Metabolomic Characterization of Hot Pepper (Capsicum annuum

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Metabolomic Characterization of Hot pepper (Capsicum annuum ‘CM334’) During Fruit Development Yu Kyung Jang, Eunsung Jung, Hyun Ah Lee, Doil Choi, and Choong Hwan Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03873 • Publication Date (Web): 14 Oct 2015 Downloaded from http://pubs.acs.org on October 21, 2015

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Journal of Agricultural and Food Chemistry

Metabolomic Characterization of Hot pepper (Capsicum annuum ‘CM334’) During Fruit Development

Yu Kyung Jang a, Eun Sung Jung a, Hyun Ah Lee b, Doil Choi b, and Choong Hwan Lee a, *

a

Department of Bioscience and Biotechnology, Konkuk University, Seoul, Republic of

Korea. b

Department of Plant Science, Seoul National University, Seoul, Republic of Korea

*Corresponding author Choong Hwan Lee Department of Bioscience and Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea Tel.: +82-2-2049-6177, Fax: +82-2-455-4291, E-mail address: [email protected]

ACS Paragon Plus Environment


Abstract Non-targeted metabolomic analysis of hot pepper (Capsicum annuum ‘CM334’) was performed at six development stages (16, 25, 36, 38, 43, and 48 days post anthesis [DPA]) to analyze biochemical changes. Distinct distribution patterns were observed in the changes of metabolites, gene expressions, and antioxidant activities by early (16–25 DPA), breaker (36– 38 DPA), and later stages (43–48 DPA). In the early stages, glycosides of luteolin, apigenin, and quercetin, shikimic acid, GABA, and putrescine were highly distributed, but gradually decreased over the breaker stage. At later stages, leucine, isoleucine, proline, phenylalanine, capsaicin, dihydrocapsaicin, and kaempferol glycosides were significantly increased. Pathway analysis revealed metabolite-gene interactions in the biosynthesis of amino acids, capsaicinoids, fatty acid chains, and flavonoids. The changes in antioxidant activity were highly reflective of alterations in metabolites. The present study could provide useful information about nutrient content at each stage of pepper cultivation.

Key words Hot pepper, Fruit development stage, Metabolomics, Antioxidant activity, Biosynthetic pathway


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1

Introduction

2

Pepper (Capsicum annum L.) belongs to the Solanaceae family, which is one of the most

3

widely consumed spices worldwide1, 2. Pepper fruits have a unique spicy flavor and contain

4

various bioactive components such as vitamins C, E, and A, β-carotene, lycopene, and

5

polyphenols. Pepper varieties are diverse and have been categorized based on size, color,

6

flavor (pungent or non-pungent), and species into jalapeno3, bell4, Black Cuban, Hongjinju,

7

Yeokgang-hongjanggun5, and Capsicum annuum, frutescens, chinense, and baccatum6.

8

General research on pepper fruits has been associated with antioxidant activity7, nutritive

9

components8, and phenolic contents7 at each development stage. In addition, most

10

metabolomic research has focused on targeted metabolite analysis that quantified levels of

11

carotenoids, capsaicinoids, ascorbic acid, and flavonoids, which are well-known

12

phytonutrients of pepper. There is a lack of research into non-targeted metabolomic

13

approaches to study pepper. Non-targeted metabolomics could explain the responses of plants

14

to various changes in environmental conditions, as reflected by changes in metabolites. This

15

approach has been applied to the fruit ripening process of many plant species, such as Oryza

16

sativa9, 10, blueberries11, and pitayas12. In pepper, phytochemicals such as flavonoids and

17

polyphenols undergo changes during development, which could influence important dietary

18

considerations regarding the consumption of pepper13.

19

Therefore, to gain insight into the development of pepper, we aimed to investigate six

20

developmental stages of hot pepper fruits using a non-targeted metabolomics approach

21

combined with interpretation of gene expression and antioxidant activity.

22 23 24

Materials and methods Chemicals



25

Acetonitrile, water, methanol, and chloroform were purchased from Fisher Scientific

26

(Pittsburgh,

PA,

USA).

Methoxyamine

hydrochloride,

N-methyl-N-(trimethylsilyl)

27

trifluoroacetamide (MSTFA), pyridine, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic

28

acid (Trolox), naringin, gallic acid, formic acid, 2,2′-azino-bis(3-ethylbenzothiazoline-6-

29

sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6,-

30

tris(2-pyridyl)-s-triazine (TPTZ), iron(III) chloride hexahydrate, acetic acid, sodium acetate,

31

ethanol, hydrochloride, Folin-Ciocalteu’s phenol reagent, sodium carbonate, diethylene

32

glycol, potassium persulfate, and other standard compounds were obtained from Sigma

33

Chemical Co. (St. Louis, MO, USA).

34 35

Plant materials and growth conditions

36

The pepper cultivar, Capsicum annuum ‘CM334’, was used for metabolic profiling and

37

RNA sequencing. Pepper plants were grown in greenhouses at the Boyce Thompson Institute,

38

NY, USA, under standard conditions (27 °C day/19 °C night; 16 h light/8 h dark). Pericarp

39

tissue of pepper fruits were harvested at 6 days post-anthesis (DPA), 16 DPA, 25 DPA, 36

40

DPA, 38 DPA (breaker stage, Br), 43 DPA, and 48 DPA (Figure 1A). Three independent

41

biological replicates of each ripening stage were prepared. All samples were frozen in liquid

42

nitrogen and then ground to a fine powder with a mortar and pestle. The samples were stored

43

at -80 °C for 3 months. The frozen samples were then packed in dry ice, delivered to Korea,

44

and stored in a deep freezer for two years before metabolome analysis.

45 46

Metabolite extraction of hot pepper fruits (Capsicum annuum ‘CM334’)

47

For metabolome analysis, each sample was dried using a freeze dryer, extracted by

48

sonication in methanol (50 mg/mL) for 10 min at room temperature, and shaken for 1 h using

49

a Twist Shaker (Biofree, Seoul, Korea). After centrifugation (37732 g, 4 °C, 15 min) (Hettich


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50

Zentrifugen, Universal 320R, Germany), the supernatant was filtered through a 0.2 µm

51

polytetrafluoroethylene (PTFE) filter and concentrated using a speed vacuum concentrator

52

(Modulspin 31, Biotron, Korea). For gas chromatography-time of flight mass spectrometry

53

(GC-TOF-MS) analysis, methyloxime derivatives were obtained by dissolving the dry

54

extracts in 50 µL of methoxyamine-HCl (20 mg/mL in pyridine) at 30 °C for 90 min. After

55

methoximation, samples were silylated at 37 °C for 30 min, by adding 50 µL of MSTFA. For

56

Ultrahigh-performance liquid chromatography linear trap quadrupole electrospray ionization

57

ion trap mass spectrometry (UHPLC–LTQ-ESI-MS/MS) analysis, dry extracts were

58

redissolved in methanol. Three biological replications and three analytical replications of

59

each hot pepper fruit sample were obtained. Similarly, for analysis of antioxidant activities,

60

50 mg of each sample was extracted using the above procedure.

61 62

RNA-sequencing Data

63

RNA-sequencing of the pericarp of CM334 was previously performed by Kim et al.14 on

64

samples harvested from six-week-old plants at a similar stage of ripening, grown under

65

standard conditions (27 °C/19 °C; 16-h light/8-h dark) in a greenhouse in Korea.

66

Comparative analysis between gene expression and metabolite changes during the fruit

67

ripening process was performed, using the reads per kilobase per million reads (RPKM) data

68

of Kim et al.14

69 70

GC-TOF-MS analysis

71

An Agilent 7890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA),

72

installed with an Agilent 7693 auto-sampler and equipped with a Pegasus® High-Throughput

73

(HT)-TOF-MS (LECO, St. Joseph, MI, USA) system, was used for GC-TOF-MS analysis.

74

Metabolites were separated on an Rtx-5MS column (30-m i.d. × 0.25-mm length, 0.25-µm



75

particle size; Restek Corp., Bellefonte, PA, USA), with helium as the carrier gas at a constant

76

flow rate of 1.5 mL/min. Each 1 µL aliquot sample was injected into the GC with a split ratio

77

of 10:1. The front inlet, transfer line, and ion source temperatures were set at 250°C, 240°C,

78

and 230°C, respectively. The oven temperature was maintained at 75°C for 2 min, then

79

increased to 300°C at 15°C/min, and then maintained at the final temperature for 3 min.

80

Electron ionization was performed at 70 eV, and mass data were collected by full scanning

81

over a mass-to-charge ratio (m/z) range of 50–600.

82 83

UHPLC-LTQ-ESI-IT-MS/MS Analysis

84

Ultrahigh-performance liquid chromatography linear trap quadrupole Electrospray

85

ionization Ion Trap mass spectrometry (UHPLC-LTQ-ESI-IT-MS) was used to analyze hot

86

pepper fruit extracts. The Thermo Fischer Scientific LTQ ion trap mass spectrometer

87

equipped with electrospray interface (Thermo Fischer Scientific, San José, CA), and the

88

DIONEX UltiMate 3000 RS Pump, RS Autosampler, RS Column Compartment, and RS

89

Diode Array Detector (Dionex Corporation, Sunnyvale, USA) were used. Each sample (10

90

µL) was injected to and separated on a Thermo Scientific Syncronis C18 UHPLC column

91

(particle size 1.7 µm; flow rate 0.3 mL/min). The mobile phase consisted of A (0.1% (v /v)

92

formic acid in water) and B (0.1% (v /v) formic acid in acetonitrile). The gradient conditions

93

were increased from 10% to 100% of solvent B over 15 min, maintained for 3 min, then

94

decreased to 10% over 4 min. Photodiode array was set at 200–600 nm for detection and

95

managed by 3D field. Ion trap was performed in negative (-), positive (+), and full-scan ion

96

modes within a range of 150–1,000 m/z. The operating parameters were as follows: Source

97

voltage, ± 5 kV; Capillary voltage, 39 V; and Capillary Temperature, 275°C. Tandem MS

98

analysis was performed by scan-type turbo data-dependent scanning, under the same

99

conditions used for MS scanning.


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100 101 102

Determination of antioxidant activities (ABTS, DPPH, FRAP) and total flavonoid contents (TFC) of hot pepper fruit (Capsicum annuum ‘CM334’)

103

The ABTS assay protocol followed the method of Re et al.15 with some modifications. The

104

stock solution was 7 mM ABTS dissolved in 2.45 mM potassium persulfate solution and

105

stored for 12 h in the dark at 4°C. The solution was diluted until the absorbance reached 0.7 ±

106

0.02 at 750 nm, using a spectrophotometer (SpectronicGenesys 6; Thermo Electron, Madison,

107

WI, USA). Extracted pepper (20 µL) was left to react with 180 µL of diluted ABTS solution

108

for 7 min in the dark at room temperature, and the absorbance was recorded at 750 nm.

109

The DPPH assay protocol was conducted according to the method of Lee et al.16 with some

110

modifications. Each sample extract (20 µL) was left to react with 180 µL DPPH ethanol

111

solution for 20 min in the dark, at room temperature and the absorbance was measured at 515

112

nm.

113

The ferric ion reducing antioxidant power (FRAP) assay was conducted according to the

114

method of Benzie and Strain17 with some modifications. The FRAP reagent was prepared by

115

mixing acetate buffer (pH 3.6); 10 mM TPTZ (in 40 mM HCl solution); and 20 mM

116

FeCl3·6H2O in a ratio of 10:1:1. Each pepper extract (10 µL) was left to react with 300 µL of

117

FRAP reagent for 6 min in the dark, at room temperature and the absorbance was measured at

118

570 nm. For all three assays, standard curves were linear between 0.0625 and 2 mM of

119

Trolox, and results were presented in µmol Trolox equivalents (TE) per gram sample extract.

120

All assays were performed in triplicate, with similar pepper extracts as those used for mass

121

spectrometry analysis.

122

The TFC protocol was conducted according to the method of Jung et al.10 with some

123

modifications. Each pepper extract (20 µL) was left to react with 20 µL 1N NaOH and 180

124

µL of 90% diethylene glycol for 60 min in the dark at room temperature, and absorbance was



125

measured at 405 nm. Standard curves were linear between 6.25 ppm and 200 ppm of naringin

126

and results were presented in ppm naringin per gram sample extract. All experiments were

127

performed in triplicate, with similar pepper extracts as those used for mass spectrometry

128

analysis.

129 130

Data processing and statistical analysis

131

GC-TOF-MS raw data files were converted to a computable document form (*.cdf) using

132

the inbuilt data processing software of the Agilent GC system program. UHPLC-LTQ-ESI-IT-

133

MS/MS raw data files were converted using the thermo file converter program in the Thermo

134

Xcalibur software (Version 2.1; Thermo Fisher Scientific Inc., USA). After acquiring the data

135

in .cdf format, the files were subjected to preprocessing alignment by the MetAlign software

136

package (http://www.metalign.nl). After alignment, the resulting peak list was compiled as a

137

Microsoft Excel (Microsoft, Redmond, WA, USA) file. The Excel file included the corrected

138

peak retention times (min), peak areas, and corresponding mass (m/z) data for further analysis.

139

Primary and secondary metabolites were represented through multivariate statistical analysis,

140

using SIMCA-P+ 12.0 software (Version 12.0; Umerics, Umea, Sweden) to compare

141

metabolite differences during pepper fruits development, by principal component analysis

142

(PCA) and partial least-square discriminant analysis (PLS-DA). Significantly different

143

metabolites between groups were selected with variable importance in the projection (VIP)

144

values > 0.7, and a p value < 0.05. In the antioxidant activity and TFC assays, significance

145

was evaluated by analysis of variance and Duncan’s multiple range tests using PASW

146

Statistica 18 (SPAA inc., Chicago, IL, USA). In addition, pairwise comparisons between

147

metabolites and antioxidant activity and TFC assays were performed by the Pearson’s

148

correlation coefficient test using PASW Statistica 18, and correlation and heat maps were

149

created using the MEV software version 4.8 (multiple array viewer).


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150 151 152

Results Primary metabolite profiling of hot pepper fruits during development by GC-TOF-MS.

153

To investigate the alterations of various metabolites in hot pepper fruits at the development

154

stages (16, 25, 36, 38 (Br), 43, and 48 DPA), multivariate analysis was conducted to

155

applicate performed on the MS spectrum data to visualize different primary metabolites. In

156

Figure 1A, the pericarp colors of hot pepper fruits changed as development progressed:

157

immature green (16 DPA), green (25 DPA), mature green (36 DPA), orange (38 DPA), red

158

(43 DPA), and dark red (48 DPA). In addition, the image of hot pepper samples was

159

previously presented in Kim et al.14 According to principal component analysis (PCA), each

160

development stage showed clear distributions by DPA and clustered into three patterns (16

161

DPA, 25-38 DPA, and 43-48 DPA) depending on the pericarp color of hot pepper. PC1

162

(27.28%) comprised fruits before breaker stage (16-36 DPA), separated from those after

163

breaker stage (38–48 DPA); and PC2 (13.29%) comprised fruits 25-36 DPA separated from

164

those 16 DPA and 43-48 DPA (Figure 1B). Significant differences in development stages

165

were determined based on VIP value (VIP > 0.7) and p-value (p < 0.05) of partial least

166

squares 1 and 2 (PLS1 and PLS 2). Metabolites were identified with their retention times and

167

mass fragment patterns in comparison to standard compounds, NIST (National Institute of

168

Standards and Technology mass search version 2.0, 2011, USA), and in-house library.

169

Primary metabolites in four categories including 16 amino acids, 1 polyamine, 7 organic

170

acids, 1 fatty acid, 7 sugars and sugar alcohols were selected and identified as significantly

171

different during various development stages (Supporting information, Table S1 and Figure

172

S2).

173 174

Secondary metabolite profiling of hot pepper fruits during development by UHPLC-



175

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LTQ-ESI-IT-MS/MS

176

The UHPLC-LTQ-ESI-IT-MS spectrum data were used to elucidate secondary metabolite

177

changes of hot pepper fruits during development. PCA score plots showed similar clear

178

grouping patterns by DPA in Figures 1B and 1C. According to PCA, 16-36 DPA were

179

significantly divided from 38-48 DPA in PC1 (18.20%), whereas 25-38 DPA were divided

180

from both 16 DPA and 43-48 DPA in PC2 (10.52%). Significantly different secondary

181

metabolites among six development periods, VIP > 0.7 and p < 0.05 of PLS 1 and PLS 2

182

were determined. A total of 16 secondary metabolites, including 8 flavonoids, 2 capsaicinoids,

183

and 6 non-identified metabolites (N.I.) were selected (Supporting information, Figure S1).

184

These secondary metabolites were tentatively identified by retention time, molecular weight,

185

UV λ max (nm), and MS/MS fragment patterns, in comparison to standard compounds and

186

references (Supporting information, Table S2).

187 188 189

Metabolite biosynthesis related gene expressions of hot pepper fruits during development.

190

Gene expression data were selected using a one-way ANOVA with p value < 0.05 and the

191

target selections that were related to the metabolic biosynthesis pathway were processed.

192

Thirty-two selected hot pepper gene expression data were used to link the metabolic

193

biosynthesis pathway, which were classified into four types as follows: 7 glycolysis stages

194

related genes; 13 tricarboxylic acid cycle related genes; 10 capsaicinoid biosynthesis related

195

genes; and 2 genes related to secondary biosynthesis. The candidate genes in the biosynthetic

196

pathway are listed in Table 1 with pepper gene IDs, annotations, and EC numbers that are

197

based on assignments in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.

198

In Table 1, 11 enzyme genes: phosphoglycerate mutase, putative (CA02g28630),

199

argininosuccinate

lyase,

putative

(CA00g01390),

phosphoserine


aminotransferase

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200

(CA00g32170),

glutamine

synthetase

201

(CA04g16520), and related capsaicinoid biosynthesis enzymes, such as phenylalanine

202

ammonia-lyase (PAL, CA09g02410), cinnamic acid 4-hydroxylase (CA00g30980), 4-

203

coumarate: coenzyme A ligase (CA00g3869), putative caffeoyl-CoA 3-O-methyltransferase

204

(CA00g52190), putative aminotransferase (AMT, CA03g07640), and acyltransferase (CS,

205

CA02g18630) showed high levels of expression during the early stages (16 and 25 DPA),

206

which subsequently decreased. Three enzyme genes, isocitrate dehydrogenase (CA10g17090),

207

serine

208

(CA00g53140) showed high expression around the breaker stage (36 and 38 DPA). During

209

the later stages (43 and 48 DPA), ten enzyme genes: myo-inositol oxygenase (CA06g14270),

210

beta-galactosidase

211

(CA01g23920), succinate dehydrogenase (CA01g05250), threonine synthase (CA06g13720),

212

and enzymes related to the fatty acid chain biosynthesis pathway, such as branched-chain-

213

amino-acid aminotransferase (CA04g13860), acyl-ACP thioesterase (CA00g30270), and

214

putative long-chain acyl-CoA synthetase (CA07g07250), and flavanone 3-hydroxylase

215

(CA00g55430), which is associated with the flavonoid synthesis pathway, showed high levels

216

of expression.

hydroxymethyltransferase

(CA00g03060),

(CA05g12940),

(CA12g17950),

L-asparaginase

and

ornithine

serine

decarboxylase

acetyltransferase

(CA00g74620),

citrate

7

synthase

217 218 219

Changes in primary and secondary metabolites of hot pepper fruit development stages and its relation to gene expression and antioxidant activities.

220

Significant differences between primary and secondary metabolites during development

221

stages are shown in a heat map (Figure 2A), where values represent fold changes normalized

222

by an average of all values at each development stage. Metabolite changes showed three

223

dissimilar patterns: high metabolite contents at the early stage (16 and 25 DPA), around

224

breaker stage (36 and 38 DPA), and the later stages (43 and 48 DPA). The levels of 10



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225

primary metabolites: succinic acid (19), D-glucopyranose (29), ethanolamine (3), GABA (12),

226

putrescine (17), oxalic acid (18), malic acid (20), shikimic acid (22), D-gluconic acid (24),

227

and maltose (32); and 11 secondary metabolites: Luteolin 8-C-hexoside (33), apigenin 7-O-

228

glucopyranoside (35), N.I. 2 (36), luteolin 7-O-apiosyl glucoside (37), kaempferol 3-O-

229

hexoside (38), N.I. 3 (39), quercetin 3-O-rhamnoside (41), N.I. 4 (42), N.I. 5 (43), chrysoeriol

230

6,8-di-C-hexoside (45), and N.I 6 (46) were high in the early stages (16 and 25 DPA), then

231

decreased gradually until 48 DPA. Secondary metabolites were slightly increased at 48 DPA.

232

Twelve metabolites: glycine (7), glyceric acid (26), L-alanine (1), L-serine (8), L-threonic acid

233

(21), L-threonine (9), glutamine (13), L-valine (2), pyroglutamic acid (11), glutamic acid (14),

234

citric acid (23), and L-asparagine (16), were high around the breaker stage (36 and 38 DPA)

235

and their compounds showed an increase until 36 or 38 DPA, followed by a decrease. In later

236

stages (43 and 48 DPA), five primary metabolites: L-leucine (4), L-isoleucine (5), L-proline

237

(6), L-aspartic acid (10), and L-phenylalanine (15) and 3 secondary metabolites (kaempferol

238

feruloyl dihexoside (44), capsaicin (47), and dihydrocapsaicin (48) showed a gradual increase.

239

To provide a better understanding of the relationships between 48 metabolite changes and 32

240

gene expressions, we proposed a biosynthetic pathway using selected metabolites and genes

241

(Figure 3). According to this biosynthetic pathway, several metabolites and their related gene

242

expressions showed both early and later stage specific distribution patterns, as well as

243

metabolite-gene interactions. These metabolites and genes were as follows: glutamic acid

244

(14), glutamine synthetase (CA05g12940), putrescine (17), ornithine decarboxylase

245

(CA04g16520), phenylalanine (15), capsaicine (47), dihydrocapsaicin (48), capsaicinoid

246

biosynthesis

247

CA03g07640, CA02g18630, and CA08g09460), valine (2), fatty acid chain synthesis

248

enzymes (CA00g30270, CA04g13860, and CA07g07250), flavonoids (33, 35, 37, 38, 40, 41,

249

44, and 45), and flavanone 3-hydroxylase (CA00g55430).

enzymes

(CA09g02410,

CA00g30980,

CA00g38690,


CA00g52190,

Page 13 of 33


250

Antioxidant activities of hot pepper by development stage were measured using three

251

different assays (ABTS, DPPH, and FRAP), the results of which were very similar, and are

252

presented in Figure 2B, with results of total flavonoid contents. Antioxidant activity was

253

highest at the early stages (16-25 DAP), continuously decreased until the breaker stage (38

254

DPA), then increased. Antioxidant activity in the later stages (43-48 DPA) was similar to that

255

of the early stages. The patterns of antioxidant activity change by development stage were

256

similar to those of metabolite distribution. Among these metabolites,

257

phenylalanine (15), apigenin 7-O-glucopyranoside (35), luteolin 7-o-apiosyl glucoside (37),

258

kaempferol 3-O-hexoside (38), luteolin 6-C-malonyl-C-pentoside (40), quercetin 3-O-

259

rhamnoside (41), and kaempferol feruloyl dihexoside (44) showed a positive correlation with

260

antioxidant activity. However, TFC levels showed a continual decrease in progressive

261

development stages until 43 DPA, and subsequent increase at 48 DPA, almost the exact

262

pattern as the heat map of flavonoids (Figure 2A).

L-proline

(6),

L-

263 264

Discussion

265

Fruits are important dietary sources for humans and animals because they possess flavor,

266

minerals, vitamins, and fiber18. Fruits undergo substantial changes in color, aroma, nutrient

267

composition, and softening during development and ripening. These changes occur because

268

of alterations in various biochemical and physiological processes, including gene expression,

269

enzyme activity, and metabolite formation, in response to environmental perturbations15, 19, 20.

270

A large number of scientific studies have focused on the development, maturation, ripening,

271

and organogenesis of fruits21. A reasonable time of harvest and consumption has not been

272

established in pepper fruits specifically, and they are sometimes consumed before complete

273

ripening7. In the present study, we used the metabolomics approach linked with genetic

274

analysis to assess various changes in hot pepper fruit development stages. The changes in



275

pericarp colors, metabolites, gene expressions, and antioxidant activities were distinct for

276

each development stage (16, 25, 36, 38, 43, and 48 DPA). From the heat map (Figure 2A),

277

gene expression levels (Table 1), and biosynthetic pathway (Figure 3), a total of 48

278

metabolites divided into 7 categories (amino acids, polyamine, organic acids, fatty acids,

279

sugars, flavonoids, and capsaicinoid); and 32 genes associated with glycolysis, the

280

tricarboxylic acid cycle, and capsaicinoid biosynthesis; were selected as variables in hot

281

pepper fruit development stages.

282

In the early stages (16 and 25 DPA), the levels of most organic acids, including oxalic acid

283

(18), malic acid (20), and succinic acid (19), as well as putrescine (17), and maltose (32) were

284

high and gradually decreased. Organic acids are generally known to contribute to taste, flavor,

285

and overall quality of fruits22. However, the compositions of organic acids are diverse

286

depending on plant species, development stage, and tissue type23. Malic and citric acids are

287

strongly affected by fruit ripening19. Putrescine is a major polyamine, produced directly from

288

ornithine by the enzymatic action of ornithine decarboxylase. In Figure 3, the expression

289

levels of ornithine decarboxylase (CA04g16520) was consistent with the metabolic levels of

290

putrescine. Our findings were consistent with those of other researchers, such as Wan et al.24

291

and Elhadi et al.25, whose teams studied putrescine intensively, because it is related to biotic

292

and abiotic stresses26, ethylene production27, plant growth25, flowering, and fruit

293

development27. Most flavonoid contents, including luteoline-8-C-hexoside (33), apigenin-7-O-

294

glucopyranoside (35), luteolin-C-6-malonyl-C-pentoside (40), quercetin-3-O-rhamnoside (41),

295

and chrysoeriol 6,8,-di-C-hexoside (45) also exhibited high levels at 16 and 25 DPA.

296

Flavonoid contents were generally influenced by the gene flavanone-3-hydroxylase

297

(CA00g55430), which showed similar alteration patterns as those of flavonoids. According to

298

Choi et al.28, flavonoids can be degraded depending on plant development, maturation, and


Page 14 of 33

Page 15 of 33


299

ripening stages. These phenomena were also observed in other plants, such as Ziziphus

300

jujube28, Artemisia annua L.29, pear30, and sweet orange31. The metabolites shikimic acid

301

(22)32, GABA (12)33, putrescine (17)34, and flavonoids35 are well known to possess

302

antioxidant activity. The levels of these metabolites were relatively high in the early stages

303

(16 and 25 DPA) and gradually decreased, which may be related to changes in antioxidant

304

activity in the early to breaker stages.

305

High levels of most amino acids except for putrescine (17), were observed around the

306

breaker stage (including 36 and 38 DPA). Citric acid (23) levels were also highest at the

307

breaker stage. Pepper fruit color generally changed from green to red during development,

308

and an orange color, defined the changing point or breaker stage36. Amino acids are natural

309

compounds in fruits and vegetables that play an important role in maintaining fruit quality

310

and nutritional values37. L-Valine (2) is an amino acid that is a precursor to one end of the

311

capsaicin chain structure38 and the valine pathway is an important element in capsaicin

312

biosynthesis39. Its related genes include branched-chain-amino-acid aminotransferase

313

(CA04g13860), acyl-ACP thioesterase (CA00g30270), and putative long-chain acyl-CoA

314

synthetase (CA07g07250), which were highly expressed after the breaker stage.

315

At the later stages (43 and 48 DPA), levels of the hydrophobic amino acids, L-leucine (4), L-

316

isoleucine (5), and L-phenylalanine (15), as well as L-proline (6) and L-aspartic acid (10),

317

kaempferol feruloyl dihexoside (38), capsaicine (47), and dihydrocapsaicin (48) were

318

significantly higher than at earlier stages.

319

phenylpropanoid biosynthetic pathway, which is related to the production of secondary

320

metabolites in plant species. In addition, phenylalanine ammonia-lyase (CA09g02410, PAL),

321

which is a key enzyme in the biosynthesis of phenolic compounds, converts phenylalanine to

322

trans-cinnamic acid in the first step of the phenylpropanoid pathway, and is a precursor in

L-phenylalanine


(15) is a precursor of the


Page 16 of 33

323

both the capsaicinoid and flavonoid biosynthesis pathways40. Levels of PAL increased rapidly

324

until 25 DPA, and then slowly decreased until 48 DPA. A similar pattern was observed by

325

Perucka and Materska41. Other key genes involved in this pathway, including cinnamic acid

326

4-hydroxylase (CA00g30980), 4-coumarate: coenzyme A ligase (CA00g38690), putative p-

327

coumarate 3-hydroxylase (CA08g09460), putative caffeoyl CoA 3-o-methyltransferase

328

(CA00g52190),

329

(CA02g18630) showed expression patterns opposite to those of

330

capsaicine (47), and dihydrocapsaicin (48). According to Materska and Perucka42,

331

capsaicinoid contents start to accumulate at an early stage and reach maximal levels during

332

the final growth stage. Similarly, levels of antioxidant activity increased in the later stages.

333

These changes can be attributed to an increase in several metabolites including L-leucine (4)41,

334

L-isoleucine

335

capsaicin (47)45, and dihydrocapsaicin (48) 45, which are also known to have antioxidant

336

activity.

(5)43,

putative

L-proline

aminotransferase

(6)

44

,

(CA03g07640),

L-phenylalanine

and

acyltransferase

L-phenylalanine

(15),

(15)41, kaempferol glycoside (44)35,

337

In conclusion, we connected significant changes in metabolites with the expression of

338

selected enzyme genes and the antioxidant activity of six stages of hot pepper (C. annuum

339

‘CM334’) fruit development. The distribution of metabolites, gene expression, and

340

antioxidant activity were distinct for the early (16 and 25 DPA), breaker (36 and 38 DPA),

341

and later stages (43 and 48 DPA). As a result, our study suggests that non-targeted

342

metabolomics and gene regulatory network analysis are suitable approaches to interpret

343

biochemical changes in the development stages of hot pepper.

344


Page 17 of 33


345

ABBREVIATIONS USED

346

DPA, days post anthesis; MSTFA, N-methyl-N-(trimethylsilyl)-trifluoroacetamide; Trolox, 6-

347

hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic

348

ethylbenzothiazoline-6-sulfonic

349

picrylhydrazyl; FRAP, ferric ion reducing antioxidant power; TFC, total flavonoid contents;

350

TPTZ, 2,4,6,-tris(2-pyridyl)-s-triazine; TE, Trolox equivalents; PCA, principal component

351

analysis; PLS-DA, partial least squares-discriminant analysis; VIP, variable importance in the

352

projection; GC-TOF-MS, gas chromatography-time of flight-mass spectrometry; UHPLC-

353

LTQ-ESI-IT-MS/MS, Ultrahigh-performance liquid chromatography linear trap quadrupole

354

electrospray ionization Ion Trap mass spectrometry; N.I., Non-identified metabolites

acid)

acid;

diammonium

salt;


ABTS, DPPH,

2,2′-azinobis(31,1-diphenyl-2-


Page 18 of 33

355

Acknowledgments

356

This work was supported by a grant from the Next-Generation BioGreen 21 Program (No.

357

PJ01109403), Rural Department Administration, Republic of Korea and was carried out with

358

the support of Basic Science Research Program through the National Research Foundation of

359

Korea

360

2014R1A2A1A11050884).

(NRF)

grant

funded

by

the

Korea

government

(MSIP)

(No.

NRF-

361 362

Supporting Information

363

GC-TOF-MS analysis chromatograms of hot pepper fruits during development; UHPLC-LTQ-

364

ESI-IT-MS/MS analysis chromatogram of hot pepper fruits during development; List of primary

365

metabolites of hot pepper fruits at different development stages using GC-TOF-MS and UHPLC-

366

LTQ-ESI-IT-MS/MS (PDF). This material is available free of charge via the Internet at

367

http://pubs.acs.org.


Page 19 of 33


368

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Page 26 of 33

Page 27 of 33


Figure Captions

Figure 1. Experimental design of hot pepper CM 334 (Capsicum annuum.) harvested at six development stages (A). Principal component analysis (PCA) score plots derived from non-targeted metabolite profiling of hot peppers analyzed by GC-TOF-MS (B) and UHPLC -LTQ-ESI-IT-MS (C). (▲ 16 DPA; ▲ 25 DPA; ▲ 36 DPA; ▲ 38 DPA (Br); ▲ 43 DPA; ▲ 48 DPA). Figure 2. Heat map of significantly changed primary (left) and secondary (right) metabolites analyzed by GC-TOF-MS and UHPLC-LTQESI-IT-MS, respectively (A); antioxidant activity tests ABTS (◆), DPPH (■), FRAP (▲), and total flavonoid contents (●) (B) during hot pepper development stages (16, 25, 36, 38, 43, and 48 DPA). Values represent fold changes normalized by an average of all values. Figure 3. The proposed biosynthetic pathway and relative metabolite contents and gene expressions in six development stages of hot peppers (16, 25, 36, 38, 43, and 48 DPA). The pathway was modified from the KEGG database (http://www.genome.jp/kegg/). EC numbers for the mentioned enzymes are as follows: 1.13.99.1 (myo-Inositol oxygenase), 3.2.1.23 (Beta-galactosidase), 5.3.1.9 (Glucose6-phosphate isomerase), 4.2.1.13 (Fructose-bisphosphate aldolase), 5.4.2.12 (Phosphoglycerate mutase), 2.6.1.52 (Phosphoserine aminotransferase), 2.1.2.1 (Serine hydroxymethyltransferase), 2.3.3.1 (Citrate synthase), 1.1.1.37 (Malate dehydrogenase), 4.2.1.2 (Fumarase), 1.3.5.1 (Succinate dehydrogenase), 1.1.1.41 (Isocitrate dehydrogenase [NADP]), 3.5.1.1 (L-Asparaginase), 4.3.2.1 (Argininosuccinate lyase),



Page 28 of 33

1.4.3.16 (L-aspartate oxidase), 2.3.1.31 (Serine acetyltransferase 7), 2.7.1.39 (Homoserine kinase), 4.2.3.1 (Threonine synthase), 6.3.1.2 (Glutamine synthetase), 2.6.1.42 (Branched-chain-amino-acid aminotransferase), 4.3.1.5 (Phenylalanine ammonia-lyase), 1.14.13.11 (Cinnamic acid 4-hydroxylase), 6.2.1.12 (4-coumarate:coenzyme A ligase), 1.14.13 (Putative p-coumarate 3-hydroxylase), 2.1.1.104 (Putative caffeoyl-CoA 3-O-methyltransferase), 1.14.11.9 (Flavanone 3-hydroxylase), 1.14.11.23 (Flavonol synthase/flavanone 3-hydroxylase), 6.2.1.3 (Putative long-chain acyl-CoA synthetase), 3.1.2.14 (Acyl-ACP thioesterase), AMT* (Putative aminotransferase), and CS* (Acyltransferase).


Page 29 of 33


Tables Table 1. List of metabolite biosynthesis related genes of hot pepper at six development stages pepper gene ID a

Annotation b

EC number c

16 DAP

25 DAP

36 DAP

38 DAP

43 DAP

48 DAP

p-value

CA05g12940 CA09g02410 CA00g30980 CA00g38690 CA00g52190 CA03g07640 CA02g18630 CA04g16520 CA00g01390 CA02g28630 CA00g32170 CA10g17090 CA12g17950 CA09g12650 CA00g53140 CA00g30270 CA01g23920 CA04g13860 CA06g13720 CA00g74620 CA00g55430 CA01g05250 CA00g03060 CA06g14270 CA07g07250 CA03g09540 CA00g97170 CA08g09460 CA09g01230 CA03g29880 CA00g62320 CA09g02560

Glutamine synthetase Phenylalanine ammonia-lyase Cinnamic acid 4-hydroxylase 4-coumarate:coenzyme A ligase Putative caffeoyl-CoA 3-O-methyltransferase Putative aminotransferase Acyltransferase Ornithine decarboxylase Argininosuccinate lyase, putative Phosphoglycerate mutase, putative Phosphoserine aminotransferase Isocitrate dehydrogenase [NADP] Serine hydroxymethyltransferase Malate dehydrogenase Serine acetyltransferase 7 (Fragment) Acyl-ACP thioesterase Citrate synthase Branched-chain-amino-acid aminotransferase Threonine synthase L-Asparaginase, putative Flavanone 3-hydroxylase Succinate dehydrogenase, putative Beta-galactosidase Myo-inositol oxygenase Putative long-chain acyl-CoA synthetase L-Aspartate oxidase, putative Homoserine kinase, putative Putative p-coumarate 3-hydroxylase Fructose-bisphosphate aldolase Fumarase Glucose-6-phosphate isomerase Flavonol synthase

6.3.1.2 4.3.1.5 1.14.13.11 6.2.1.12 2.1.1.104 AMT CS 4.1.1.17 4.3.2.1 5.4.2.12 2.6.1.52 1.1.1.41 2.1.2.1 1.1.1.37 2.3.1.31 3.1.2.14 2.3.3.1 2.6.1.42 4.2.3.1 3.5.1.1 1.14.11.9 1.3.5.1 3.2.1.23 1.13.99.1 6.2.1.3 1.4.3.16 2.7.1.39 1.14.13.4.1.2.13 4.2.1.2 5.3.1.9 1.14.11.23

2.70 1.31 1.54 1.52 1.49 1.77 3.85 1.78 1.60 1.40 1.31 1.51 0.85 1.03 0.30 0.31 0.69 0.25 0.71 0.54 0.47 0.61 1.03 0.08 0.63 0.75 0.81 0.85 0.97 1.07 1.10 1.25

2.00 1.95 2.10 1.96 1.82 1.98 1.36 1.10 0.99 1.11 0.20 0.72 0.91 0.85 0.35 0.33 0.58 0.29 0.74 0.94 0.91 1.04 0.00 0.17 0.91 0.98 0.89 1.23 1.13 0.90 1.06 1.03

0.74 1.37 1.08 1.14 1.37 1.56 0.80 0.51 1.13 0.88 0.89 1.80 1.24 0.87 0.48 0.59 0.84 0.18 0.82 0.91 0.72 0.96 0.00 0.07 0.97 1.27 1.00 1.04 1.08 0.92 1.15 1.36

0.23 0.52 0.45 0.42 0.42 0.63 0.00 0.75 0.96 0.77 0.78 1.39 1.14 1.23 1.92 1.58 1.20 0.30 1.06 1.09 0.63 0.46 0.00 0.13 1.08 0.99 1.22 1.05 0.99 1.01 0.96 1.05

0.18 0.68 0.65 0.60 0.46 0.06 0.00 1.31 0.84 1.06 1.03 0.44 0.96 1.30 1.52 1.87 1.60 1.40 1.05 1.06 0.77 0.56 0.51 0.08 1.18 0.91 1.21 1.09 1.31 1.30 0.99 1.24

0.15 0.17 0.18 0.36 0.44 0.00 0.00 0.55 0.48 0.77 0.79 0.14 0.90 0.73 1.43 1.33 1.09 3.58 1.61 1.45 2.50 2.36 4.46 5.47 1.25 1.10 0.87 0.74 0.51 0.80 0.74 0.07

0.0000 0.0014 0.0000 0.0000 0.0001 0.0000 0.1825 0.0066 0.0008 0.0422 0.0081 0.0025 0.0058 0.0000 0.0023 0.0016 0.0000 0.0045 0.0000 0.0000 0.0000 0.0005 0.0400 0.0012 0.0000 0.0035 0.0021 0.1847 0.0002 0.0066 0.0102 0.0085

Values represent fold changes normalized by an average of all values at each development stage. a Gene number is approximately the same as tomato (International Tomato Annotation Group (iTAG) v2.3; 34,771 genes) and potato (Potato Genome Sequencing Consortium (PGSC) v3.4; 39,031 genes), which suggests a similar gene number in Solanaceae plants. Genes selected by p < 0.05. b Biological information in sequences. c Enzyme commission number for enzyme, KEGG database (http://www.genome.jp/kegg/) (Nonexistent EC numbers marked *). Colors indicate the changes in hot pepper fruit gene expressions during development stages. The custom color schemes are as follows: lower limit value = 0 (blue), midpoint value = 1 (white), and upper limit = 2 (red).



Figures Figure 1.


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Figure 2.



Figure 3.


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Table of Contents Graphics


Metabolomic Characterization of Hot Pepper (Capsicum annuum

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