Accumulation of Rutin and Betulinic Acid and Expression of

Subscriber access provided by CMU Libraries - http://library.cmich.edu

Article

ACCUMULATION OF RUTIN AND BETULINIC ACID AND EXPRESSION OF PHENYLPROPANOID AND TRITERPENOID BIOSYNTHETIC GENES IN MULBERRY (Morus alba L.) Shicheng Zhao, Chang Ha Park, Xiaohua Li, Yeon Bok Kim, Jingli Yang, Gyoo Byung Sung, Nam Il Park, Soonok Kim, and Sang Un Park J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03221 • Publication Date (Web): 07 Sep 2015 Downloaded from http://pubs.acs.org on September 14, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

Journal of Agricultural and Food Chemistry

1

ACCUMULATION OF RUTIN AND BETULINIC ACID AND

2

EXPRESSION

3

TRITERPENOID BIOSYNTHETIC GENES IN MULBERRY

4

(Morus alba L.)

OF

PHENYLPROPANOID

AND

5

Shicheng Zhao,† Chang Ha Park,† Xiaohua Li,† Yeon Bok Kim, ‡ Jingli Yang∥, Gyoo

6

Byung Sung⊥, Nam Il Park§, Soonok Kim,# and Sang Un Park†,*

7 8

†

9

Yuseong-gu, Daejeon 305-764, Korea.

Department of Crop Science, Chungnam National University, 99 Daehak-ro,

10

‡

11

Science (NIHHS), Rural Development Administration (RDA), Bisanro 92, Eumseong,

12

Chungbuk, 369-873, Korea

13 14 15 16 17 18 19 20

Department of Herbal Crop Research, National Institute of Horticultural and Herbal

∥

State Key Laboratory of Forest Genetics and Tree Breeding, Northeast Forestry

University, 26 Hexing Road, Harbin 150040, China ⊥

Department of Agricultural Biology, National Academy of Agricultural Science,

Rural Development Administration, Wanju 565-851, Korea §

Deptartment of Plant Science, Gangneung-Wonju National University 7 Jukheon-gil,

Gangneung-si, Gangwon-do 210-702, Korea #

Biological and Genetic Resources Assessment Division, National Institute of

Biological Resources, Incheon 404-170, Korea

21 22 23

*

24

S. U. Park

25 26 27

Department of Crop Science, Chungnam National University, 99 Daehak-Ro, Yuseong-Gu, Daejeon, 305-764, Korea. Phone: +82-42-821-5730. Fax: +82-42-822-2631. E-mail: [email protected]

To whom correspondence should be addressed

ACS Paragon Plus Environment


28

ABSTRACT

29

Mulberry (Morus alba L.) is used in traditional Chinese medicine and is the sole food

30

source of the silkworm. Here, nine cDNAs encoding phenylpropanoid biosynthetic

31

genes and 21 cDNAs encoding triterpene biosynthetic genes were isolated from

32

mulberry. The expression levels of genes involved in these biosynthetic pathways, and

33

the accumulation of rutin, betulin, and betulinic acid, important secondary metabolites,

34

were investigated in different plant organs. Most phenylpropanoid and triterpene

35

biosynthetic genes were highly expressed in leaves and/or fruit, and most genes were

36

downregulated during fruit ripening. The accumulation of rutin was more than 5-fold

37

higher in leaves than in other organs, and more betulin and betulinic acid were found

38

in roots and leaves than in fruit. By comparing the contents of these compounds with

39

gene expression levels, we speculate that MaUGT78D1 and MaLUS play important

40

regulatory roles in the rutin and betulin biosynthetic pathways.

41

KEYWORDS: mulberry, rutin, betulin, betulinic acid, gene expression

42


Page 2 of 31

Page 3 of 31


43

INTRODUCTION

44

In plants, the phenylpropanoid biosynthesis pathway produces many physiologically

45

active secondary metabolites, such as flavonoids, lignins, isoflavonoids, and

46

anthocyanins,1 which perform a wide array of important functions. Flavonoids are

47

especially important in plant growth and development; they help to determine flower

48

and fruit color, protect against UV irradiation, and facilitate nitrogen fixation.

49

Flavonoids also have human health benefits including anticancer and antioxidant

50

properties.2 Rutin is an important flavonoid compound with functions that include

51

anticancer, anti-aging, and dietary effects. Rutin and rutin biosynthesis genes have

52

been well studied in buckwheat.3

53

Triterpenoids are another group of plant secondary metabolites. The pharmaceutical

54

and physiological activities of triterpenoids are diverse and important and include

55

antinociceptive, antidiabetic, anti-HIV, and antioxidant properties.4-7 Betulin and

56

betulinic acid are important triterpenoids that are distributed in plant bark. There has

57

been increasing study of betulin and betulinic acid because of their effectiveness

58

against a variety of tumors.8-10

59

Phenylpropanoid biosynthesis starts with the formation of the aromatic amino acid

60

phenylalanine (Figure 1a). Phenylalanine ammonia lyase (PAL) is the first enzyme in

61

the phenylpropanoid pathway and catalyzes the conversion of phenylalanine into

62

cinnamic acid. Then, cinnamate 4-hydroxylase (C4H), catalyzes the transcinnamic

63

acid hydroxylate into p-coumaric acid; 4-coumarate-CoA ligase (4CL) then catalyzes



Page 4 of 31

64

the conversion of p-coumaric acid to p-coumaroyl-CoA, which is the precursor for

65

many phenylpropanoid products, such as lignins and flavonoids. Subsequently,

66

chalcone synthase (CHS) catalyzes the production of a naringenin chalcone, which is

67

the precursor for all flavonoids. In the next step, chalcone isomerase (CHI) converts

68

naringenin chalcone to naringenin. Afterward, naringenin is converted to

69

dihydrokaempferol by flavone 3-hydroxylase (F3H); then, flavonoid 3′-hydroxylase

70

(F3′H) catalyzes the transformation of dihydrokaempferol to quercetin. Finally,

71

quercetin is converted to isoquercitrin and rutin by flavonol 3-O-glucosyltransferase

72

(F3GT)

73

respectively.

74

Triterpene biosynthesis is derived from the mevalonic acid (MVA) pathway and the

75

2-C-methyl-D-erythritol 4-phosphate (MEP) pathway at the initial period (Figure 1b).

76

These pathways generate the same products: isopentenyl diphosphate (IPP) and

77

dimethylallyl diphosphate (DMAPP). Then, geranyl diphosphate synthase (GPPS)

78

catalyzes the condensation of IPP and DMAPP to form geranyl pyrophosphate (GPP).

79

GPP then unites with another molecule of IPP to from farnesyl pyrophosphate (FPP)

80

catalyzed by farnesyl pyrophosphate synthetase. Next, squalene synthase (SQS) and

81

squalene epoxide (SQE) catalyze two NADPH-requiring reactions to form squalene

82

and 2,3-oxidosqualene, respectively; 2,3-oxidosqualene is the common precursor of

83

triterpenes

84

2,3-oxidosqualene is converted to lupeol by lupeol synthase (LUP), and betulinic acid

85

is derived from lupeol followed by successive oxidation at the C-28 position by the

and

flavonol-3-O-glucoside

through

different

pathways.

L-rhamnosyltransferase

In

betulinic


acid

(UGT78D1),

biosynthesis,

Page 5 of 31


86

cytochrome P450 family.

87

Mulberry (Morus alba L., Moraceae) is used in traditional Chinese medicine and is

88

the sole food source of the silkworm. The medicinal value of mulberry derives from

89

the many important secondary metabolites compounds it produces and includes

90

protection against liver damage and antidiabetic, antioxidant, anticancer and

91

anti-fever effects.11-17 Secondary metabolites produced by M. alba include flavonoid

92

compounds18-20 such as anthocyanin and rutin,16, 21 which have strong antioxidant and

93

anti-inflammatory effects.22, 23

94

Mulberry contains many more phenolic and flavonoid compounds than other fruits

95

and vegetables.24 Some studies have assessed the rutin content in mulberry leaves,25, 26

96

but the contents of betulin and betulinic acid have not been evaluated for this plant.

97

Although the draft genome sequence has been reported for another mulberry species,

98

Morus notabilis,27 and most phenylpropanoid and triterpene biosynthetic genes have

99

been identified, currently, the genetic background is not clear for M. alba. In a

100

previous study, our group sequenced the transcriptome of M. alba L. by using an NGS

101

sequencing platform (unpublished), which is the first report of transcriptome analysis

102

for M. alba. Here, we investigated the expression levels of genes related to

103

phenylpropanoid and triterpene biosynthesis with reference to this transcriptome data

104

and analyzed the rutin and betulinic acid contents in different organs by using

105

quantitative real-time PCR (qRT-PCR) and high-performance liquid chromatography

106

(HPLC), respectively. Our results are expected to provide baseline information toward

107

elucidating the mechanism of astragaloside biosynthesis in M. alba.



108

MATERIALS AND METHODS

109

Plant Materials. M. alba plants were grown at the experimental farm of National

110

Academy of Agricultural Science, Rural Development Administration (Wanju, Korea).

111

The fruits, flowers, leaves, stems and roots were excised from mature plants. The

112

samples were immediately frozen in liquid nitrogen and stored at -80 °C and/or

113

freeze-dried for RNA isolation and/or HPLC analysis.

114

RNA Isolation and cDNA Synthesis. Samples (200 mg) of different organs of M.

115

alba L. were ground with a mortar and pestle in liquid nitrogen. RNA was isolated

116

separately using a Plant Total RNA Mini Kit (Geneaid, Taiwan) according to the

117

manufacturer’s instructions. The quality and concentration of total extracted RNA

118

were determined by 1.2% formaldehyde RNA agarose gel electrophoresis and

119

NanoVue Plus spectrophotometer analysis (code No. 28956058, GE Healthcare, UK),

120

respectively. For cDNA synthesis, 1 µg of total RNA was reverse transcribed (RT)

121

using a ReverTra Ace-R kit (Toyobo, Osaka, Japan) and oligo (dT) 20 primer

122

according to the manufacturer’s protocol. A 20-fold dilution of 20 µL of the resulting

123

cDNA was used as a template for quantitative real-time PCR.

124

Sequence Analysis. To identify the phenylpropanoid and triterpene biosynthetic gene

125

sequences in our mulberry NGS database, all generated sequences were blasted with

126

related phenylpropanoid biosynthetic genes in GenBank using BioEdit version 7.0.9.0.

127

The deduced amino acid sequences of the phenylpropanoid and triterpene biosynthetic

128

genes were analyzed for homology by blasting the GenBank database. Identified gene

129

sequences were submitted to GenBank.


Page 6 of 31

Page 7 of 31


130

Quantitative Real-Time Polymerase Chain Reaction. Based on the published gene

131

sequences of MaPAL1, MaPAL2, MaPAL3, MaC4H1, MaC4H2, Ma4CL2, Ma4CL7-1,

132

Ma4CL7-2, Ma4CL9, MaCHS1, MaCHS2, MaCHS3, MaCHS9, MaCHI1, MaCHI2,

133

MaCHI3, MaF3H, MaF3′H1, MaF3′H2, MaF3GT, and MaUGT78D1 (accession

134

numbers

135

biosynthesis, and MaAACT1, MaAACT2, MaHMGS, MaHMGR1, MaHMGR2,

136

MaMVK, MaPMK, MaMVD, MaIDI, MaDXS, MaDXR, MaMCT, MaCMK, MaMCS,

137

MaHDS, MaIDS, MaGPPS, MaFPPS, MaSQS, MaSQE, MaLUP (accession numbers

138

KR080458–KR080471, KR132245–KR132251), real-time PCR primers were

139

designed

140

(Supplementary file Table S1). The expression of these genes was calculated by

141

relative quantification with the actin housekeeping gene (accession number KJ616403)

142

of M. alba as a reference. For quantification of the standard, PCR products amplified

143

from cDNA were purified, and the concentration of the products was measured to

144

calculate the number of cDNA copies. Quantitative real-time PCR (qRT-PCR) was

145

performed in a 20 µL reaction mixture including 5 µL of template cDNA, 10 µL of 1×

146

SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan), 0.5 µL of each

147

primer (10 µL), and DEPC-treated water. Thermal cycling conditions were as follows:

148

95 °C for 15 min and 41 cycles of 95 °C for 20 s, 55 °C for 40 s, and 72 °C for 30 s.

149

The PCR reactions were performed on a CFX96 Real-Time system (Bio-Rad

150

Laboratories, Hercules, CA). PCR products were analyzed with the Bio-Rad CFX

151

Manager 2.0 software. Three replications per sample were used for the real-time PCR

KJ616395–KJ616404,

using

the

Primer

KT630878-KT630889)

3

website

for

phenylpropanoid

(http://frodo.wi.mit.edu/primer3/)



152

analysis, and values were expressed as means ± SD.

153

High Performance Liquid Chromatography Analysis. Different organs of M. Alba

154

L. were freeze-dried at –80 °C for 48 h and then ground into a fine powder using a

155

mortar and pestle. Rutin was released from the samples (50 mg) by adding 3 mL of 80%

156

methanol containing 0.1% ascorbic acid (w/v) at 60 °C for 1 h. After centrifuging

157

(3000 ×g) the extract, the supernatant was filtered with a 0.22 µm Acrodisc syringe

158

filter (Pall Corp.; Port Washington, NY) and analyzed by HPLC. The

159

phenylpropanoids were separated on a C18 column (250 × 4.6 mm, 5 µm; RStech,

160

Daejeon, Korea) using an Agilent 1100 HPLC system (Agilent Technologies, Massy,

161

France) equipped with a photodiode array detector. The mobile phase consisted of

162

methanol, water, and 0.2% acetic acid, and the column was maintained at 30 °C. The

163

flow rate was maintained at 1.0 mL/min, the injection volume was 20 µL, and the

164

detection wavelength was 280 nm. Pentacyclic triterpene were released from the M.

165

alba samples (500 mg) by adding 5 mL 95% methanol containing 250 µL 1%

166

hydrogen chloride, sonicating for 15 min, and then allowing to stand for 30 min. After

167

centrifugation and filtration, pentacyclic triterpenes were separated on a ProntoSIL

168

120-5 C18 ace-EPS (150 × 4.6 mm, 5 µm; ProntoSIL, Bollinger, Germany) using a

169

Futex NS-4000 HPLC system (Futex Chromatography, Daejeon, Korea). The mobile

170

phase consisted of acetonitrile and water (9:1); the column was maintained at 25 °C,

171

the flow rate was 1.0 mL/min, the injection volume was 20 µL, and the detection

172

wavelength was 210 nm. The concentrations of compounds were determined by using

173

a standard curve. All samples were analyzed in triplicate. Values were expressed as


Page 8 of 31

Page 9 of 31


174

means ± SD.

175

RESULTS AND DISCUSSION

176

Sequence Analyses of Phenylpropanoid and Triterpene Biosynthetic Genes from

177

Mulberry. Full-length cDNAs encoding PAL1, PAL2, C4H1, C4H2, 4CL7-1, 4CL7-2,

178

CHS3, CHI1, CHI3 AACT1, AACT2, HMGS, PMK, MVD, IDI, DXS, MCS, HDS,

179

IDS, GPPS, SQE, and LUS, and partial-length cDNA clones encoding PAL3, 4CL2,

180

4CL9, CHS1, CHS2, CHS9, CHI2, F3H, F3′H1, F3′H2 F3GT, UGT78D1, HMGR1,

181

HMGR2, MVK, DXR, MCT, CMK, FPPS, and SQS were identified. They were

182

confirmed for homology with the BLAST program and designated as MaPAL1 (722

183

amino acids [aa]), MaPAL2 (749 aa), MaPAL3 (344 aa), MaC4H1 (525 aa), MaC4H2

184

(507 aa), Ma4CL2 (365 aa), Ma4CL7-1 (547 aa), Ma4CL7-2 (539 aa), Ma4CL9 (471

185

aa), MaCHS1 (257 aa), MaCHS2 (259 aa), MaCHS3 (399 aa), MaCHS9 (218 aa),

186

MaCHI1 (228 aa). MaCHI2 (308 aa), MaCHI3 (208 aa), MaF3H (228 aa), MaF3′H1

187

(354 aa), MaF3′H2 (177 aa), MaF3GT (339 aa), MaUGT78D1 (447 aa), MaAACT1

188

(403 aa), MaAACT2 (416 aa), MaHMGS (464 aa), MaHMGR1 (548 aa), MaHMGR2

189

(586 aa), MaMVK (356 aa), MaPMK (507 aa), MaMVD (421 aa), MaIDI (295 aa),

190

MaDXS (714 aa), MaDXR (363 aa), MaMCT (243 aa), MaCMK (254 aa), MaMCS

191

(248 aa), MaHDS (740 aa), MaIDS (461 aa), MaGPPS (380 aa), MaFPPS (182 aa),

192

MaSQS (408 aa), MaSQE (524 aa), and LcLUS (754 aa). The data provided in Tables

193

1 and 2 showed that the mulberry phenylpropanoid and triterpene biosynthetic genes

194

exhibited high identity with other orthologous genes.

195

Expression of Phenylpropanoid and Triterpene Biosynthetic Genes in Mulberry.



196

The expression of phenylpropanoid and triterpene biosynthetic genes was analyzed in

197

roots, stems, and leaves, and in three fruit stages (unripe: fruits-1, semi-ripe: fruits-2,

198

and fully ripe: fruits-3) by qRT-PCR (Figure 2). The expression patterns of

199

phenylpropanoid biosynthetic genes differ among species.28-32 In M. alba, the

200

expression level of three isoform PAL genes were different, MaPAL1 was expressed at

201

the highest levels in stems and fully ripe fruits, however, MaPAL2 and MaPAL3 were

202

highly expressed in leaves. The expression level of MaC4H1 was the highest in the

203

stems than in the other organs, while the expression level of MaC4H2 was similar in

204

all mulberry organs. The expression levels of Ma4CL2 and Ma4CL9 were high in

205

unripe fruits; Ma4CL7-1 was highly expressed in the leaves, and Ma4CL7-2 was

206

highly expressed in the roots. MaCHS1 showed high expression level in fruits;

207

MaCHS2 was highly expressed in the leaves and unripe fruits, intermediately

208

expressed in semi- and fully ripe fruits, and showed low expression in the roots and

209

stems; however, MaCHS9 was highly expressed in the root. MaCHI1 was highly

210

expressed in the leaves, whereas MaCHI2 and MaCHI3 was mainly expressed in the

211

fruits. The expression patterns of MaF3H, MaF3′H1, and MaF3GT were similar, with

212

relatively higher expression in fruits than in roots, stems, and leaves. However,

213

MaF3′H2 was different with MaF3′H1, with the highest expression level in roots.

214

MaUGT78D1 showed significantly higher expression in leaves than in other organs.

215

During fruit ripening, the expression of MaPAL2, MaPAL3, MaC4H2, Ma4CL,

216

MaCHS2, MaCHS3, MaCHS9 and MaCHI3 declined gradually. In contrast, the

217

expression of MaPAL1, MaCHS1, MaCHI, MaF3H, MaF3′H, and MaF3GT increased


Page 10 of 31

Page 11 of 31


218

gradually as fruit ripened. In the triterpene biosynthetic pathway, most genes were

219

highly expressed in leaves (Figure 3). During the fruit development, the expression of

220

most triterpene biosynthetic genes decreased, including that of MaAACT2, MaHMGS,

221

MaHMGR1, MaHMGR2, MaMVK, MaMVD, MaDXS, MaDXR, MaMCT, MaCMK,

222

MaMCS, MaHDS, MaIDS, MaGPPS, and MaLUS. MaIDI, MaPMK were expressed at

223

the same level in the three fruit-ripening stages. In contrast, MaAACT1, MaFPPS,

224

MaSQS, and MaSQE showed increased expression during ripening. Possibly, these

225

genes were relative with fruit maturation.

226

There are various explanations for the diverse expression levels of phenylpropanoid

227

biosynthetic genes among plant species, including the existence of different gene

228

isoforms in different organs. Xu et al.33 cloned three isoforms of PAL in Scutellaria

229

baicalensis and found different transcript levels in different organs. Expression of

230

isoform SbPAL1 was highest in stems, SbPAL2 was highest in leaves, and SbPAL3

231

expression was highest in roots. Li et al.28 compared two isoforms of 4CL in

232

buckwheat and found that Fe4CL1 was most strongly expressed in roots, while

233

Fe4CL2 had the highest expression in stems. Biotic and abiotic environmental factors

234

can also influence phenylpropanoid gene expression due to the protective function of

235

phenylpropanoid compounds. Ozone can modify phenylpropanoid and lignin

236

expression pathways in leaves and stems of poplar.34 Gene expression in leaves was

237

stimulated by ozone exposure, but in stems, gene expression was decreased by ozone.

238

Xu et al.33 also found that different concentrations of methyl jasmonate (a plant

239

hormone) and wounding treatments affected the transcript levels of phenylpropanoid



240

genes.

241

A few reports have examined differences in transcription levels of triterpene

242

biosynthetic genes in different plant organs. Huang et al.35 investigated the expression

243

of amyrin synthase (AS) and amyrin oxidase (AO) in Catharanthus roseus and found

244

that both genes were more highly expressed in leaves. Here, we isolated 21 triterpene

245

biosynthesis-related genes and examined their expression levels in different organs.

246

Similarly, we found that most genes were highly expressed in leaves. The

247

transcription level of MaLUS was very high in leaves and roots, which was related to

248

higher levels of betulin and betulinic acid in these organs. We propose that MaLUS is

249

the key regulator of betulin and betulinic acid accumulation in mulberry; however,

250

further studies are needed to confirm this.

251

Rutin, Betulin, and Betulinic Acid Content. Mulberry fruit have been used for

252

centuries as food, tea, and traditional medicine in southwestern Asia. The same plant

253

materials used for qRT-PCR were used for the HPLC analysis of rutin, botulin, and

254

betulinic acid accumulation (Figure 4). Rutin content in various species have been

255

shown to differ depending on environmental factors and growth stages28, 36. Our

256

results indicated that larger amounts of rutin (6–7-fold higher) were detected in leaves

257

than that in fruits, and that rutin content decreased during fruit maturation (Figure 4).

258

Small quantities of rutin were detected in the stems. As mentioned above,

259

MaUGT78D1 was highly expressed in leaves (Figure 2); we speculate that

260

MaUGT78D1 may play an important regulatory role in the rutin biosynthetic

261

pathways, which would require further confirmation by transformation experiments.


Page 12 of 31

Page 13 of 31


262

UGT78D1, a flavonol-specific glycosyltransferase, has been shown to be responsible

263

for transferring rhamnose or glucose to the 3-OH position in vitro37.

264

The natural compound, betulinic acid, has been shown to have potent anticancer

265

activity in cancer cells, and thus, is a new and promising experimental anticancer

266

agent for the treatment of human cancers38. Large amounts of betulin were found in

267

the roots and leaves (Figure 5), whereas only a little was detected in the stems and

268

fruits (average of approximately 0.3 µg/mg DW). Leaves contained the highest levels

269

of betulinic acid, followed by the roots and stems; little or no betulinic acid was

270

detected in the fruit (Figure 5).

271

In conclusion, we conducted detailed investigations of the phenylpropanoid and

272

triterpenoid biosynthetic pathways in the mulberry plant. To our knowledge, this is the

273

first study on expression of triterpenoid biosynthetic pathway genes in mulberry. We

274

identified 18 phenylpropanoid and 21 triterpenoid biosynthetic genes with different

275

isoforms showing varied levels of expression. In addition, we found abundant rutin in

276

mulberry leaves, and abundant betulin and betulinic acid in the roots and leaves.

277

Because of the significant medicinal functions of these compounds, we believe that

278

our results will provide the basic data for research on the significant health benefits

279

and medicinal potential of mulberry roots and leaves. Furthermore, the molecular

280

characterization of genes involved in the phenylpropanoid and triterpene biosynthesis

281

broadens our understanding of the molecular mechanisms in these biosynthesis

282

pathways in Morus alba.



283

ACKNOWLEDGEMENT

284

This research was supported by Basic Science Research Program through the National

285

Research Foundation of Korea (NRF) funded by the Ministry of Education

286

(NRF-2014R1A1A4A01008939)

287

288

Notes

289

The authors declare no competing financial interest.

290

291

SUPPORTING INFORMATION

292

Supplementary file Table S1: Primers used for qRT-PCR. This material is available

293

free of charge via the Internet at http://pubs.acs.org.

294


Page 14 of 31

Page 15 of 31


295

REFERENCES

296

1.

Vogt, T., Phenylpropanoid Biosynthesis. Mol Plant 2010, 3, 2-20.

297

2.

Vijayalakshmi, A.; Kumar, P. R.; Priyadarsini, S. S.; Meenaxshi, C., In Vitro Antioxidant and

298

Anticancer Activity of Flavonoid Fraction from the Aerial Parts of Cissus quadrangularis Linn. against

299

Human Breast Carcinoma Cell Lines. J Chem-Ny 2013.

300

3.

301

Flavonoid Biosynthesis Genes and Accumulation of Phenolic Compounds in Common Buckwheat

302

(Fagopyrum esculentum). J Agr Food Chem 2010, 58, 12176-12181.

303

4.

304

of the antinociceptive action of the ethanolic extract and the triterpene 24-hydroxytormentic acid

305

isolated from the stem bark of Ocotea suaveolens. Planta Med 1999, 65, 50-5.

306

5.

307

natural medicines. IV. Aldose reductase and qlpha-glucosidase inhibitors from the roots of Salacia

308

oblonga Wall. (Celastraceae): structure of a new friedelane-type triterpene, kotalagenin 16-acetate.

309

Chem Pharm Bull (Tokyo) 1999, 47, 1725-9.

310

6.

311

constituents from Cynomorium songaricum and related triterpene derivatives on HIV-1 protease. Chem

312

Pharm Bull (Tokyo) 1999, 47, 141-5.

313

7.

314

ganoderma lucidum. Phytother Res 1999, 13, 529-31.

315

8.

316

M., Study of the betulin enriched birch bark extracts effects on human carcinoma cells and ear

Li, X. H.; Il Park, N.; Xu, H.; Woo, S. H.; Park, C. H.; Park, S. U., Differential Expression of

Beirith, A.; Santos, A. R.; Calixto, J. B.; Hess, S. C.; Messana, I.; Ferrari, F.; Yunes, R. A., Study

Matsuda, H.; Murakami, T.; Yashiro, K.; Yamahara, J.; Yoshikawa, M., Antidiabetic principles of

Ma, C.; Nakamura, N.; Miyashiro, H.; Hattori, M.; Shimotohno, K., Inhibitory effects of

Zhu, M.; Chang, Q.; Wong, L. K.; Chong, F. S.; Li, R. C., Triterpene antioxidants from

Dehelean, C. A.; Soica, C.; Ledeti, I.; Aluas, M.; Zupko, I.; A, G. L.; Cinta-Pinzaru, S.; Munteanu,



317

inflammation. Chemistry Central journal 2012, 6, 137.

318

9.

319

Betulin complex in gamma-cyclodextrin derivatives: properties and antineoplasic activities in in vitro

320

and in vivo tumor models. Int J Mol Sci 2012, 13, 14992-5011.

321

10. Wick, W.; Grimmel, C.; Wagenknecht, B.; Dichgans, J.; Weller, M., Betulinic acid-induced

322

apoptosis in glioma cells: A sequential requirement for new protein synthesis, formation of reactive

323

oxygen species, and caspase processing. The Journal of pharmacology and experimental therapeutics

324

1999, 289, 1306-12.

325

11. Liu, J. D., Study of the etiology of low-fever among female silk-weaving workers: extrinsic

326

allergic alveolitis due to mulberry silk dust exposure. Zhonghua yu fang yi xue za zhi [Chinese journal

327

of preventive medicine] 1985, 19, 354-7.

328

12. Kim, H.; Yoon, Y. J.; Shon, J. H.; Cha, I. J.; Shin, J. G.; Liu, K. H., Potent inhibition of human

329

liver CYP3A activity by mulberry juice. Drug Metab Rev 2006, 38, 50-50.

330

13. Hemmati, A. A.; Jalai, M. T.; Rashidi, I.; Hormozi, T. K., Effects of aqueous extract of black

331

mulberry (Morus nigra) on liver and kidney of diabetic mice. Toxicol Lett 2008, 180, S48-S49.

332

14. Heo, S. I.; Jin, Y. S.; Jung, M. J.; Wang, M. H., Antidiabetic properties of 2,5-dihydroxy-4,3

333

'-di(beta-D-glucopyranosyloxy)-trans-stilbene from mulberry (Morus bombycis Koidzumi) root in

334

streptozotocin-induced diabetic rats. J Med Food 2007, 10, 602-607.

335

15. Wang, Y. H.; Xiang, L. M.; Wang, C. H.; Tang, C.; He, X. J., Antidiabetic and Antioxidant Effects

336

and Phytochemicals of Mulberry Fruit (Morus alba L.) Polyphenol Enhanced Extract. PloS one 2013,

337

8.

338

16. Jiang, D. Q.; Guo, Y.; Xu, D. H.; Huang, Y. S.; Yuan, K.; Lv, Z. Q., Antioxidant and anti-fatigue

Soica, C.; Dehelean, C.; Danciu, C.; Wang, H. M.; Wenz, G.; Ambrus, R.; Bojin, F.; Anghel, M.,


Page 16 of 31

Page 17 of 31


339

effects of anthocyanins of mulberry juice purification (MJP) and mulberry marc purification (MMP)

340

from different varieties mulberry fruit in China. Food Chem Toxicol 2013, 59, 1-7.

341

17. Naowaratwattana, W.; De-Eknamkul, W.; De Mejia, E. G., Phenolic-containing organic extracts of

342

mulberry (Morus alba L.) leaves inhibit HepG2 hepatoma cells through G2/M phase arrest, induction

343

of apoptosis, and inhibition of topoisomerase IIalpha activity. J Med Food 2010, 13, 1045-56.

344

18. Radojkovic, M. M.; Zekovic, Z. P.; Vidovic, S. S.; Kocar, D. D.; Maskovic, P. Z., Free radical

345

scavenging activity and total phenolic and flavonoid contents of mulberry (Morus spp. L., Moraceae)

346

extracts. Hem Ind 2012, 66, 545-550.

347

19. Jia, D. D.; Li, S. F.; Gu, Z. P., Preparative Isolation of Flavonoids from Mulberry (Morus Alba L.)

348

Leaves by Macroporous Resin Adsorption. J Food Process Eng 2011, 34, 1319-1337.

349

20. Kumar, R. V.; Babu, G. S.; Chauhan, S.; Srivastava, A.; Rao, Y.; Kumar, D., Total phenolics and

350

flavonoids content in ripened and unripened fruits of different mulberry (Morus alba) varieties. Indian

351

J Agr Sci 2012, 82, 277-279.

352

21. Lee, Y.; Lee, D. E.; Lee, H. S.; Kim, S. K.; Lee, W.; Kim, S. H.; Kim, M. W., Influence of auxins,

353

cytokinins, and nitrogen on production of rutin from callus and adventitious roots of the white

354

mulberry tree (Morus alba L.). Plant Cell Tiss Org 2011, 105, 9-19.

355

22. Lotito, S. B.; Frei, B., Consumption of flavonoid-rich foods and increased plasma antioxidant

356

capacity in humans: cause, consequence, or epiphenomenon? Free radical biology & medicine 2006,

357

41, 1727-46.

358

23. Guardia, T.; Rotelli, A. E.; Juarez, A. O.; Pelzer, L. E., Anti-inflammatory properties of plant

359

flavonoids. Effects of rutin, quercetin and hesperidin on adjuvant arthritis in rat. Farmaco 2001, 56,

360

683-7.



361

24. Lin, J.-Y.; Tang, C.-Y., Determination of total phenolic and flavonoid contents in selected fruits

362

and vegetables, as well as their stimulatory effects on mouse splenocyte proliferation. Food Chem 2007,

363

101, 140-147.

364

25. Yan, J. W.; Wang, M.; Lu, J. D., Determination of rutin, quercetin, and chlorogenic acid in

365

mulberry leaves by capillary zone electrophoresis. Anal Lett 2004, 37, 3287-3297.

366

26. Li, J. R.; Han, X. X., Ultrahigh-pressure extraction for mulberry leaf rutin. Chinese J Anal Chem

367

2008, 36, 365-368.

368

27. He, N.; Zhang, C.; Qi, X.; Zhao, S.; Tao, Y.; Yang, G.; Lee, T. H.; Wang, X.; Cai, Q.; Li, D.; Lu,

369

M.; Liao, S.; Luo, G.; He, R.; Tan, X.; Xu, Y.; Li, T.; Zhao, A.; Jia, L.; Fu, Q.; Zeng, Q.; Gao, C.; Ma,

370

B.; Liang, J.; Wang, X.; Shang, J.; Song, P.; Wu, H.; Fan, L.; Wang, Q.; Shuai, Q.; Zhu, J.; Wei, C.;

371

Zhu-Salzman, K.; Jin, D.; Wang, J.; Liu, T.; Yu, M.; Tang, C.; Wang, Z.; Dai, F.; Chen, J.; Liu, Y.; Zhao,

372

S.; Lin, T.; Zhang, S.; Wang, J.; Wang, J.; Yang, H.; Yang, G.; Wang, J.; Paterson, A. H.; Xia, Q.; Ji, D.;

373

Xiang, Z., Draft genome sequence of the mulberry tree Morus notabilis. Nat Commun 2013, 4, 2445.

374

28. Li, X.; Kim, J. K.; Park, S. Y.; Zhao, S.; Kim, Y. B.; Lee, S.; Park, S. U., Comparative analysis of

375

flavonoids and polar metabolite profiling of tanno-original and tanno-high rutin buckwheat. J Agric

376

Food Chem 2014, 62, 2701-8.

377

29. Tuan, P. A.; Park, N. I.; Li, X.; Xu, H.; Kim, H. M.; Park, S. U., Molecular Cloning and

378

Characterization of Phenylalanine Ammonia-lyase and Cinnamate 4-Hydroxylase in the

379

Phenylpropanoid Biosynthesis Pathway in Garlic (Allium sativum). J Agr Food Chem 2010, 58,

380

10911-10917.

381

30. Tuan, P. A.; Park, W. T.; Xu, H.; Park, N. I.; Park, S. U., Accumulation of Tilianin and Rosmarinic

382

Acid and Expression of Phenylpropanoid Biosynthetic Genes in Agastache rugosa. J Agr Food Chem


Page 18 of 31

Page 19 of 31


383

2012, 60, 5945-5951.

384

31. Zhao, S.; Li, X.; Cho, D. H.; Arasu, M. V.; Al-Dhabi, N. A.; Park, S. U., Accumulation of

385

kaempferitrin and expression of phenyl-propanoid biosynthetic genes in kenaf (Hibiscus cannabinus).

386

Molecules 2014, 19, 16987-97.

387

32. Zhao, S.; Tuan, P. A.; Li, X.; Kim, Y. B.; Kim, H.; Park, C. G.; Yang, J.; Li, C. H.; Park, S. U.,

388

Identification of phenylpropanoid biosynthetic genes and phenylpropanoid accumulation by

389

transcriptome analysis of Lycium chinense. Bmc Genomics 2013, 14, 802.

390

33. Xu, H.; Park, N. I.; Li, X.; Kim, Y. K.; Lee, S. Y.; Park, S. U., Molecular cloning and

391

characterization of phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and genes involved in

392

flavone biosynthesis in Scutellaria baicalensis. Bioresour Technol 2010, 101, 9715-22.

393

34. Richet, N.; Tozo, K.; Afif, D.; Banvoy, J.; Legay, S.; Dizengremel, P.; Cabane, M., The response

394

to daylight or continuous ozone of phenylpropanoid and lignin biosynthesis pathways in poplar differs

395

between leaves and wood. Planta 2012, 236, 727-737.

396

35. Huang, L.; Li, J.; Ye, H.; Li, C.; Wang, H.; Liu, B.; Zhang, Y., Molecular characterization of the

397

pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus. Planta 2012, 236, 1571-81.

398

36. Kim, H. J.; Park, K. J.; Lim, J. H., Metabolomic analysis of phenolic compounds in buckwheat

399

(Fagopyrum esculentum M.) sprouts treated with methyl jasmonate. J Agric Food Chem 2011, 59,

400

5707-13.

401

37. Ren, G.; Hou, J.; Fang, Q.; Sun, H.; Liu, X.; Zhang, L.; Wang, P. G., Synthesis of flavonol

402

3-O-glycoside by UGT78D1. Glycoconjugate journal 2012, 29, 425-32.

403

38. Fulda, S., Betulinic Acid for cancer treatment and prevention. Int J Mol Sci 2008, 9, 1096-107.

404



Page 20 of 31

405 406

FIGURE LEGENDS

407

Figure 1. Schematic of rutin biosynthesis (a) and betulin and betulinic acid

408

biosynthesis (b). PAL, phenylalanine ammonia lyase; C4H, cinnamic acid

409

4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; CHI,

410

chalcone isomerase; F3H, flavone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase;

411

F3GT,

412

L-rhamnosyltransferase;

413

hydroxymethylglutaryl-CoA

414

3-hydroxy-3-methylglutaryl-coenzyme A reductase; MVK, mevalonate kinase; PMK,

415

phosphomevalonate kinase; MVD, diphosphomevalonate decarboxylase; DXS,

416

1-deoxyxylulose-5-phosphate

417

reductoisomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase;

418

CMK,

419

2-C-methyl-D-erythritol

420

4-hydroxy-3-methylbut-2-enyl

421

4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IDI, Isopentenyl-diphosphate

422

delta-isomerase; GPPS, geranyl diphosphate synthase; FPPS, farnesyl diphosphate

423

synthase; SQS, squalene synthetase; SQE, squalene epoxidase; LUS, lupeol synthase.

424

Figure 2. Expression levels of phenylpropanoid biosynthetic genes in different organs

425

of mulberry (Morus alba L.) calculated by relative quantification with the mulberry

426

actin housekeeping gene. Each value is the mean of three replicates, and error bars

427

indicate standard deviation.

428

Figure 3. Expression levels of triterpene biosynthetic genes in different organs of

429

mulberry (Morus alba L.) calculated by relative quantification with the mulberry actin

430

housekeeping gene. Each value is the mean of three replicates, and error bars indicate

431

standard deviations.

flavonol

3-O-glucosyltransferase; AACT,

UGT78D1,

acetyl-CoA

flavonol-3-O-glucoside

acetyltransferase;

synthase;

synthase;

DXR,

HMGR,

1-deoxy-D-xylulose-5-phosphate

4-diphosphocytidyl-2-C-methyl-D-erythritol 2,4-

HMGS,

cyclodiphosphate diphosphate


kinase;

MCS,

synthase;

HDS,

synthase;

IDS,

Page 21 of 31


432

Figure 4. Accumulation of rutin in different organs of mulberry (Morus alba L.).

433

Each value is the mean of three replicates, and error bars indicate standard deviation.

434

Figure 5. Accumulation of betulin and betulinic acid in different organs of mulberry

435

(Morus alba L.). Each value is the mean of three replicates, and error bars indicate

436

standard deviation.

437



Page 22 of 31

Table 1. Comparison of phenylpropanoid biosynthetic genes of Morus alba L. with the most orthologous genes L. chinense

Length

(Accession no.)

(amino acids)

MaPAL1

722

MaPAL2

749

MaPAL3

344

MaC4H1

525

MaC4H2

507

Ma4CL2

Ma4CL7-1

Ma4CL7-2

Ma4CL9

MaCHS1

MaCHS2

MaCHS3

365

547

539

471

257

259

399

Orthologous genes (Accession no.) Morus notabilis PAL (XM_010113926)

Identity (%) 97

Ricinus communis PAL (XM_002531631)

84

Nelumbo nucifera PAL (XM_010264565)

84

Morus notabilis PAL(XM_010109218)

97

Boehmeria nivea PAL (KP100114) Jatropha curcas PAL (XM_012226984)

87 86

Morus notabilis PAL (XM_010091807) Jatropha curcas PAL (XM_012221611)

99 78

Nelumbo nucifera PAL (XM_010263680) Morus notabilis C4H (XM_010108644)

78 98

Lonicera japonica C4H (JX068605) Theobroma cacao C4H (XM_007014477)

81 84

Morus notabilis C4H (XM_004249173) Jatropha curcas C4H (XM_012222786)

99 92

Rubus occidentalis C4H (FJ554629) Morus notabilis 4CL2 (XM_010089179)

91 99

Vitis vinifera 4CL (JN858959) Prunus persica 4CL (XM_007219446)

85 84

Morus notabilis 4CL7 (XM_010103319) Prunus mume 4CL7 (XM_008243188)

97 73

Cucumis melo 4CL7 (XM_008444488) Morus notabilis 4CL7 (XM_010113516)

67 97

Malus domestica 4CL7 (XM_008386493) Betula pendula 4CL (KM099197)

81 80

Morus notabilis 4Cl9 (XM_010106939) Prunus mume 4CL9 (XM_008241147)

81 70

Malus domestica 4CL9 (XM_008394619) Morus notabilis CHS (XM_010100833)

68 99

Vaccinium ashei CHS (AB694902) Hypericum perforatum CHS (AF461105)

93 93

Citrus sinensis CHS2 (XP_006468926) Clausena lansium CHS (KP064028)

86 86

Elaeis guineensis CHS2 (XM_010931189) Citrus limon CHS (KP720587)

87 66

Populus alba CHS (DQ371803) Cochlearia danica CHS (AF144532)

65 64 98 84 84 95

MaCHS9

218

Morus notabilis CHS9 (XM_010090374) Ricinus communis CHS (XM_002529211)

MaCHI1

228

Vitis vinifera CHS (XM_002276617) Morus notabilis CHI (XM_010103211)


Page 23 of 31


MaCHI2

308

MaCHI3

208

MaF3H

228

MaF3’H1

354

MaF3’H2

177

MaF3GT

339

MaUGT78D1

447

Prunus cerasifera CHI (KP772274) Camellia nitidissima CHI (HQ269805)

57 57

Morus notabilis CHI (KF438043) Prunus avium CHI1 (GU990525)

98 80

Populus trichocarpa CHI (XM_002315222) Morus notabilis CHI (XM_010105346)

77 98

Actinidia chrysantha CHI (KM453235) Gossypium hirsutum CHI (GU295063)

77 77

Morus notabilis F3H1 (KF438044) Dimocarpus longan F3H (EF468104)

99 92

Vitis vinifera F3H (XM_002275527) Morus notabilis F3H (XM_010107524)

91 97

Prunus avium F3’H1 (GU990527) Vitis vinifera F3’H3 (AB213604)

74 73

Morus notabilis F3’H (XM_010107523) Prunus persica F3’H (KJ484546)

97 73

Vitis vinifera F3’H (AJ880357) Morus notabilis UGT85A1 (XM_010089319)

73 95

Theobroma cacao UGT85A2 (XM_007047847) Prunus mume UGT85A5 (XM_008235943)

78 72

Morus notabilis UGT85A2 (XM_010100315) Prunus mume UGT85A2 (XM_008221095)

99 74

Theobroma cacao UGT85A2 (XM_007042858)

73



Page 24 of 31

Table 2. Comparison of triterpene biosynthetic genes of Morus alba L. with the most orthologous genes L. chinense

Length

Orthologous genes

Identity

(Accession no.)

(amino acids)

(Accession no.) Morus notabilis AACT1 (XM_010090057)

(%)

MaAACT1

403

MaAACT2

MaHMGS

MaHMGR1

MaHMGR2

MaMVK

MaPMK

416

464

548

586

356

507

MaMVD

421

MaIDI

295

MaDXS

714

MaDXR

363

MaMCT

243

MaCMK

254

98

Camellia oleifera AACT (GU594059)

92

Sesamum indicum AACT1 (XM_011102210) Morus notabilis AACT2 (XM_010093715)

91 99

Prunus mume AAC1 (XM_008226416) Cicer arietinum AACT2 (XM_004507531)

84 83

Morus notabilis HMGS (XM_010109749) Ricinus communis HMGS (XM_002509646)

98 88

Jatropha curcas HMGS (XM_012231634) Morus notabilis HMGR1 (XM_010096207)

88 97

Betula platyphylla HMGR (KJ452334) Hevea brasiliensis HMGR1 (P29057)

85 81

Morus notabilis HMGR2 (XM_010092425) Prunus mume HMGR1 (XM_008237609)

98 82

Cucumis melo HMGR1 (XM_008452963) Morus notabilis MVK (XM_010107540)

80 99

Prunus mume MVK (XM_008248266) Jatropha curcas MVK (XM_012233688)

83 82

Morus notabilis PMK (XM_010112088) Prunus mume PMK (XM_008239928)

91 84

Jatropha curcas PMK (XM_012236623) Morus notabilis MVD (XM_010090485)

82 95

Astragalus membranaceus MVD (KF355964) Glycine max MVD (XM_003555822)

86 87

Morus notabilis IDI2 (XM_010112783) Pueraria montana IDI (AY315650)

99 89

Glycine soja IDI2 (KN645832) Morus notabilis DXS (XM_010115150)

88 98

Prunus mume DXS2 (XM_008238129) Ricinus communis DXS (XM_002532338)

84 85

Morus notabilis DXR (XM_010102910) Populus trichocarpa DXR (EU693020)

99 92

Hevea brasiliensis DXR (AB294701) Morus notabilis MCT (XM_010090393)

91 87

Populus trichocarpa MCT (EU693021) Prunus mume MCT (XM_008220406)

82 71

Morus notabilis CMK (XM_010095512) Prunus mume CMK (XM_008229149)

99 81


Page 25 of 31


MaMCS

248

MaHDS

740

MaIDS

461

MaGPPS

380

MaFPPS

182

MaSQS

408

MaSES

MaLUS

524

754

Cicer arietinum CMK (XM_004499380) Morus notabilis MCS (XM_010114326)

78 96

Fragaria vesca MCS (XM_004298785) Prunus mume MCS (XM_008240732)

88 74

Morus notabilis HDS (XM_010099896) Vitis vinifera HDS (XM_002285094)

99 90

Theobroma cacao HDS1 (XM_007016661) Morus notabilis IDS (XM_010097923)

91 97

Prunus mume IDS (XM_008238530) Hevea brasiliensis IDS (AB294708)

85 85

Morus notabilis GPPS (XM_010096985) Corylus avellana GPPS (EF553534)

95 77

Elaeagnus umbellata GPPS (FJ827762) Morus notabilis FPPS1 (XM_010105650)

78 99

Cucumis sativus FPPS1 (XM_004149361) Nelumbo nucifera FPPS1 (XM_010246724)

89 88

Morus notabilis L484 (XM_010111466) Cicer arietinum SQS (XM_004507443)

98 85

Diospyros kaki SQS (FJ687954) Theobroma cacao SQE1 (XM_007018355)

85 86

Cucumis melo SQE (XM_008454464) Jatropha curcas SQE (XM_012212770)

86 83

Eucalyptus grandis LUS (XM_010050218) Morus notabilis LUS (XM_010096904)

74 72

Theobroma cacao LUS1 (XM_007014650)

73



Figure 1


Page 26 of 31

Page 27 of 31


Figure 2



Figure 3


Page 28 of 31

Page 29 of 31


Rutin 7 5.96 6

(mg/g)

5 4 3 2 1

1.09

1.07

Fruits-1

Fruits-2

0.80

0.43 0.01

0 Roots

Stems

Leaves

Figure 4


Fruits-3


Betulin 2.5

(mg/g)

2

1.88 1.67

1.5 1 0.31

0.5

0.34

0.32

0.35

Fruits-1

Fruits-2

Fruits-3

0 Roots

Stems

Leaves

(mg/g)

Betulinic acid 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

0.70

0.40 0.24 0.10

0.10 0

Roots

Stems

Leaves

Fruits-1

Fruits-2

Figure 5


Fruits-3

Page 30 of 31

Page 31 of 31


Table of Contents Graphics


Accumulation of Rutin and Betulinic Acid and Expression of

Recommend Documents