the pESC-His vector. Primer sequences were shown in Supplemental table 1. The

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plasmid containing the sequence of CsSAMT and the pESC-His vector were

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digested using the respective restriction enzymes (EcoR I and Spe I), and the ORF of

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CsSAMT was inserted into the pESC-His vector to obtain the recombinant

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pESC-His–CsSAMT expression vector. After the confirmation of the ligated target

161

fragment, the recombinant expression vector was transformed into the competent

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yeast strain InVscI for expression.

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For comparison, the pESC-His vector was used as a control. Transformed cells

164

were cultured overnight at 30 °C in SD (Synthetic Dropout Medium)-His medium

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containing 2% glucose, and the cells were further cultured at 30 °C for 24 h 12. The

166

enzyme assay was performed according to the same procedure used for the enzyme

167

assay of CsSAMT in E. coli, except that the only substrate used was SA. The GC–MS

168

condition was the same as that applied when SA served as the substrate.

169

Construction of transformation vectors and transient expression in tobacco

170

Specific Gateway primers GTF1 and GTP2 based on the sequence of the amplified

171

cDNA fragment of CsSAMT were designed using Primer Premier 5.0 and were then

172

synthesized by Sangon (primer sequences were shown in Supplemental table 1). The

173

mixture for the PCR was prepared using the following components: ddH2O, 16 µL;

174

Prime STAR Max Premix, 30 µL; GTF primer (10 µM), 1.0 µL; GTR primer (10 µM),

175

1.0 µL; and the plasmids containing the cDNA sequence of CsSAMT, 2.0 µL. The

176

specific fragments were purified from agarose gels by using the AxyPrep DNA Gel 9



177

Extraction Kit (Axygen).

178

The purified cDNA was cloned into the entry vector pDONR207 using the

179

Gateway method. After the confirmation, the entry vector was carried out to introduce

180

the ORF into the destination vector pK7GWF2.0 containing the enhanced green

181

fluorescent protein (EGFP) gene. The recombinant expression vector and only the

182

pK7GWF2.0 vector used as the control were separately transferred to the

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Agrobacterium tumefaciens strain EHA105 through electroporation. The tobacco leaf

184

disks were cut into 0.5 cm × 0.5 cm pieces by using a blade, and the expression of

185

EGFP was then observed under a confocal laser scanning microscope (Olympus,

186

Japan) 13.

187

DNA sequence of CsSAMT

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Total genomic DNA was isolated from two leaves with one bud of the tea plant C.

189

sinensis Shuchazao according to the SDS method 14. Based on the cDNA sequence of

190

CsSAMT, pairs of specific primers were designed using Primer Premier 5.0. The

191

primer sequences were shown in Supplemental table 1. The mixture for the PCR was

192

prepared using the following components: ddH2O, 10 µL; dNTP (2 mM), 10 µL;

193

KOD FX, 1.0 µL; 2× PCR Buffer, 25 µL; primers (10 µM), 1.5 µL each; and DNA,

194

1.0 µL. The PCR was run using the following program: denaturing at 94 °C for 2 min;

195

30 cycles of denaturing at 94 °C for 10 s; annealing at Tm; extension at 68 °C for 1

196

min/kb; and final extension at 72°C for 10 min. Each reaction tube was run on a 1.0%

197

agarose gel, and the specific bands were sequenced subsequently.

198

Ligation of genomic DNA to GenomeWalker™ adaptors 10


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199

The total genomic DNA isolated from the tea leaves, as described in the preceding

200

section, was applied to amplify the promoter of CsSAMT by using genome walking

201

with the BD GenomeWalker™ Kit of Clontech. The total genomic DNA was digested

202

by the restriction enzymes Stu I, Pvu II, EcoR V, and Dra I separately

203

reaction tube was run on a 1.0% agarose gel to determine whether digestion was

204

complete. A total of four ligation reactions were performed to set up four libraries:

205

DNA library-Stu I, DNA library-Pvu II, DNA library-EcoR V, and DNA library-Dra

206

I.

207

Cloning the promoter of CsSAMT

15

. Each

208

One pair of specific primers (GSP1 and GSP2) was designed using Primer Premier

209

5.0 after bioinformatics analysis of the DNA sequence of CsSAMT, and the primers

210

were then synthesized by Sangon. The adaptor primers were AP1 and AP2 provided

211

by BD GenomeWalker™ Kit. The 5’ region flanking the DNA sequence of CsSAMT

212

was amplified using nested-PCR with the primers of GSP1/AP1 followed by

213

GSP2/AP2 16. The primer sequences were shown in Supplemental table 1.

214

The primary PCR was performed separately using 2 µL of DNA library-Stu I, DNA

215

library-Pvu II, DNA library-EcoR V, and DNA library-Dra I as templates and 10 µM

216

primer GSP1/AP1 in a 25-µL reaction volume. After primary PCR, the second

217

nested-PCR was performed using the same procedure as that for the first PCR.

218

However, different templates and primers were used: 2.0 µL of the first PCR product

219

diluted 50 times and 10 µM primer GSP2/AP2 were added to the second reaction

220

mixture. Each reaction tube was run on a 1.2% agarose gel to determine whether 11



221

specific bands were present. Next, the specific fragments were purified from agarose

222

gels by using an AxyPrep DNA Gel Extraction Kit (Axygen). The purified amplified

223

fragment was cloned into the pEASY-Blunt vector, and the positive clone was

224

obtained through sequencing and identifying.

225

Quantitative analysis using real-time quantitative PCR

226

Total RNA was extracted from different tea organs (bud, first leaf, second leaf,

227

old leaf, stem, and root) by using the RNA Prep Pure Plant Kit (Axygen). cDNA was

228

transcribed using the PrimeScript RT Reagent Kit (Takara, Japan). Real-time

229

quantitative PCR (qRT-PCR) was performed using 2 µL of cDNA and 0.4 µM of

230

each primer in a 20-µL reaction volume with SYBR Premix Ex Taq™ II (Takara,

231

Japan). GAPDH (GAPDHF and GAPDHR) was used as an internal control, and the

232

specific primers (QTF and QTR) used for qRT-PCR analysis were designed based

233

on the ORF of CsSAMT. The primer sequences were shown in Supplemental table 1.

234

Each PCR was performed in three replicates, and the expression level in the third

235

leaf was used as a control. According to the threshold cycle (Ct), the changes in gene

236

expression were quantiﬁed using the 2-∆∆Ct method 11.

237 238

Results and discussion

239

Variation of MeSA in tea leaves during the withering for white tea processing

240

White tea has a unique processing method: fresh tea leaves → withering (sun

241

withering or indoor withering) → drying (air drying, solar drying, or machine drying)

242

→ white tea 17. The processing steps of panning, rolling and shaking are not required 12


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243

for white tea, thus the withering process exerts an important effect on aroma

244

formation for white tea. In this study, the retention index of MeSA was 1191, which

245

was calculated against those of n-paraffin. And the identification of the MeSA in tea

246

samples was confirmed by the reported retention index (1190) 18, the retention time of

247

the standard of MeSA, the mass spectrum, and the total ion chromatogram of the

248

MeSA standard. We found MeSA content increased almost linearly with the withering

249

time (Figure 2), and the MeSA content was 2.5-fold higher in tea samples withered

250

for 72 h than withered for 36 h. After 72 h withering, MeSA content in the samples

251

was approximately 1.08 µg/g (calculated from the standard curve of MeSA,

252

supplemental figure 2A). However, MeSA was not detected in the control tea samples,

253

whose fresh leaves were directly treated by steam fixation, but could be detected in

254

fresh tea leaves (trace amount, data not shown). The hardly detectable amount of

255

MeSA in the directly fixed tea samples was probably due to the high temperature

256

during steam fixation might evaporate the trace amount of MeSA. MeSA is an

257

important component of white tea aroma; the mechanism underlying the formation of

258

MeSA should be elaborated 4. During the withering process, the formation of MeSA

259

can be catalyzed by SAMT. In this study, a CsSAMT isolated from tea leaves and the

260

encoded protein was also characterized.

261

cDNA sequence of CsSAMT and phylogenetic relationship analysis

262

After the bioinformatic analysis from our tea transcriptome database, an 1104-bp

263

fragment of CsSAMT was amplified from tea leaves by using the specific primers

264

(Figure 3). The fragment with the initiation codon ATG and the termination codon 13



265

TAG was acquired after PCR amplification and sequencing. The sequence was

266

submitted to NCBI and GenBank accession No. MG459470 was assigned afterwards.

267

The deduced translation product of CsSAMT consisted of 367 amino acids, with a

268

molecular weight of 41297.38 Da, a GC content of 43.03%, and a theoretical

269

isoelectric point of 5.224. Regarding phylogenetic analysis, the sequence alignment

270

of presently known SAMTs by ClustalW revealed 42%–100% homology to other

271

SAMTs at the amino acid level (Figure 4). The amino acid sequence of CsSAMT

272

showed the highest homology with SAMTs from C. japonica. The SAMT from C.

273

breweri was shown to have a crystal structure, which led to the influence of the

274

dimer formation by its N-terminal sequence. Many conserved residues were found at

275

the N- and C-termini, and the C-terminal domain was demonstrated to be primarily

276

involved in substrate binding

277

SAM/SAH-binding residues in CsSAMT, because of SAM-binding sites are highly

278

conserved, while the Q-25, SSYSLMW, MRAV, and IW residues are amino acid

279

side chains in contact with SA in SAMT and substrates in other members of this

280

methyltransferase family 19, 21.

281

Expression of CsSAMT in E. coli and GC-MS analysis of enzyme products

19, 20

. The S-22, D-57, SF, and DL residues are

282

To confirm the enzyme activity of CsSAMT encoded by the cDNA sequence of

283

CsSAMT, recombinant pET-32a(+)–CsSAMT was expressed in E. coli and was

284

analyzed using an in vitro functional enzyme assay; the pET-32a(+) vector was used

285

as the control. The putative ORF of CsSAMT in E. coli was induced by adding IPTG

286

(1.0 mM) and incubating at various temperatures (16, 28, and 37°C). The analysis 14


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287

showed that inducing the expression of recombinant pET-32a(+)–CsSAMT resulted

288

in the production of a protein with a molecular weight of approximately 60 kD,

289

which was consistent with the molecular weight predicted by DNAStar software

290

(Figure 5). In the reaction of MeSA formation by SMAT, SA was used as the

291

substrate, and SMAT was derived from bacterial lysates containing the recombinant

292

protein pET-32a(+)–CsSAMT after induction. After that, GC–MS was used to

293

identify

294

pET-32a(+)–CsSAMT. The identification of the MeSA was confirmed by the

295

reported retention index

296

spectrum, and the total ion chromatogram of the MeSA standard. In our research,

297

volatile products catalyzed by CsSAMT were verified to MeSA and methyl benzoate

298

by GC–MS determination (Figure 6A and 6B). For investigating substrate specificity,

299

SA was replaced with the following substrates: benzoic acid, cinnamic acid, vanillic

300

acid, caffeic acid, and jasmonic acid. However, the enzyme activity of CsSAMT was

301

only detected when benzoic acid as substrate, and generated a small amount product

302

of methyl benzoate (Figure 6C). Methyltransferases can transfer a methyl group

303

from a methyl donor, S-adenosyl-L-methionine, to the carboxyl group or ring

304

nitrogen of a substrate, forming S-adenosyl-L-homocysteine and methyl esters or

305

N-methylated compounds 22. The family of SAMTs has a preference for SA 23, and

306

the enzyme activity of CsSAMT was verified by the substrates of SA and BA, with

307

SAM as methyl donor in this research.

308

Enzyme assay of purified protein, pH and temperature optima

the

metabolites

produced

upon

incubation

of

substrates

with

18

, the retention time of the standard of MeSA, the mass

15



309

After the expression of CsSAMT in E. coli, protein expressed in supernatant was TM

310

purified by HisTrap

HP. The purified CsSAMT was found in the SDS-PAGE

311

analysis (Fig. 7A), and the purified protein from the supernatant was subsequently

312

used for the enzyme assay. After the GC-MS analysis, the purified CsSAMT could

313

catalyze the biosynthesis of MeSA, but display a lower activity level compared to

314

the crude protein (Fig. 7C). CsSAMT had a pH optimum of 7.5. At pH 6.0-6.5,

315

activity of CsSAMT was not detected, while at pH 8.0 it was about 70% of optimal

316

activity, and at pH 7.0, it was less than 50% of the optimal activity. CsSAMT was

317

almost 100% stable for 30 min at 20-30 °C and 50% stable for 30 min at 10 °C, but

318

after 30 min incubation at higher than 40 °C it completely lost 70% activity

319

(supplemental figure 2B, C). Similar patterns of pH and temperature optima were

320

found in a SAMT from the annual California plant Clarkia breweri 8. It also showed

321

a pH optimum of 7.5 and 75% at pH 8.0, while at pH 6.0, activity was still 80% of

322

the optimal activity. And this SAMT was 100% stable for 30 min at 20 °C and 80%

323

stable for 30 min at 30 °C, and after 30 min incubation at 40 °C it completely lost

324

activity 8.

325

Expression of CsSAMT in yeast and GC-MS analysis of enzyme products

326

Since the eukaryotic expression system has not been employed to express SAMTs

327

up to now, the expression of the recombinant CsSAMT in yeast was performed,

328

together with a control of the pESC-His vector in this study. The pESC vectors

329

containing the GAL1 and GAL10 yeast promoters in opposing orientation are a series

330

of epitope-tagging vectors designed for the expression and functional analysis of 16


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Page 17 of 42


331

eukaryotic genes in S. cerevisiae. The gene of interest can be inserted in front of the

332

epitope sequence to generate C-terminal tagging or can be inserted after the epitope

333

sequence for N-terminal tagging 24.

334

In our established yeast system using S. cerevisiae to validate the in vitro function

335

of CsSAMT involved in aroma compound formation, SA was used as a substrate in

336

this enzymatic reaction. The identification of MeSA formation was confirmed by the

337

reported retention index

338

spectrum, and the total ion chromatogram of the MeSA standard. Compared with

339

CsSAMT expressed in E. coli, only a smaller amount of MeSA with no methyl

340

benzoate was generated by the recombinant CsSAMT in yeast (Supplemental figure

341

1).

342

Subcellular localization of CsSAMT

18

, the retention time of the standard of MeSA, the mass

343

In order to investigate the subcellular localization of CsSAMT, the fragment of

344

CsSAMT was transferred into vectors containing compatible recombination sites (attB

345

× attP or attL × attR) in reactions mediated by Gateway

346

recombination vector and the empty vector pK7GWF2.0 used as a control were

347

transformed into tobacco leaf disks through Agrobacterium-mediated transformation.

348

EGFP-fused CsSAMT was observed in the chloroplast of the tobacco leaf cell.

349

However, EGFP-fused pK7GWF2.0 was observed at the tobacco leaf cell edge

350

(Figure 8). The immunogold localization of the BAMT protein in Snapdragon petals

351

provided in vivo evidence for its cytosolic location

352

methyltransferases are cytoplasmic enzymes, while our subcellular localization

13

. Subsequently, the

25

, suggesting that carboxyl

17



Page 18 of 42

353

investigations revealed that CsSAMT was located in the chloroplast in the tobacco

354

leaf cell. The different subcellular localization was probably due to distinct functions

355

of the enzymes in different species.

356

DNA and promoter sequences of CsSAMT

357

The DNA sequence of CsSAMT was amplified and was found to comprise four

358

exons and three introns (Table 1). The identified DNA sequence is similar to that of

359

N-methyltransferase gene families 26.

360

For obtaining the promoter sequence, genome walking is a widely used method 15.

361

It is crucial to use an appropriate genome walker and design appropriate nested

362

gene-specific primers. For a more effective nested-PCR, the primers GSP1 and GSP2

363

used in our cloning procedure were designed based on the DNA sequence of CsSAMT,

364

and finally a 2675-bp fragment was targeted after the nested-PCR amplification

365

The DNA and promoter sequences were submitted to NCBI and GenBank accession

366

No. MG459471 was assigned afterwards. Bioinformatics analysis of this 2675-bp

367

sequence

368

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

369

cis-acting enhancers were associated with various functional elements. The cis-acting

370

element conferred high transcription ability; moreover, the 5UTR Py-rich stretch,

371

CAAT box, and TATA (TATAAAT) box, in addition to other special motifs or

372

elements, conferred functions such as SA responsiveness, MeJA responsiveness, light

373

responsiveness, defense, and stress responsiveness. These elements are listed in

374

Supplemental table 2. Some previous reports have shown that SAMT genes are

through

18


16

.

PlantCARE

Page 19 of 42


27-29

375

involved in defense and biotic or abiotic stresses

, and the expression of SAMT

376

genes is thought to be induced by mechanical wounding and treatment with SA and

377

methyl jasmonate

378

the damage of Ectropis oblique 32. In addition, the expression and enzyme activity of

379

SAMTs from Antirrhinum majus, Stephanotis floribunda, and Nicotiana suaveolens

380

have been shown to oscillate during a light/dark cycle 23, 25, 33, 34.

381

Analysis of CsSAMT expression in different tea organs using qRT-PCR

30, 31

. In C. sinensis, the expression of SAMT could be induced by

382

The expression levels of CsSAMT in different tea organs (bud, first leaf, second leaf,

383

old leaf, stem, and root) were detected using qRT-PCR, and the expression level in the

384

old leaf was used as a control (Figure 9). The results showed that the maximum

385

expression levels of CsSAMT were found in the second leaf, whereas the expression in

386

old leaf was the lowest, together with the expression levels of CsSAMT in the bud and

387

first leaf were almost similar. The high expression levels of CsSAMT in young tea

388

leaves, might lead to the high content of MeSA, which can protect tender tissues from

389

insects 35.

390

The traditional manufacturing process of white tea is only two stages of withering

391

and drying. After these two stages, the specific flavor and aroma were generated to

392

express the characteristic properties for white tea. As for a specific aroma compound

393

for white tea, the MeSA content was almost in a linear growth phenomenon during the

394

withering process. In order to investigate the enzymatic reaction for producing MeSA

395

by SAMT with SA in tea, a 1104-bp fragment of CsSAMT was amplified from tea

396

leaves and its enzyme activity of CsSAMT in prokaryotic and eukaryotic expression 19



397

systems was identified. The full length and promoter fragment of CsSAMT was

398

obtained after genome walking and nested-PCR amplification. The obtained results

399

might give a new understanding for tea aroma formation, provide a further study for

400

tea aroma optimization and regulation, and have great significance for improving the

401

specific quality for tea, especially for white tea. For the further investigation, it would

402

play an important role in plant defense mechanism, because MeSA is one of the

403

volatile organic compounds and releases the floral scent.

404 405

Funding sources

406

This study was supported by the Natural Science Foundation of Anhui Province

407

project 1608085QC60, the National Natural Science Foundation of China (NSFC)

408

project 31300576, 31570692, and the Changjiang Scholars and Innovative Research

409

Team in University (IRT_15R01).

410 411

Supporting Information Available

412

Supplemental table 1

413

The primer sequences for PCR amplification in this study. All the primers used

414

in this research were shown as the functions, the primer names and the sequences (5’

415

→ 3’).

416 417

Supplemental table 2

418

Regulatory elements in the cloned CsSAMT promoter sequence. The site name, 20


Page 20 of 42

Page 21 of 42


419

the organism, sequence and the function were shown.

420 421

Supplemental figure 1

422

GC-MS analysis of the volatile product of recombinant CsSAMT in yeast

423

(Saccharomyces cerevisiae). The established yeast system using S. cerevisiae to

424

validate the in vitro function of CsSAMT involved in aroma compound formation,

425

SA was used as a substrate in this enzymatic reaction.

426

A, Partial GC chromatogram from a reaction with salicylic acid as the substrate and

427

lysates of cells with the recombinant pESC-His–CsSAMT after galactose induction;

428

B, Partial GC chromatogram from a reaction with salicylic acid as the substrate and

429

lysates of cells with the recombinant pESC-His after galactose induction; C, GC

430

chromatogram of the MeSA standard. GC mass spectrum of the characteristic peak

431

was identified as that of MeSA, as indicated by the arrow (retention time = 26.7 min);

432

D, GC mass spectrum of enzymatic product of recombinant pESC-His–CsSAMT; E,

433

methyl salicylic standard, the authentic standard spectrum of methyl salicylic.

434 435

Supplemental figure 2

436

The standard curve of methyl salicylate and the detection of the optima pH and

437

temperature. For the pH optima, assays were performed at 30 °C for 30 min using

438

100 mM PBS buffer with pH levels ranging from 6.0 to 8.0. For the temperature

439

optima, reactions were performed at pH 7.5 for 30 min, with the temperature ranging

440

from 10 to 60 °C. Data represent the means ± standard deviation (n = 3). 21



Page 22 of 42

441

A, The standard curve of methyl salicylate established by GC-MS; B, The enzyme

442

assays performed at 30 °C with different pH ranging from 6.0–8.0 for 30 min and

443

determined by GC-MS; C, The enzyme assays performed at pH 7.5 with different

444

temperatures ranging from 10–60 °C for 30 min and determined by GC-MS.

445 446

References

447

1.

448

Insect-Plant Interactions; Bernays, E. A.; CRC Press: Boca Raton, Florida, USA,

449

1994; Vol. 5, 47-82.

450

2. Hippauf, F.; Michalsky, E.; Huang, R.; Preissner, R; Barkman, T. J.; Piechulla, B.

451

Enzymatic,

452

o-methyltransferases

453

Plant Mol. Biol. 2010, 72(3), 311-330.

454

3. Sharma, P.; Ghosh, A.; Tudu, B.; Bhuyan, L. P.; Tamuly, P.; Bhattacharyya, N.;

455

Bhattacharyya, R. Detection of linalool in black tea using a quartz crystal

456

microbalance sensor. Sens. Actuators, B. 2014, 190(190), 318-325.

457

4. Yu, S. J.; Li, X. L.; Wang, T. T.; Jin, X. Y. Research progress on white tea flavor

458

and its withering processing. J. Tea Comm. 2015, 42(4), 14-18.

459

5. Zhao, Y. Q.; Zhou, S. J.; Peng, P. H.; Pan, H. T.; Zhang, Q. X. Research advances

460

in metabolic regulation and genetic engineering of floral scent. J. Trop. Subtrop. Bot.

461

2011, 19(4), 381-390.

462

6. Ying, K.; Ming, S.; Pan, H. T. Advances in metabolism and regulation of floral

Dobson,

H

E

M.

expression

Floral

and

after

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structural duplication

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divergences and

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scent. J. Beijing Forestry University, 2012, 34(2), 146-154.

464

7. Zhang, Q.; Tian, Y. Y.; Meng, Y.; Li, Y. M.; Wang, H. J.; Wang, L. M. Research

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advance in genetic engineering of floral fragrance. J. Henan Agric. Sci. 2014, 43(4),

466

11-16.

467

8. Ross, J. R.; Nam, K. H.; D'Auria, J. C.; Pichersky, E. S-Adenosyl-L-methionine:

468

salicylic acid carboxyl methyltransferase, an enzyme involved in floral scent

469

production and plant defense, represents a new class of plant methyltransferases.

470

Arch. Biochem. Biophys. 1999, 367(1), 9-16.

471

9. Zhu, M.; Li, E.; He, H. Determination of volatile chemical constitutes in tea by

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simultaneous distillation extraction, vacuum hydrodistillation and thermal desorption.

473

Chromatographia. 2008, 68(7), 603-610.

474

10. Pott, M. B.; Hippauf, F.; Saschenbrecker, S.; Chen, F.; Ross, J.; Kiefer,

475

I.; Slusarenko, A.; Noel, J. P.; Pichersky, E.; Effmert, U.; Piechulla, B. Biochemical

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and structural characterization of benzenoid carboxyl methyltransferases involved in

477

floral scent production in Stephanotis floribunda and Nicotiana suaveolens. Plant

478

Physiol. 2004, 135(4), 1946-55.

479

11. Deng, W. W.; Wu, Y. L.; Li, Y. Y.; Tan, Z.; Wei, C. L. Molecular cloning and

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characterization of hydroperoxide lyase gene in the leaves of tea plant (Camellia

481

sinensis). J. Agric. Food Chem. 2016, 64(8), 1770-1776.

482

12. Zhou, Y.; Zeng, L. T.; Gui, J. D.; Liao, Y. Y.; Li, J. L.;Tang, J. C.; Qing, M.; Fang,

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D.;Yang, Z. Y. Functional characterizations of β-glucosidases involved in aroma

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compound formation in tea (Camellia sinensis). Food Res. Int. 2017, 96, 206-214. 23



Karimi,

M.;

Inzé,

D.;

Depicker,

A.

GATEWAY(TM)

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Agrobacterium-mediated plant transformation. Trends Plant Sci. 2002, 7(5),

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193-195.

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14. Chen, L.; Chen, D.; Gao, Q.; Yang, Y.; Yu, F. Isolation and appraisal of genomic

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DNA from tea plant [Camellia sinensis (L.) O. Kuntze]. J. Tea Sci. 1997, 17(2),

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177-181.

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15. Laboratories C. GenomeWalker™ Universal Kit User Manual. Clontech

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Laboratories, 2007.

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16. Satyanarayana, K. V.; Kumar, V.; Chandrashekar, A.; Ravishankar, G. A. Isolation

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of promoter for N-methyltransferase gene associated with caffeine biosynthesis in

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Coffea canephora. J. Biotechnol. 2005, 119(1), 20-25.

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17. Guo, L.; Guo, Y. L.; Liao, Z. M.; Lin, Z. Research advance in aroma components

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of white tea. J. Food Saf. Food Qual. 2015, 6(9), 3580-3586.

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18. Pino, J.; Sauri-Duch, E.; Marbot, R. Changes in volatile compounds of Habanero

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chile pepper (Capsicum chinense, Jack. cv. Habanero) at two ripening stages. Food

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Chem. 2006, 94(3), 394-398.

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19. Zubieta, C.; Ross, J. R.; Koscheski, P.; Yang, Y.; Pichersky, E.; Noel, J. P.

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Structural basis for substrate recognition in the salicylic acid carboxyl

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methyltransferase family. Plant Cell. 2003, 15(8), 1704-16.

504

20. Noel, J. P.; Dixon, R. A.; Pichersky, E.; Zubieta, C.; Ferrer, J. L. Chapter two

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Structural, functional, and evolutionary basis for methylation of plant small

506

molecules. Recent Adv. Phytochem. 2003, 37(03), 37-58. 24


vectors

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21. Koeduka, T.; Kajiyama, M.; Suzuki, H.; Furuta, T.; Tsuge, T.; Matsui, K.

508

Benzenoid biosynthesis in the flowers of Eriobotrya japonica: molecular cloning

509

and

510

methyltransferase. Planta. 2016, 244(3), 1-12.

511

22. Huang, R. Studies on enzyme functional evolution in the SABATH multigene

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family using phylogenetic and biochemical approaches. Master's Theses. 2012, 52.

513

23. Effmert, U.; Saschenbrecker, S.; Ross, J.; Negre, F.; Fraser, C. M.; Noel, J. P.;

514

Dudareva, N. Piechulla, B. Floral benzenoid carboxyl methyltransferases: From in

515

vitro to in planta function. Phytochemistry. 2005, 66(11), 1211-30.

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24. Sung, H.; Han, K. C.; Kim, J. C.; Oh, K. Wan.; Yoo, H. S.; Hong, J. T.; Chung, Y.

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B.; Lee, C. k.; Lee, K. S.; Song, S. A set of epitope-tagging integration vectors for

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functional analysis in Saccharomyces cerevisiae. FEMS Yeast Res. 2005, 5(10), 943.

519

25. Kolosova, N.; Sherman, D.; Karlson, D.; Dudareva, N. Cellular and subcellular

520

localization of S-Adenosyl-L-Methionine: Benzoic acid carboxyl methyltransferase,

521

the enzyme responsible for biosynthesis of the volatile ester methylbenzoate in

522

Snapdragon flowers. Plant Physiol. 2001, 126(3), 956-64.

523

26. Jin, J.; Yao, M.; Chunlei, M. A.; Jiangqiang, M. A.; Liang, C. Cloning and

524

sequence analysis of the N-methyltransferase gene family involving in caffeine

525

biosynthesis of tea plant. J. Tea Sci. 2014, 34(2), 188-194.

526

27. Fukami, H.; Asakura, T.; Hirano, H.; Abe, K.; Shimomura, K.; Yamakaw, T.

527

Salicylic acid carboxyl methyltransferase induced in hairy root cultures of Atropa

528

belladonna after treatment with exogeneously added salicylic acid. Plant Cell

functional

characterization

of

p-methoxybenzoic

25


acid

carboxyl


529

Physiol. 2002, 43(9), 1054-8.

530

28. Negre, F.; Kolosova, N.; Knoll, J.; Kish, C. M.; Dudareva, N. Novel

531

S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase, an enzyme

532

responsible for biosynthesis of MeSA and methyl benzoate, is not involved in floral

533

scent production in Snapdragon flowers. Arch. Biochem. Biophys. 2002, 406(2),

534

261-270.

535

29. Chen, F.; D'Auria, J. C.; Tholl, D.; Ross, J. R.; Gershenzon, J.; Neol, J. P.;

536

Pichersky, E. An Arabidopsis thaliana gene for methylsalicylate biosynthesis,

537

identified by a biochemical genomics approach, has a role in defense. Plant J. Cell

538

Mol. Biol. 2003, 36(5), 577-88.

539

30. Van Poecke, R. M.; Posthumus, M. A.; Dicke, M. Herbivore-induced volatile

540

production by Arabidopsis thaliana leads to attraction of the parasitoid Cotesia

541

rubecula: chemical, behavioral, and gene-expression analysis. J. Chem. Ecol. 2001,

542

27(10), 1911-1928.

543

31. Shulaev, V.; Silverman, P.; Raskin, I. Airborne signalling by methyl salicylate in

544

plant pathogen resistance. Nature. 1997, 385(6618), 718-721.

545

32. Cao, S. X.; Cheng, X.; Jiang, Z. Z.; Sheng, L.; Shangguan, M. Z.; Deng, W. W.;

546

Wei, C. L.; Differential genes expression in tea plant (Cameilla sinensis L.) induced

547

by Ectropis oblique feeding based on cDNA-AFLP. Chin. Agric. Sci. 2013, 46(19),

548

4119-4130.

549

33. Pott, M. B.; Effmert, U. T. A.; Piechulla, B. Transcriptional and post-translational

550

regulation of S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase 26


Page 26 of 42

Page 27 of 42


551

(SAMT) during Stephanotis floribunda, flower development. J. Plant Physiol. 2003,

552

160(6), 635-643.

553

34. Altenburger, R.; Matile, P. Circadian rhythmicity of fragrance emission in

554

flowers of Hoya carnosa R. Br. Planta. 1988, 174(2), 248

555

35. Miao, J.; Li, G. P.; Han, B. Y. Recent developments in effect and mechanism of

556

salicylic acid and methyl salicylate on plant resistance to pests. Chin. J. Trop. Crops.

557

2007, 28(1), 111-114.

558 559

Figure captions

560

Figure 1. Partial pathway for the biosynthesis of phenylpropanoid/benzenoids.

561

The red box indicates the biosynthesis of methyl salicylic from salicylic acid.

562 563

Figure 2. Variation of methyl salicylic obtained in tea leaves during the

564

withering for white tea processing.

565

The content of the target analyte at different wilting times (6, 12, 24, 36, and 72 h)

566

was calculated based on the standard curve (supplemental figure 2A). “0 h”

567

corresponds to the sample directly fixed using steam (the control). Data represent the

568

mean ± standard deviation of three independent experiments.

569 570

Figure 3. Agarose gel electrophoresis of PCR amplification product.

571

M: Marker; l–5: PCR amplification product obtained using cDNA as a template.

572 27



573

Figure 4. Phylogenetic tree of amino acid sequences of SAMT in different plant

574

species.

575

Phylogenetic analysis of CsSAMT and related, functionally characterized SAMTs in

576

the following plants: Medicago truncatula, Antirrhinum majus, Camellia japonica,

577

Populus x beijingensis, Populus tomentosa, Clarkia breweri, Hoya carnosa,

578

Stephanotis floribunda, Atropa belladonna, Datura wrightii, Solanum lycopersicum,

579

Nicotiana alata, Nicotiana benthamiana, Nicotiana suaveolens, and Oryza sativa. The

580

neighbor-joining phylogenetic tree was generated using MEGA 6.0 software. The

581

proteins included in the tree are represented by their GenBank accession number.

582 583

Figure 5. SDS-PAGE analysis of recombinant CsSAMT expressed in E. coli.

584

CsSAMT was ligated into the pET-32a(+) vector and expressed in E. coli [BL21

585

(DE3)]. After IPTG-induced protein expression, all cells or cell lysates after

586

sonication were subjected to SDS-PAGE. The gel was stained using Coomassie blue

587

R-250. M: protein molecular mass marker; 1: total crude protein from uninduced

588

cells was transformed using the pET-32a(+) vector alone; 2: total crude protein from

589

induced cells was transformed using the pET-32a(+) vector alone; 3: total crude

590

protein from uninduced cells was transformed using pET-32a(+)–CsSAMT; 4: total

591

crude protein from induced cells was transformed using pET-32a(+)–CsSAMT; 5:

592

suspension of total crude protein from induced cells was transformed using

593

pET-32a(+)–CsSAMT after sonication; and 6: precipitation of total crude protein

594

from induced cells was transformed using pET-32a(+)–CsSAMT after sonication. 28


Page 28 of 42

Page 29 of 42


595

The bands of pET-32a(+)-CsSAMT are indicated by arrows in Lanes 4–6.

596 597

Figure 6. GC-MS analysis of the volatile product of recombinant CsSAMT in E.

598

coli.

599

A: Aa, pET-32a(+)–CsSAMT: Partial GC chromatogram from a reaction with

600

salicylic acid as the substrate and lysates of cells with the recombinant

601

pET-32a(+)–CsSAMT after IPTG induction; Ab, pET-32a(+): Partial GC

602

chromatogram from a reaction with salicylic acid as the substrate and lysates of cells

603

with the recombinant pET-32a(+) after IPTG induction; Ac, standard: GC

604

chromatogram of MeSA. GC mass spectrum of the characteristic peak was identified

605

as that of MeSA, as indicated by the arrow (retention time = 26.7 min); Ad,

606

pET-32a(+)–CsSAMT: GC mass spectrum of enzymatic product of recombinant

607

pET-32a(+)–CsSAMT; Ae, methyl salicylic standard, the authentic standard

608

spectrum of methyl salicylic.

609

B: Ba, pET-32a(+)–CsSAMT: Partial GC chromatogram from a reaction with

610

salicylic acid as the substrate and lysates of cells with the recombinant

611

pET-32a(+)–CsSAMT after IPTG induction; Bb, pET-32a(+): Partial GC

612


613

with the recombinant pET-32a(+) after IPTG induction; Bc, standard: GC

614

chromatogram of methyl benzoate. GC mass spectrum of the characteristic peak was

615

identified as that of methyl benzoate, as indicated by the arrow (retention time = 20

616

min); Bd, pET-32a(+)–CsSAMT: GC mass spectrum of enzymatic product of 29



617

recombinant pET-32a(+)–CsSAMT; Be, methyl benzoate standard, the authentic

618

standard spectrum of methyl benzoate.

619

C: Ca, pET-32a(+)–CsSAMT: Partial GC chromatogram from a reaction with

620

benzoate acid as the substrate and lysates of cells with the recombinant

621

pET-32a(+)–CsSAMT after IPTG induction; Cb, pET-32a(+): Partial GC

622


623

with the recombinant pET-32a(+) after IPTG induction; Cc, standard: GC

624

chromatogram of methyl benzoate. GC mass spectrum of the characteristic peak was

625

identified as that of methyl benzoate, as indicated by the arrow (retention time = 20

626

min); Cd, pET-32a(+)–CsSAMT: GC mass spectrum of enzymatic product of

627

recombinant pET-32a(+)–CsSAMT; Ce, methyl benzoate standard, the authentic

628

standard spectrum of methyl benzoate.

629 630

Figure 7. SDS-PAGE analysis of the purified CsSAMT and activity assay.

631

A, SDS-PAGE analysis of the recombinant CsSAMT expressed in E. coli and the

632

purified CsSAMT: lane 1, total crude protein from uninduced cells was transformed

633

using pET-32a(+)–CsSAMT; 2, total crude protein from induced cells was

634

transformed using pET-32a(+)–CsSAMT; 3, precipitation of total crude protein from

635

induced cells was transformed using pET-32a(+)–CsSAMT after sonication; 4,

636

suspension of total crude protein from induced cells was transformed using

637

pET-32a(+)–CsSAMT after sonication; 5, the purified CsSAMT from suspension of

638

total crude protein. The bands of pET-32a(+)-CsSAMT are indicated by arrows in 30


Page 30 of 42

Page 31 of 42


639

Lanes 3–5.

640

B, Partial GC chromatogram from a reaction with salicylic acid as the substrate

641

catalyzed by the purified-pET-32a(+).

642

C, Partial GC chromatogram from a reaction with salicylic acid as the substrate

643

catalyzed by the purified-CsSAMT.

644

D, Partial GC chromatogram of the MeSA standard. GC mass spectrum of the

645

characteristic peak was identified as MeSA, indicated by arrows (retention time =

646

26.7 min).

647

E, GC mass spectrum of enzymatic product of the purified-CsSAMT.

648

F, Methyl salicylic standard, the authentic standard spectrum of methyl salicylic.

649 650

Figure 8. Subcellular localization of CsSAMT in tobacco leaf disks.

651

Subcellular localization analysis of (a) pK7GWF2.0 and (b) pK7GWF2.0–CsSAMT;

652

(c) leaf containing pK7GWF2.0–CsSAMT was in a bright field; (d) leaf containing

653

pK7GWF2.0–CsSAMT was merged. EGFP: enhanced green fluorescent protein.

654 655

Figure 9. Relative expression levels of CsSAMT in different tea organs, as

656

determined through qRT-PCR.

657

GAPDH was used as an internal control. The expression levels of CsSAMT are

658

shown in the buds, first leaf, second leaf, old leaf, stem, and root. The expression

659

level in old leaf was used as the control. Data represent the means ± standard

660

deviation (n = 3). 31



Page 32 of 42

Table 1: The length of intron/exon in the genomic sequence of CsSAMT mismatch q-start

q-end

s-start

s-end

1

75

1

75

0

73

484

996

1406

262

3

478

739

1951

2212

368

0

737

1104

5975

6342

q-name

s-name

alignment

Exon 1

DNA

75

0

Exon 2

DNA

411

Exon 3

DNA

Exon 4

DNA

32


Page 33 of 42


Figure 1

33


The content of methyl salicylate in tea leaves ( µ g/g)


Page 34 of 42

1.5

1.0

0.5

0.0 0

6

12

24

36

72

The withering time (Hours)

Figure 2

34


Page 35 of 42


Figure 3

35



Page 36 of 42

Figure 4

36


Page 37 of 42


Figure 5

37



Ba

pET-32a(+)-CsSAMT

Aa

pET-32a(+)-CsSAMT

6000000

pET-32a(+)-CsSAMT

Ca

80000

Page 38 of 42

8000000

60000 6000000 20000

4000000

20000

26.7

0 10

20

4000000

20

40000 2000000

2000000

0 30

40

50

60

19.5 20 20.5

70

Ab pET-32a(+)

21

0

21.5

Bb pET-32a(+)

20 10

20

30

40

50

60

70

40

50

60

70

Cb pET-32a(+)

80000

6000000

8000000 60000

4000000

2000000

20000

6000000

40000

4000000

20000

0 10

20

Ac

30

26.7

50

40

60

70

19.5 20 20.5

Bc

Standard

10000000

4000000

21

0

21.5

0

30

20

40

50

10000000

6000000

8000000 6000000

2000000 19.5 20 20.5

70

21

0

21.5

pET-32a(+)-CsSAMT

pET-32a(+)-CsSAMT

Bd

120.0

Cd

105.0

9000

152.0

5000

65.0 50.0 20

40

60

137.0 80

100 120 140 160

m/z-->

Methyl salicylate standard

Ae

3000

51.0

m/z-->

20 40 60 80 100120140160180200

8000 92.0

6000

Methyl benzoate standard 105.0

20 m/z-->

136.0

119.9

207.0

20 40 60 80 100120140160180200 m/z-->

Ce

Methyl benzoate standard 105.0

9000

7000 152.0

65.0 41.0 53.0 81.0 40

60

80

7000

77.0

77.0 136.0

5000

4000

0

70

120.0 9000

2000

60

51.0

207.1 1000 0

1000 0

Be

50

77.0

5000

136.0

3000

0

40

105.0

77.0

4000 2000

30

pET-32a(+)-CsSAMT

7000

7000

92.0

20

9000

8000 6000

10

Retention (min)

Retention (min)

Retention (min)

Ad

Methyl benzoate

4000000

0

60

30

Standard

12000000

8000000

2000000 10

20 20

14000000

Methyl benzoate

4000000 2000000

10

Cc

20 Standard

12000000

Methyl salicylate

6000000

2000000

0

5000

136.0

3000

51.0 51.0 206.8 119.9 206.8 1000 119.9 1000 0 0 20 40 60 80 100 120 140 160 180 200 100 120 140 160 20 40 60 80 100120140160180200 m/z--> m/z--> 3000

109.0 137.0

Figure 6

38


Page 39 of 42


A

20

B

(kD)

700000

pET-32a(+)

AU

500000 26.7

300000 100000

60

20 20

C

25 25

35 35

30

700000

AU

50 40

pET-32a(+)-CsSAMT

500000 300000

26.7

100000

20

D

25

30

35

26.7

AU

6000000


4000000 2000000

1000000 0

20

25

30

35

Retention time (min)

pET-32a(+)-CsSAMT

E

120.0

AU

8000 6000

92.0

152.0

4000 65.0

2000 0 m/z-->

40

50

60

70

80

90

100

110

120

130

140

150

160


120.0

F AU

9000 92.0

7000

152.0

5000

3000 1000 0 m/z-->

65.0 44.0 40

53.0 50

81.0 60

70

80

136.9 90

100

110

120

130

140

150

160

Figure 7

39



Page 40 of 42

Figure 8

40


Page 41 of 42


Figure 9

41



Graphic for table of contents

42


Page 42 of 42

Functional characterization of salicylic acid carboxyl

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