Involvement of Three CsRHM Genes from Camellia sinensis in UDP

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Food and Beverage Chemistry/Biochemistry

Involvement of Three CsRHM Genes from Camellia sinensis in UDP-rhamnose Biosynthesis Xinlong Dai, Guifu Zhao, Tianming Jiao, Yingling Wu, Xinmin Li, Kan Zhou, Liping Gao, and Tao Xia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01870 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

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Involvement of Three CsRHM Genes from Camellia sinensis in UDP-rhamnose

2

Biosynthesis

3 4

Xinlong Dai 1, 2, Guifu Zhao 1, Tianming Jiao1, Yingling Wu1, Xinmin Li2

5

Kang Zhou2 Liping Gao2*and Tao Xia1*

6 7

1

8

230036, China

9

2

State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, Anhui

School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, China

10 11 12

Corresponding author:

13

Tao Xia, State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei,

14

Anhui 230036, China

15

Tel: 86-551-65786003, Fax: 86-551-65785833, E-mail: [email protected]; Liping Gao, School of Life

16

Science, Anhui Agricultural University, 130 West Changjiang Rd, Hefei, Anhui 230036 China

17

Tel: 86-551-65786232, Fax: 86-551-65785729, [email protected]

18

ORCID

19

Tao Xia: 0000-0003-0814-2567

20

Liping Gao: 0000-0002-0348-4608

21 22 23 24 25 26 27


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Abstract UDP-rhamnose synthase (RHM), the branch-point enzyme controlling the

30

nucleotide

sugar

interconversion

pathway,

converts

31

UDP-rhamnose. As a rhamnose residue donor, UDP-L-rhamnose is essential for the

32

biosynthesis of pectic polysaccharides and secondary metabolites in plants. In this

33

study, three CsRHM genes from tea plants (Camellia sinensis) were cloned and

34

characterized. Enzyme assays showed that three recombinant proteins displayed RHM

35

activity and were involved in the biosynthesis of UDP-rhamnose in vitro. The

36

transcript profiles, metabolite profiles, and mucilage location suggest that the three

37

CsRHM genes likely contribute to UDP-rhamnose biosynthesis and may be involved

38

in primary wall formation in C. sinensis. These analyses of CsRHM genes and

39

metabolite profiles provide a comprehensive understanding of secondary metabolite

40

biosynthesis and regulation in tea plants. Moreover, our results can be applied for the

41

synthesis of the secondary metabolite rhamnoside in future studies.

42

Keywords: UDP-rhamnose synthase, Camellia sinensis, recombinant protein, enzyme assays,

43

expression analysis

44 45 46 47 48 49 50 51 52 53 54 55


UDP-D-glucose

into


56

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1. Introduction

57

Tea is one of the three most popular nonalcoholic beverages worldwide, in which

58

the high content of phenolic compounds provides the health benefits.1 The flavonoids

59

in tea are the main flavor components and functional ingredients and include acylated

60

glycosylated flavonols, O-glycosylated flavonols, and C-glycosylated flavones.2

61

Flavonol glycosides, which are often converted from aglycones in a reaction catalyzed

62

by UDP-glycosyltransferases involving various nucleotide sugar donors (including

63

UDP-glucose, UDP-galactose, UDP-rhamnose, UDP-xylose, UDP-mannose, and

64

UDP-glucuronic acid),3-5 play a role in inducing a tea infusion’s silky, mouth-drying,

65

and mouth-coating sensation at very low threshold concentrations.6, 7 However, in tea

66

plants, the genes involved in the biosynthesis of nucleotide sugars such as

67

UDP-L-rhamnose (UDP-Rha) remain unknown. In addition, the new shoots of tea

68

plants are used as the raw material for making tea. The tenderness of the new shoots is

69

crucial for tea quality and is determined by the degree of lignification of the cell wall

70

and the content of pectin.

71

Pectins include three main classes of polysaccharides: homogalacturonan (HGA),

72

rhamnogalacturonan-I (RG-I), and rhamnogalacturonan-II (RG-II).8 HGA plays a

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critical role in pectin cross-linking, which is a component of A. thaliana mucilage

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consisting of individual (1,4)-α-linked galacturonic acid (GalA) residues.9-11 RG-I is

75

the primary component

76

(1,2)-α-L-rhamnosyl and (1,4)-α-GalA.8,12 RG-II, a dimer covalently cross-linked by a

of seed

mucilage,

consisting


of

the

alternating

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borate diester bond, is a component of a structurally complex polysaccharide that

78

exists in primary cell walls.13,14

79

As a rhamnose residue donor, UDP-Rha is required for the biosynthesis of pectic

80

polysaccharides of plant cell walls8 and various secondary metabolites (flavonoids,

81

terpenoids, and saponins) in plants.15 In tea shoots, numerous phenolic compounds

82

accumulate, including various flavonol glucosides and flavonol rhamnosides.16,17 Their

83

content is directly related to the bitter taste of tea. Nguema-Ona reported that

84

L-rhamnose participates in the biosynthesis of some O-linked glycoproteins involved

85

in growth, morphogenesis, and responses to various stresses.18

86

The biosynthesis of UDP-Rha occurs in bacteria, fungi, and plants. However, the

87

biosynthetic pathway of UDP-Rha in the new shoots of tea plants remains unclear. In

88

bacteria,

89

thymidylyltransferase; EC 2.7.7.24), RmlB (coding dTDPD-glucose 4,6-dehydratase;

90

EC 4.2.1.46), RmlC (coding dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase; EC

91

5.1.3.13), and RmlD (coding dTDP-6-deoxy-L-lyxo-4-hexulose reductase; EC

92

1.1.1.133) genes has been identified to be responsible for the biosynthesis of

93

dTDP-rhamnose (dTDP-Rha)19-22 (Fig. 1A). In fungi, the main nucleotide diphosphate

94

rhamnose form of UDP-Rha is synthesized in a two-step reaction catalyzed by two

95

enzymes:

96

UDP-4-keto-6-deoxyglucose-3,5-epimerase-4-reductase (U4k6dG-ER)23 (Fig. 1B).

a

gene

cluster

including

RmlA

(coding

UDP-glucose-4,6-dehydratase

glucose-1-phosphate

(UG4,6-Dh)

and

97

In plants, the biosynthesis of UDP-Rha is catalyzed by a trifunctional enzyme in

98

a three-step reaction, with UDP-D-glucose (UDP-Glc) as a substrate and NAD+ and



99

NADPH as cofactors14,24 (Fig. 1C). An excellent study in A. thaliana demonstrated

100

that three proteins of UDP-rhamnose synthase (RHM) are trifunctional enzymes that

101

convert UDP-Glc to UDP-Rha.25,26 Although biochemical evidence supporting the role

102

of RHM in UDP-Rha biosynthesis has been provided in A. thaliana,25 comprehensive

103

research on the function of RHM in vitro and in vivo is still lacking. Conversely, many

104

researchers are focusing on herbs such as A. thaliana but not on woody plants.

105

In this study, we cloned and characterized three homologous CsRHM genes from

106

tea plants (Camellia sinensis). Biochemical evidence showed that the proteins are

107

encoded by three CsRHM genes and exhibit RHM activity in vitro. Transcript profiles,

108

metabolite profiles, and mucilage location suggested that the CsRHM genes are likely

109

to be involved in the biosynthesis of UDP-Rha compounds and might participate in the

110

formation of the primary cell wall in tea plants.

111

2. Materials and Methods

112

2.1 Plant materials and chemicals

113

Tea plants (C. sinensis (L.) O. Kuntze cv. “Shuchazao”), including buds, first

114

leaves, second leaves, third leaves, fourth leaves, mature leaves, old leaves, young

115

stems, tender roots, and flowers, were collected from the experimental tea garden of

116

Anhui Agricultural University (Anhui, China) during early spring for use in this study.

117

Samples were divided into three parts: one part was used for analyzing the total RNA

118

extracted and gene expression, a second for determining the total nucleotide sugar

119

extracted, and a third for histochemical staining assays in tea plants.


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The nucleotide sugar donor UDP-Glc and ruthenium red were obtained from

121

Sigma-Aldrich (www.sigmaaldrich.com/israel.html). NAD+ and NADPH were

122

obtained from Solarbio (Shanghai, China). E.coli DH5α and BL21 (DE3) (TransGen

123

Biotech, Beijing, China) were used as the host strain and expression strain for

124

prokaryotic expression, respectively.

125

2.2 Analysis of CsRHM gene sequences

126

In this study, to screen the genes involved in the biosynthesis of UDP-Rha in tea

127

plants, three CsRHM sequences were identified from tea plant transcriptome data

128

using sequence homology search. In addition, using the same method, protein

129

sequences from other species (including bacteria and fungi) participating in the

130

biosynthesis

131

(https://www.ncbi.nlm.nih.gov/).

of

UDP-Rha

were

obtained

from

the

NCBI

website

132

The protein sequences of the three CsRHM genes were aligned with those of

133

corresponding genes from other species using DNAMAN 7.0 (Lynnon, Canada). An

134

unrooted phylogenetic tree was constructed using MEGA 5.0 based on the ClustalW

135

multiple alignment through the neighbor-joining method with 1000 bootstrap

136

replications.

137

2.3 cDNA cloning

138

Total RNA was extracted from tea plants using RNAiso-mate for Plant Tissue

139

(Takara, Dalian, China; Code: D325A) and RNAiso Plus (Takara, Dalian, China;

140

Code: D9108B), according to the manufacturer’s instructions. Subsequently, cDNA

141

was reverse transcribed from total RNA using PrimeScript RT Master Mix (Takara,



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142

Dalian, China; Cat: RR036A). The 5′-RACE and 3′-RACE based on ESTs from C.

143

sinensis (Bioproject ID PRJNA343349) were performed using a SMARTer™ RACE

144

cDNA Amplification Kit (Clontech, USA; Cat. Nos 634923 and 634924). The full

145

lengths of the three candidate genes were obtained by assembling them based on the 5′

146

and 3′ sequences. Subsequently, the ORF sequence of the three candidate genes was

147

amplified using Phusion® High-Fidelity DNA Polymerase (New England Biolabs,

148

MA, USA) with gene-specific primers from C. sinensis. To determine the exact

149

sequences, the polymerase chain reaction (PCR)-amplified product was cloned into the

150

PESY-T1 vector (TransGen, Beijing, China). All primer sequences used in this study

151

are listed in Table S1.

152

2.4 Heterologous expression and recombinant protein purification

153

Full-length coding sequences were subcloned into the expression vector

154

pMAL-c2X (New England Biolabs) at the BamHI and PstI restriction sites under the

155

control of a tac promoter. The identity of the cloned gene was confirmed through

156

sequence

157

5′-TGCGTACTGCGGTGATCAAC-3′

158

5′-CTGCAAGGCGATTAAGTTGG-3′ (http://www.lifetechnologies.com/). The three

159

recombinant

160

pMAL-c2X-CsRHMc were transformed into E. coli NovaBlue (DE3) competent cells

161

(Novagen, Schwalbach, Germany). The expression strain harboring the recombinant

162

plasmids was grown at 37 °C in 200 mL Luria–Bertani medium containing 100

163

µg·mL−1 ampicillin and 2 g·L−1 glucose. Subsequently, protein expression was

analysis

with

plasmids

the

of

following

sequencing and

pMAL-c2X-CsRHMa,


primers:

pMAL-C2X-F pMAL-C2X-R

pMAL-c2X-CsRHMb,

and

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164

induced

by

adding

isopropyl-β-D-thiogalactopyranoside

(IPTG)

at

a

final

165

concentration of 0.5 mM when the OD600 reached approximately 0.4–0.6. This culture

166

was incubated at 28 °C for 24 h. The cells were harvested by centrifugation and stored

167

at −20 °C overnight. Fusion proteins were purified through affinity chromatography

168

using maltose-binding protein (MBP) resin (New England Biolabs), according to the

169

manufacturer’s protocol. The identity of the obtained protein fraction was confirmed

170

by electrophoresis on a 12% SDS polyacrylamide gel stained. The purified

171

recombinant protein was used for the activity assay in vitro.

172

2.5 UDP-Rha synthesis assay in vitro

173

During the initial screening, each reaction mixture (total volume of 100 µL)

174

contained 100 mM Tris–HCl (pH 7.5), 3 mM UDP-Glc or 3 mM UDP-Gal, 3 mM

175

NAD+, 3 mM NADPH, and 15 µg of purified fusion proteins. Each reaction mixture

176

was incubated at 35 °C for 60 min. To determine the optimum pH for the catalytic

177

activity of the three purified fusion proteins with 3 mM UDP-Glc as a substrate, assays

178

were performed at pH 4.0–11.0 with 0.5 pH increments using different buffer

179

solutions, including 100 mM acid-sodium citrate buffer (pH 5.0–8.0), 100 mM

180

Tris–HCl buffer (pH 7.0–10.0), 100 mM phosphate buffer (pH 7.0–9.5), and 100 mM

181

Na2CO3/NaHCO3 buffer (pH 9.0–11.0). The effect of temperature on catalytic activity

182

was determined at a series of temperatures (20, 25, 30, 35, 40, and 45 °C) in phosphate

183

buffer of pH 9.5 for 30 min.

184

To analyze the kinetic parameters (Km and Vmax) of UDP-Glc for three

185

recombinant proteins, namely CsRHMa, CsRHMb, and CsRHMc, purified enzymes



186

(20 µg) were incubated in reaction mixtures containing 3 mM NAD and 3 mM

187

NADPH (as cofactors) and 100 mM phosphate buffer (pH 7.5) in a final volume of

188

100 µL. The concentration of the tested substrate UDP-Glc ranged from 0 to 4 mM.

189

Each assay mixture was incubated at 35 °C for 15 min.

190

All of the reaction mixtures were terminated through heat treatment (100 °C) for

191

10 min after incubation and were repeated in triplicate. The enzyme reaction samples

192

were centrifuged at 14,720 x g for 10 min and analyzed using high-performance liquid

193

chromatography (HPLC), as previously described.25 The kinetic parameters Km and

194

Kcat of UDP-Glc for CsRHMa, CsRHMb, and CsRHMc proteins were calculated using

195

Hyper 32 (http:// hyper32.software.informer.com/).

196

2.6 HPLC and nuclear magnetic resonance analysis

197

The HPLC system from Agilent Technologies (RHMo Alto, CA, USA) was used

198

in this study and comprised a Venusil XBP C18 reverse phase column (4.6 × 251 mm,

199

Agela Technologies), quaternary pump with a vacuum degasser, thermostated column

200

compartment, and autosampler; the protocol of the HPLC system was described

201

previously.25 The mobile phase consisted of 20 mM of triethylamine acetate buffer

202

(pH 7.5) with a flow rate of 0.5 mL·min−1. The HPLC-purified enzyme products from

203

UDP-Glc of CsRHMb were lyophilized and dissolved in 500 µL of 99.97% D2O. The

204

proton nuclear magnetic resonance (1H-NMR) and carbon-13 nuclear magnetic

205

resonance (13C-NMR) spectra of the products of UDP-Rha were acquired on a Bruker

206

AVANCE AV 600-MHz nuclear magnetic resonance (NMR) spectrometer from

207

Agilent Technologies (RHMo Alto, CA, USA) at 22 °C. For each sample,


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208

high-resolution

one-dimensional

water-suppressed

and

two-dimensional

209

water-suppressed correlation spectroscopy, total correlation spectroscopy (TOCSY),

210

and nuclear overhauser effect spectroscopy (NOESY) experiments were performed.

211

The data were processed and analyzed using MestReNova v. 5.2.5.

212

2.7 Gene expression analysis

213

Gene-specific primers for semiquantitative reverse transcription PCR (RT-PCR)

214

analysis were verified through the efficiency and specificity of amplicons using

215

melting curve analysis and are listed in Table S1. The conditions for the quantitative

216

RT-PCR (RT-qPCR) used in the study were described in a previous report.27 The

217

semiquantitative RT-PCR analysis of three CsRHM genes expressions in buds, first

218

leaves, second leaves, third leaves, fourth leaves, mature leaves, old leaves, young

219

stems, tender roots, and flowers was performed. The housekeeping gene,

220

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a calibrator in all

221

RT-qPCR measurements. The resultant data were expressed as the mean of three

222

replicates.

223

2.8 Extraction and analysis of nucleotide sugars

224

Total nucleotide sugars in tea plants (content in 2 g of buds, first leaves, second

225

leaves, third leaves, fourth leaves, mature leaves, old leaves, young stems, tender roots,

226

and flowers) were extracted as follows. Step 1: the samples with 5:1 (m/m)

227

polyvinylpolypyrrolidone were ground in liquid nitrogen using a pestle and mortar.

228

Step 2: the fine powders were washed with 2 mL of water and pelleted by

229

centrifugation at 14,720 x g for 15 min. Step 3: after centrifugation, the residues were



230

re-extracted twice with 1 mL of water. Step 4: the pooled supernatant was extracted

231

three times with an equal volume of ethyl acetate and then centrifuged at 14,720 x g

232

for 10 min. Step 5: after centrifugation, the supernatants (ethyl acetate phase) were

233

removed, and the water phase was washed with 70% aqueous ethanol. Step 6: the

234

mixed sample was gently swirled for 10 min at room temperature and then centrifuged

235

at 14,720 x g for 10 min. Step 7: after centrifugation, the supernatants were collected

236

and freeze-dried. Finally, the samples were dissolved in 0.2 mL of water and stored at

237

–20 °C before HPLC analysis.

238

2.9 Tissue slicing and staining

239

Tea tissues slices were obtained from fresh organs (including second leaves, old

240

leaves, young stems, tender roots, and upper and lower epidermis of second leaves) as

241

supporter samples. A fresh plant organ was cut into 0.5 cm × 0.5 cm sections. The

242

supporter sample containing the tea organ or epidermis was sliced by freehand

243

sectioning and observed under a light microscope (XQT-2, COIC).28,29 Images of the

244

section were recorded before and after staining. The section was stained with 0.01%

245

(w/v) ruthenium red.30,31 Sufficient reagent was added to one side of the section and

246

absorbed into it with tissue paper on the opposite side for about 5 min. After staining,

247

the excess reagent on the surface of the section was completely removed with tissue

248

paper. The stained tissues were observed under a light microscope, and the mucilage

249

appeared pink.

250

2.10 Accession numbers


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251


The GenBank accession numbers and sequence data used in this study are listed

252

in Supplementary sequence information.

253

3. Results

254

3.1 Cloning and sequence analysis of CsRHM genes

255

In this study, three CsRHM sequence fragments were screened from the tea

256

transcriptome sequencing database (Bioproject ID PRJNA343349) through homology

257

search using AtRHM as the homologous sequence. Subsequently, the sequences of

258

full-length cDNA were amplified using PCR with gene-specific primers (Table S1),

259

and the proteins of the three candidate genes were named CsRHMa, CsRHMb, and

260

CsRHMc. The predicted protein molecular weights (MWs) and isoelectric point values

261

are listed in Table S2.

262

To investigate the evolutionary relationships of CsRHMs with other plant RHMs,

263

a phylogenetic tree, including 267 plant RHM homologous candidates, was

264

constructed using the neighbor-joining method. Phylogenetic analysis revealed that,

265

except for an unresolved polytomy root (including alga, pteridophyta, and bryophyta),

266

the angiosperm RHMs were divided into two groups (cluster I and cluster II) (Fig. 2

267

and Fig. S1). CsRHMa was grouped into cluster I, which consisted of monocot RHMs

268

(HvRHM from Hordeum vulgare subsp, BdRHM from Brachypodium distachyon,

269

OsRHM from Oryza sativa, and SbRHM from Sorghum bicolor) and dicot RHMs

270

(CcRHM from Coffea canephora, NnRHM from Nelumbo nucifera, JrRHM from

271

Juglans regia, and GhRHM from Gossypium hirsutum Linn). CsRHMb and CsRHMc

272

were grouped into cluster II, which consisted of dicot RHMs from Vitis vinifera,



273

Glycine max, Gossypium hirsutum, Ricinus communis, and A. thaliana (Fig. 2 and Fig.

274

S1).

275

Multiple sequence alignment revealed that the angiosperm RHMs were highly

276

homologous at the protein level. Although CsRHMa, CsRHMb, and CsRHMc were

277

distributed in different branches of the phylogenetic tree, the three CsRHM proteins

278

showed 84.2%–89.8% sequence identity. Moreover, the CsRHM proteins and

279

AtRHMs displayed 80.7%–87.4% similarity (Table S3). Plant RHM proteins not only

280

shared high identity on the amino acid level, but also contained two functional units

281

(the N-terminal and the C-terminal) (Fig. 3 and Fig. S2). In vitro enzyme analysis

282

revealed that the N-terminal region of AtRHM2 displayed UDP-Glc 4,6-dehydratase

283

activity, and that the C-terminal region displayed both UDP-4K6DG 3,5-epimerase

284

and UDP-4KR 4-keto-reductase activity.25 Similar to other plant RHMs, the three

285

CsRHMs also contained two functional units (Fig. 3 and Fig. S2), and each unit

286

harbored a putative NAD(P)(H)-binding motif (GxxGxxG/A) and a conserved

287

catalytic triad (YxxxK) motif (Fig. 3 and Fig. S2). The above mentioned analysis

288

suggests that the three CsRHMs are trifunctional enzymes that convert UDP-Glc to

289

UDP-Rha.

290

3.2 Identification of CsRHMs catalyzing UDP-L-rhamnose biosynthesis

291

The ORF sequences of the three CsRHM genes were first expressed with a MBP

292

tag in E. coli. Subsequently, the three recombinant proteins were purified through

293

affinity chromatography using amylose resin. The MWs of the three recombinant

294

CsRHM proteins were all approximately 114 kDa, and degradation fragments were


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295

not detectable in the SDS-PAGE gel (Fig. S3). This was consistent with the predicted

296

MWs of CsRHMa (~75.75 kDa), CsRHMb (~75.91 kDa), and CsRHMc (~75.65 kDa)

297

plus the MBP tag (~42.5 kDa).

298

To evaluate the biochemical activities of the three recombinant proteins, a

299

standard enzyme activity assay was performed at 35 °C for 1 h with a mixture

300

containing 3 mM NAD+, 3 mM NADPH, 3 mM UDP-Glc or 3 mM UDP-Gal, and 20

301

µg of the recombinant proteins. Subsequently, the enzyme products were analyzed

302

using reversed-phase HPLC. The results of HPLC analysis indicated that compared

303

with the control, a product peak (defined as product A) was detected through UV260

304

absorbance at the retention time of 13.2 min in the catalyzed reactions (Fig. 4A).

305

To confirm the identity of the product, approximately 10 mg of product A was

306

collected through prep-HPLC from a scaled-up reaction assay of the rCsRHMb protein.

307

Subsequently, the product was lyophilized and dissolved again in 500 µL of 99.95%

308

D2O; the chemical structure was analyzed using 1H-NMR and 13C-NMR spectroscopy.

309

The results of NMR spectroscopic analysis of product A are summarized in Table 1

310

and Fig. S4. Representative spectral signals δH 5.980 (1H,d,8.4) and δH 7.961 (1H,d,8.4)

311

corresponded to the H5 and H6 of uracil. δH 6.000 (1H,d,3.6), δH 4.387 (1H,dd, 3.0,1.6),

312

δH 4.240 (1H,dd,3.0,1.8), δH 4.291 (1H,dd, 2.4,2.4), and δH 4.208 (1H,d,2.4)

313

corresponded to the H1–5 of ribose, respectively. Similarly, δH 5.210 (1H,d,1.6), δH

314

4.102 (1H,dd, 3.0,1.6), δH 3.643 (1H,dd,3.0,2.4), δH 3.385 (1H,dd, 9.6,7.8), δH 3.453

315

(1H,dd, 6.0,3.0), and δH 1.326 (3H,d,6.0) corresponded to the H1–6 of rhamnose,

316

respectively. Moreover, the representative spectral signals δC 165.99, δC 151.47, δC



317

141.50, and δC 102.48 corresponded to the C2, C4, C5, and C6 of uracil residues,

318

respectively; δC 95.33, δC 72.68, δC 83.12, δC 42.07, and δC 10.44 corresponded to the

319

C1, C2, C3, C4, and C5 of ribose residues, respectively; and δC 102.16, δC 72.08, δC

320

72.65, δC 73.66, δC 64.74, and δC 16.68 corresponded to the C1, C2, C3, C4, C5, and C6

321

of rhamnose residues, respectively. In addition, two-dimensional TOCSY and NOESY

322

(Fig. 4B) spectra showed that the representative spectral signals H1, H2, H3, H4, H5,

323

and H6 corresponded to the chemical structure of rhamnose.25 These chemical shift

324

values and coupling constants for product A were consistent with the chemical

325

structure of UDP-Rha based on a previous study.25 Overall, these results showed that

326

all three CsRHM proteins exhibited the capacity for converting UDP-Glc to UDP-Rha

327

in vitro.

328

For the biochemical characterization of the three rCsRHM proteins, the optimum

329

pH, optimum temperature, and kinetic parameters were obtained by measuring

330

enzyme activities with UDP-Glc as a substrate. These results showed that rCsRHMa

331

and rCsRHMb displayed maximum activity from pH 8.0 to 9.5 (Fig. 5A,B) and

332

temperature 35 to 50 °C (Fig. 5E,F). However, rCsRHMc showed lower pH optima

333

(7.5–8.5) and temperature optima (25–35 °C) than rCsRHMa and rCsRHMb (Fig.

334

5C,G).

335

Kinetic parameters were calculated using an appropriate non-linear regression

336

software Hyper 32.32 The Km values of the three rCsRHM proteins were 448.5 ± 62,

337

201.0 ± 33, and 969.2 ± 137 µM, with Vmax values of 83.33 ± 15, 112.11 ± 17, and


Page 16 of 38

Page 17 of 38


338

55.56 ± 11 µM·min−1, respectively (Table 2). Kinetic analysis showed that rCsRHMa

339

and rCsRHMb displayed higher catalytic efficiency than rCsRHMc.

340

3.3 Expression patterns of CsRHM genes in tea plants

341

In this study, to characterize the functions of CsRHMa, CsRHMb, and CsRHMc,

342

their relative expression levels in different tea organs (including buds, first leaves,

343

second leaves, third leaves, fourth leaves, mature leaves, old leaves, young stems,

344

tender roots, and flowers) were analyzed through semiquantitative RT-PCR analysis.

345

Fig. 6B shows the differential expression levels of the three CsRHM genes in

346

different tissues. The three genes (i.e., CsRHMa, CsRHMb, and CsRHMc) displayed

347

similar expression profiles and were highly expressed in tender tissues (including first,

348

second, and third leaves; young stems; and tender roots; Fig. 6B). Although the

349

expression pattern of CsRHMc was similar to the patterns of CsRHMa and CsRHMb,

350

the transcript levels of CsRHMa (30 cycles) and CsRHMb (30 cycles) were

351

predominantly higher than that of CsRHMc (36 cycles) in all tea organs (Fig. 6B).

352

Intriguingly, CsRHMb was not only detected in young leaves, stems, and roots but

353

also specifically expressed in flowers (Fig. 6B).

354

3.4 Metabolite profiles of nucleotide sugars and mucilage

355

The major nucleotide sugar compounds UDP-Glc, UDP-Gal, and UDP-Rha in

356

different tea organs were measured through HPLC. The results indicated that

357

accumulations of UDP-Glc, UDP-Gal, and UDP-Rha were highest in buds and young

358

leaves (first, second, and third leaves), followed by young stems and tender roots;

359

however, the lowest accumulation was found in mature leaves, old leaves, and flowers



360

(Fig. 6C), which was relatively consistent with the transcript profile of the three

361

CsRHM genes.

362

Mucilage is a complex mixture composed of pectic polysaccharides; it can be

363

stained with ruthenium red and conveniently visualized under a light microscope. In

364

this study, to investigate the accumulation profiles and histochemical localization of

365

mucilage in tea plant samples, sections of different tea organs (including second leaves,

366

old leaves, young stems, tender roots, and the upper and lower epidermis of second

367

leaves) obtained from freehand sectioning were stained with ruthenium red (Fig. 6D).

368

After staining, the red-stained mucilage was visualized using a light microscope (Fig.

369

6D). Ruthenium red staining showed that mucilage could be detected easily in almost

370

all tissues. Compared with mucilage accumulation in old leaves, mucilage

371

accumulation was higher in tender organs, such as second leaves, young stems, and

372

tender roots. Moreover, the red-stained mucilage was specifically accumulated in the

373

intercellular space and primary cell wall (Fig. 6D). Overall, the transcript profiles of

374

the three CsRHM genes were consistent with the metabolite patterns of nucleotide

375

sugar compounds and the accumulation profiles of mucilage (Fig. 6B,C). These results

376

suggest that the three CsRHM genes may contribute to the biosynthesis of UDP-Rha

377

and may be involved in the formation of primary walls in C. sinensis.

378

4. Discussion

379

4.1 Protein motif analysis of the plant RHM family

380

In bacteria, a gene cluster consisting of RmlA, RmlB, RmlC, and RmlD genes is

381

responsible for the biosynthesis of dTDP-Rha from dTDP-Glc19-22 (Fig. 1A). In fungi,


Page 18 of 38

Page 19 of 38


382

two genes UG4,6-Dh and U4k6dG-ER are responsible for the biosynthesis of

383

UDP-Rha from UDP-Glc23 (Fig. 1B). In plants, RHM1, RHM2/MUM4, and RHM3

384

genes from A. thaliana encode a trifunctional enzyme.25 Recombinant AtRHM

385

proteins display UDP-Glc 4,6-dehydratase; UDP-4K6DG 3,5-epimerase; and

386

UDP-4KR 4-keto-reductase activities and convert UDP-Glc to UDP-Rha in vitro.25 In

387

this study, we expressed and purified three CsRHM proteins from E. coli and found

388

that the three proteins showed trifunctional activity and converted UDP-Glc to

389

UDP-L-rhamnose in vitro.

390

Takuji Oka, Nemoto, and Jigami proved that the N-terminal region of RHM2

391

(1–370 amino acids) shows UDP-Glc 4,6-dehydratase activity, and that the C-terminal

392

region of RHM2 (371–667 amino acids) shows both UDP-4K6DG 3,5-epimerase and

393

UDP-4KR 4-keto-reductase activities.25 Amino acid sequence alignment (Fig. S2)

394

revealed that RmlB and UG4,6-Dh were similar to the N-terminal region of RHM and

395

CsRHMs, and all of them contained one NADPH-binding motif (GxxGxxG) and one

396

NAD+ binding site (YxxxK). The aforementioned similar structures and conserved

397

sites maybe the reason why they have similar function with dehydratase. Similarly,

398

U4k6dG-ER of fungi displayed higher identity with the C-terminal region of RHM

399

and CsRHMs and contained one NADPH-binding motif (GxxGxxG) and one NAD+

400

binding site (YxxxK) (Fig. S2). Such high sequence similarity explains the similarity

401

in epimerase and reductase functions.

402

4.2 CsRHMs are probably responsible for the biosynthesis of UDP-Rha in tea

403

plants



404

Page 20 of 38

UDP-Rha is not only a rhamnose donor in the biosynthesis of various

405

L-rhamnose-containing

natural

compounds

(flavonoids,

terpenoids,

and

406

saponins)15,33,34 but also an essential compound for the biosynthesis of the primary cell

407

wall components RG-I and RG-II.8,35

408

A. thaliana epidermal cells in the seed coat provide a classical model for

409

exploring genes involved in cell wall production.36 Upon hydration, the mature seed

410

coat releases a sticky capsule of mucilage, which can be stained with ruthenium red

411

and conveniently visualized under a light microscope.37 In addition, mucilage

412

represents a readily accessible source of pectins that is not required for normal plant

413

growth and development under laboratory conditions.31 Hence, the produced mucilage,

414

which is composed of cell wall polysaccharides, provides a valuable system to

415

investigate the genes related to pectin biology.36-40

416

A previous study demonstrated the overexpression of the AtRHM1 gene, which

417

caused the rhamnose content to significantly increase in the leaf cell wall of transgenic

418

lines compared with that of wild-type lines.41 Defective seed mucilage in the MUM4-1

419

and MUM4-2 mutants and rhm2 T-DNA insertion mutants suggested that the

420

AtRHM2/MUM4 gene is responsible for the biosynthesis of UDP-Rha in vivo, and that

421

the AtRHM2 protein is essential for the biosynthesis of seed coat mucilage, which is

422

mainly composed of pectins.26,35

423

In this study, an expression analysis demonstrated that CsRHMa, CsRHMb, and

424

CsRHMc genes shared similar expression profiles and were highly expressed in the

425

tender tissues of tea plants (Fig. 6B). Additionally, metabolite profiles showed that


Page 21 of 38


426

UDP-Rha accumulated at low levels in mature leaves and old leaves but at a very high

427

level in shoots (Fig. 6C). The tender tissues of tea plants accumulate a large amount of

428

the flavonol rhamnoside. In addition, as a rhamnose residue donor, UDP-Rha is

429

required for the biosynthesis of rhamnoside. In this study, the expression profiles of

430

the three CsRHM genes were correlated with the accumulation patterns of main

431

nucleotide diphosphate sugars (including UDP-Glc, UDP-Gal, and UDP-Rha) (Fig.

432

6B,C). We suggest that the CsRHMs are likely to participate in the formation of these

433

secondary metabolites; however, the content of the secondary metabolites is directly

434

related to the bitter taste of tea. The results revealed that the ruthenium red-stained

435

mucilage accumulated in larger quantities in shoots and was specifically located in the

436

intercellular space and primary cell wall (Fig. 6D). Thus, enzyme activity analysis,

437

transcript profiles, metabolite profiles, and mucilage location suggest that the CsRHM

438

genes are likely to be involved in the biosynthesis of UDP-Rha and participate in the

439

formation of primary cell walls in tea plants.

440

ABBREVIATIONS USED

441

UDP, uridine diphosphate; RHM, UDP-rhamnose synthase; UDP-Glc, UDP-D-glucose;

442

UDP-Rha, UDP-rhamnose; HGA, homogalacturonan; RG-I, rhamnogalacturonan-I; RG-II,

443

rhamnogalacturonan-II; IPTG, isopropyl-β-D-thiogalactopyranoside; NMR, nuclear magnetic

444

resonance; TOCSY, total correlation spectroscopy; NOESY, nuclear overhauser effect spectroscopy;

445

GAPDH,

446

chromatography; MWs, molecular weights; MBP, Maltose-binding protein;

447

AUTHOR INFORMATION

glyceraldehyde-3-phosphate

dehydrogenase;

HPLC,


high-performance

liquid


448

Corresponding Authors

449

Telephone: 86-551-65786003. Fax: 86-551-65785833. E-mail: [email protected].

450

Telephone: 86-551-65786232. Fax: 86-551-65785729. E-mail: [email protected]

451

Author Contributions

452

Conceived and designed the study: Liping Gao, Tao Xia, Xinlong Dai. Drafted the manuscript:

453

Liping Gao, Xinlong Dai, Yajun Liu, Yinglin Wu. Performed the experiments: Xinlong Dai, Guifu

454

Zhao, Tianming Jiao. Analyzed the data: Liping Gao, Xinlong Dai, Xiaolan Jiang. Contributed

455

reagents, materials, and analysis tools: Xinlong Dai,Yinglin Wu. All authors have read and approved

456

the final manuscript.

457

Notes

458

The authors declare no competing financial interests.

459

ACKNOWLEDGMENTS

460

This work was supported by the Natural Science Foundation of China (31470689 and

461

31570694). The Natural Science Foundation for Higher Education of Anhui Province (KJ2017A441)

462

and the Natural Science Foundation of Suzhou University (2016jb02). Anhui Major Demonstration

463

Project for the Leading Talent Team on Tea Chemistry and Health, the Chang-Jiang Scholars and the

464

Innovative Research Team in University (IRT1101). The funders had no role in the study design, data

465

collection and analysis, decision to publish, or preparation of the manuscript. We thank Prof. Jingwei

466

Hu for assistance with NMR spectroscopic analysis. This manuscript was edited by Wallace

467

Academic Editing.

468 469


Page 22 of 38

Page 23 of 38

470


Supporting Information.

471

Figure S1. RHM family phylogenetic tree; Figure S2. Multiple sequence alignment of the RHM

472

proteins from various species at the amino acid level; Figure S3. SDS-PAGE of full-length rCsRHM

473

proteins; Figure S4. NMR spectroscopic analysis of product A; Table S1. Specific primers used in

474

this study; Table S2. Basic information of three CsRHM genes; Table S3. Sequence identities of

475

CsRHM protein.

476

References

477

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chromatographic profiles of green and fermented teas. J. Agric. Food Chem. 2008, 56,

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pathways in plants. Plant Mol. Biol. 2001, 47, 95-113.

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glycolipids and glycans | Frontiers Research Topic. Plant Physiol.

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Diphosphate Glucoses in Phaseolus aureus. Plant Physiol. 1970, 45, 782-786.



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Kerr, I. D.; Whitfield, C.; Naismith, J. H., A structural perspective on the enzymes that

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convert dTDP-d-glucose into dTDP-l-rhamnose. Biochem. Soc. Trans. 2003, 31,

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sucrose synthetase reaction. Nature. 1965, 206, 825-825.

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typhimurium (strain LT2). Mol. Microbiol. 2010, 5, 695-713.

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Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in Pathogenic Fungi

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Magnaporthe grisea and Botryotinia fuckeliana. J. Biol. Chem. 2012, 287, 879.

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UDP-D-glucose by an enzyme preparation of red campion (Silene dioica (L) Clairv)

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leaves. FEBS Lett. 1978, 91, 281-284.

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RHM2/MUM4,

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UDP-L-rhamnose conversion. J. Biol. Chem. 2007, 282, 5389-5403.

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Synthesis and Is Required for the Development of the Seed Coat in Arabidopsis. Plant

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Physiol. 2004, 134, 286-295.

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Xia, T., The R2R3-MYB, bHLH, WD40, and related transcription factors in flavonoid

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staining free-hand sections and whole-mount samples. Ann Bot (Lond). 2005, 96,

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STEREOCHEMISTRY OF ITS PECTIC STAIN. Am J Bot. 1970, 57, 172-175.

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Arabidopsis seed coat. Plant Physiol. 2000, 122, 345-355.

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(32) Zorn H, and Li QX., Trends in Food Enzymology. J. Agric. Food Chem. 2017,

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65, 4.

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bisglycosides using Escherichia coli harboring two glycosyltransferases. Appl.

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Microbiol. Biotechnol. 2013, 97, 5275-5282.

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M.; Ophir, R.; L, W. S.; Eyal, Y., The molecular and enzymatic basis of

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rhamnosyltransferases under domestication. Plant J. 2013, 73, 166-178.

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(35) Western, T. L.; Samuels, A. L.; Haughn, G. W., MUCILAGE-MODIFIED4

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Encodes a Putative Pectin Biosynthetic Enzyme Developmentally Regulated by

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APETALA2, TRANSPARENT TESTA GLABRA1, and GLABRA2 in the

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Arabidopsis Seed Coat. Plant Physiol. 2004, 134, 296-306.

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(36) Haughn, G. W.; Western, T. L., ArabidopsisSeed Coat Mucilage is a Specialized

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Cell Wall that Can be Used as a Model for Genetic Analysis of Plant Cell Wall

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Structure and Function. Front. Plant Sci. 2012, 3, 64.

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(37) North, H.M.; Berger, A.; Saezaguayo, S.; Ralet, M. C., Understanding

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polysaccharide production and properties using seed coat mutants: future perspectives

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for the exploitation of natural variants. Ann. Bot. 2014, 114, 1251.

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(38) Arsovski, A. A.; Haughn, G. W.; Western, T. L., Seed coat mucilage cells of as a

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model for plant cell wall research. Plant Signal Behav. 2010, 5, 796-801.

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(39) Western, T. L. W. L., Changing spaces: the mucilage secretory cells as a novel

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system to dissect cell wall production in differentiating cells. Can. J. Bot. 2006, 84,

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622-630.

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(40) Tamara, L., The sticky tale of seed coat mucilages: production, genetics, and role

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in seed germination and dispersal. Seed Sci. Res. 2012, 22, 1-25.

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(41) Wang, J.; Ji, Q.; Ling, J.; Shen, S.; Fan, Y.; Zhang, C., Overexpression of a

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cytosol-localized rhamnose biosynthesis protein encoded by Arabidopsis RHM1 gene

594

increases rhamnose content in cell wall. Plant Physiol. Biochem. 2009, 47, 86-93.

595 596 597 598


Page 28 of 38

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599

FIGURE CAPTION

600

Figure 1. Chemical reaction and structural formula of the dTDP-Rha and UDP-Rha

601

biosynthetic pathways in bacteria, fungi, and plants. (A) In bacteria, dTDP-Rha is produced from

602

dTDP-Glc by three proteins: dTDP-Glc 4,6-dehydratase (RmlB), dTDP-4K6DG 3,5-epimerase

603

(RmlC), and dTDP-4KR 4-keto-reductase (RmlD). (B) In fungi, UDP-Rha is produced from

604

UDP-Glc by two proteins 4,6-dehydratase (UG4,6-Dh) and bifunctional 3,5-epimerase and

605

4-reductase (U4k6dG-ER). (C) In plants, UDP-Rha is produced from UDP-Glc by a trifunctional

606

RHM protein. UDP-Rha is further used in the synthesis of cell wall polysaccharides and

607

L-rhamnose-containing natural compounds.

608

Figure 2. RHM family phylogenetic tree. An unrooted phylogenetic tree was constructed using

609

MEGA 5.0 software through the neighbor-joining method. These RHM sequences were clustered

610

into five groups, including alga (blue), pteridophyta (purple), bryophyta (light blue), plant cluster I

611

(red), and plant cluster II (green). Three CsRHMs are clustered in plant cluster I and plant cluster II

612

and are indicated with triangles. Sequence information of the RHMs is shown in Table S2.

613

Figure 3. Multiple alignment of the amino acid sequences of CsRHMs with AtRHM2.

614

Conserved residues between the CsRHMs and AtRHM2 are indicated with a black column. The

615

putative highly conserved NAD(P)+ cofactor-binding (GxxGxxG/A) and active-site catalytic couple

616

(YxxxK) motifs are indicated above the sequence alignment.

617

Figure 4. Molecular identification of the enzyme reaction products of CsRHM proteins. (A)

618

HPLC analysis of the products from UDP-Glc of three purified rCsRHM proteins. The substrates and

619

corresponding products were detected through HPLC on UV260 nm absorbance. (a-1), control. (a-2),

620

(a-3), and (a-4) indicate the enzyme reaction products of the three CsRHM recombinant proteins



621

rCsRHMa, rCsRHMb, and rCsRHMc, respectively. (a-5), UDP-Rha used as a standard. (B)

622

Structural identification of product A from UDP-Glc of the three CsRHM proteins. (b-1), TOCSY

623

spectra of product A. (b-2), NOESY spectra of product A. (b-3), the chemical structure of UDP-Rha.

624

Figure 5. Optimum condition of reactions catalyzed by three rCsRHMs. Each reaction mixture

625

(100 µL) was incubated with 3 mM UDP-Glu, 3 mM NAD, and 3 mM NADPH at different ranges of

626

pH (5.0–11.0) and temperature (20–65 °C) for 30 min. (A) Effect of reaction pH on activities of the

627

rCsRHMa. (B) Effect of reaction pH on activities of the rCsRHMb. (C) Effect of reaction pH on

628

activities of the rCsRHMc. (E) Effect of reaction temperature on activities of the three rCsRHMa. (F)

629

Effect of reaction temperature on activities of the three rCsRHMb. (G) Effect of reaction temperature

630

on activities of the three rCsRHMc. Data are presented as the average mean of three independent

631

trials ± SD. Buffer 1: 100 mM acid-sodium citrate buffer (pH 5.0–8.0); Buffer 2: 100 mM Tris–HCl

632

buffer (pH 7.0–8.0); Buffer 3: 100 mM phosphate buffer (pH 7.0–10.0); and Buffer 4: 100 mM

633

Na2CO3/NaHCO3 buffer (pH 9.0–11.0).

634

Figure 6. Expression profiles of three CsRHM genes and accumulation profiles of nucleotide

635

sugars in different tea organs. (A) The tea samples (including buds, first leaves, second leaves,

636

third leaves, fourth leaves, mature leaves, old leaves, young stems, tender roots, and flowers) used

637

for gene expression and total nucleotide sugar content analysis. (B) The transcript analysis of

638

CsRHMs in various tissues. (C) The content analysis of UDP-Gal, UDP-Glc, and UDP-Rha in

639

different organs; all data points are the mean of three biological replicates, and each error bar

640

indicates the SD. (D) Histochemical localization of mucilage in different tea organs and tea callus.

641

Pink represents mucilage-accumulating areas after 0.01% (w/v) ruthenium red staining; (a and g, b

642

and h, c and i, and d and j) the transverse sections of the second leaves, old leaves, young stems, and


Page 30 of 38

Page 31 of 38


643

tender roots, respectively, before and after staining; (e and k as well as f and i) the upper and lower

644

epidermis sections, respectively, before and after staining; (m–r) the high magnification image of

645

different plant sections after staining. Abbreviations: is, intercellular space; pcw, primary cell wall.

646 647 Table 1. NMR spectroscopic data of UDP-Rha synthesized from UDP-Glu in a reaction 648 catalyzed by CsRHM (D2O, δ in ppm, J in Hz) 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683

Table 2. Kinetic parameters of recombinant CsRHMa, CsRHMb, and CsRHMc proteins



684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727

Figure 1.


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728


Figure 2. 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755

756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771



772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815

Figure 3.


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816 817


Figure 4.

818 819 820 821 822 823 824 825 826 827 828 829 830 831



832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875

Figure 5.


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876 877 878 879


Figure 6.

880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895



896 897 898

TOC graphic

899 900 901 902 903 904 905 906 907 908


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Involvement of Three CsRHM Genes from Camellia sinensis in UDP

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