Volatile Compound and Gene Expression Analyses Reveal Temporal

Subscriber access provided by Olson Library | Northern Michigan University

Article

Volatile Compound and Gene Expression Analyses Reveal Temporal and Spatial Production of LOX-derived Volatiles in Pepino (Solanum muricatum Aiton) Fruit and LOX Specificity Carolina Contreras, Wilfried G. Schwab, Mechthild Mayershofer, Mauricio González-Agüero, and Bruno Giorgio Defilippi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01569 • Publication Date (Web): 01 Jul 2017 Downloaded from http://pubs.acs.org on July 2, 2017

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 36

Journal of Agricultural and Food Chemistry

Volatile Compound and Gene Expression Analyses Reveal Temporal and Spatial Production of LOX-derived Volatiles in Pepino (Solanum muricatum Aiton) Fruit and LOX Specificity

Carolina Contreras1, Wilfried Schwab2, Mechthild Mayershofer2, Mauricio GonzálezAgüero1, and Bruno G. Defilippi1.

1

Institute for Agricultural Research, INIA-La Platina, Postharvest Unit. Santa Rosa 11610,

Santiago, Chile. 2

Technical University of Munich, Center of Life and Food Science Weihenstephan,

Biotechnology of Natural Products, Liesel-Beckmann-Str. 1. 85354 Freising, Germany.

Corresponding author: Bruno Defilippi. Institute for Agricultural Research, INIA-La Platina, Postharvest Unit. Santa Rosa 11610, Santiago, Chile. Fax: (+56) 225779104, Phone: (+56) 225779161, E-mail: [email protected]

ACS Paragon Plus Environment


1

ABSTRACT

2

Lipoxygenase (LOX) is an important contributor to aroma compounds in most fresh

3

produce; however, little is known about the LOX pathway in pepino (Solanum muricatum

4

Aiton) fruit. We explored the LOX aroma compounds produced by the flesh and the peel

5

and identified eight putative LOX genes expressed in both tissues during fruit growth and

6

development during two consecutive seasons. This study shows that pepino produces C5,

7

C6, and C9 LOX-derived compounds. Odorant C9 volatiles were produced during

8

immature stages with a concomitant decrease when the fruit ripens, whereas C5 and C6

9

compounds were formed throughout ripening. Trans-2-hexenal and its alcohol were

10

produced in the peel, but not detected in the flesh. The expression of three genes, SmLOXD

11

(putative 13-LOX), SmLOXB and SmLOX5-like1(putative 9-LOXs), increased during fruit

12

ripening. These genes may account for aroma volatiles in pepino. Here, we discuss the

13

possible roles of individual LOX genes in pepino.

14

15

16

17

18

KEYWORDS: Pepino, Solanum muricatum, lipoxygenase, aroma, LOX genes

19

20

21


Page 2 of 36

Page 3 of 36


22

INTRODUCTION

23

Pepino (Solanum muricatum Aiton) is a fragrant and sweet fruit, with a juicy melting flesh,

24

suitable for use in desserts or salads depending on the pepino cultivar and its

25

sweetness/acidity characteristics.1 The pepino is a perennial herbaceous species, native to

26

the Andean region and has been domesticated since the Pre-Columbian era.2,3 It belongs to

27

the Solanaceae family and is a close relative of the tomato (Solanum lycopersicum) and the

28

potato (Solanum tuberosum).4

29

The pepino fruit is particularly aromatic and has been reported to synthesize volatiles

30

derived from different precursors such as amino acids, lipids and carotenoids.5 These

31

volatile compounds are mainly composed of terpenes, aldehydes, alcohols and esters

32

among other exotic notes (mesifuran, lactones, and β-damascenone).1 After the terpenoids,

33

which are derived from the cytosolic mevalonic pathway (MVA) and the plastidial

34

methylerythritol phosphate (MEP) pathways,6 the most abundant volatiles found in pepino

35

are C6 compounds such as (E)-2-hexenal, hexanal and hexyl acetate 7,8 mostly derived from

36

the lipoxygenase (LOX) pathway.

37

The LOX pathway has been well documented in the generation of C6 and C9 volatiles.9 C6

38

volatiles such as hexanal, (E)-2-hexenal, (Z)-3-hexenal and their corresponding alcohols

39

and esters, are the most common volatiles produced by the LOX pathway. Moreover, they

40

are found in essentially all fresh produce but are especially important in the aroma profile

41

of relevant commercial produce such as tomatoes and apples.10,11 Whereas C9 volatiles,

42

such as (Z,Z)-3,6-nonadienal, (E)-2-nonenal and (Z)-3-nonenal and their corresponding

43

alcohols and esters, are commonly produced in high abundance by cucumber 12 and melon

44

13

fruit.



45

Compared to C6 and C9 compounds, the synthesis of C5 volatiles by the LOX pathway has

46

not yet been fully elucidated. A pathway for the C5 volatile synthesis has been proposed to

47

occur under low oxygen conditions,14 which involves two separate LOX reactions: the

48

generation of a hydroperoxide 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid

49

(13-HPOT) followed by an alkoxyl radical. This alkoxyl radical would undergo β-scission

50

(non-enzymic cleavage and a purely chemical event) to generate the C5 alcohols.14 Fall et

51

al. 15 reported on a variety of plant leaves which produce under freeze-damaged or

52

senescing conditions, substantial amounts of C5 volatiles compared with C6 volatiles,

53

mainly due to two possible reasons: i) low oxygen conditions in frozen and dried senescent

54

leaf tissues are the result of high levels of LOX present in leaves and the rapid LOX-

55

oxygen-dependent conversion and/or ii) freezing may inactivate the alcohol dehydrogenase

56

(ADH) system in leaves, affecting the alcohols and their acetate esters, and pushing the 13-

57

HPOTs toward the C5 volatile formation. Recently, Shen et al. 16 demonstrated that the

58

same lipoxygenase that acts on the substrate for the production of C6 volatiles leads to the

59

formation of 5-carbon compounds. Thus, they concluded that C5 synthesis is independent

60

of the hydroperoxide lyase (HPL) branch since 13-HPOT could also be catalyzed by the

61

same LOX. In tomato, the most common C5 volatiles found (and relevant for consumer

62

liking) are 1-penten-3-one, (E)-2-pentenal, 3-pentanone, 1-pentanol, and 1-penten-3-ol. 17

63

Moreover, 1-penten-3-ol and (E)-2-pentenol, and their derivatives 1-penten-3-one and (E)-

64

2-pentenal accumulate in vitro in soybeans,14 ozone-treated lima bean plants,18 LOX-

65

depleted mutants of potato and HPL-depleted mutants of Arabidopsis.19,20 To our

66

knowledge, no C5 LOX-derived compounds have ever been reported in pepino fruit.


Page 4 of 36

Page 5 of 36

67


The LOX family is divided into two main groups based on the reactions they

68

catalyze: the 9-LOX and the 13-LOX group but dual functional (9- and 13- specific) LOX

69

has also been reported.21 This classification operates according to substrate specificity.

70

Thus, a LOX acting at the C9 position of the fatty acid is termed a 9-lipoxygenase (9-LOX)

71

and a LOX acting at the C13 position is termed a 13-lipoxygenase (13-LOX). These

72

reactions generate 9- and 13-hydroperoxides of the fatty acid substrate, respectively.9

73

Further, if the substrate is linolenic acid the derived hydroperoxide is 13-HPOT, but if the

74

substrate is linoleic acid, the product is 13(S)-hydroperoxy-9(Z),11(E) -octadecadienoic

75

acid (13-HPOD). Considerable evidence regarding the expression of LOX family genes has

76

been reported, indicating that there are specialized LOX genes for the generation of

77

volatiles. In tomato, TomloxC, one of the six LOX genes identified, is required for C6

78

aldehyde formation.22 Similar studies demonstrated the same for potato LOX-H1 19 and

79

apple MdLOX1a.21

80

Among fruit tissues, it has been shown that epidermal tissues produce a greater amount of

81

volatiles than flesh (or mesocarp).23,24 There are also higher levels of LOX-derived C6

82

volatiles in the peel of tomatoes,24,25 apples,23,26 and kiwifruit 27 in contrast to the flesh of

83

the fruit. However, it is unclear whether this higher capacity for aroma production by the

84

peel is due to the abundance of fatty acid substrates, the higher overall metabolic activity of

85

the peel, higher specific LOX enzyme activity or induced LOX gene expression.

86

87

The objective of this study was to investigate the LOX-derived volatiles synthesized in the

88

peel and flesh of pepino fruit and to analyze their relationship with the candidate LOX

89

genes found in pepino. The study provides the first insights into the LOX pathway of



90

pepino species, and the mechanism by which the flesh and peel differentially produce the

91

aroma volatiles.

92

93

MATERIALS AND METHODS

94

Chemicals. The organic solvent diethyl ether (˃ 99.7% GC purity) and the internal

95

standard 1-heptanol (˃ 99.5% GC purity) were purchased from Sigma-Aldrich

96

(Taufkirchen, Germany).

97

Plant material. Pepino fruits were collected between January and March 2015, and

98

between February and April 2016, from a commercial orchard located in Ovalle

99

(30°33’22.854’’S 71°38’43.004’’W), northern Chile. Sound plants with similar vigor

100

characteristics were selected in the field, and 150 uniform pepino fruits were labeled

101

approximately 10 days after fruit set (Stage 1). Twenty-five labeled fruits were harvested

102

from the field every 2-3 weeks (Stages 2, 3, 4 and 5) during fruit development until ~90

103

days after fruit set (Stage 6). Stages 1 through 4 corresponded to unripe and immature fruit,

104

stage 5 was a fully ripe fruit coincident with the commercial harvest, and stage 6

105

corresponded to overripe or senescent fruit. All fruits showing insect damage or external

106

defects were removed from the experiment. The collected fruits were hand-picked, placed

107

in fruit trays to avoid bruising and abrasion during transportation, and transported to the

108

Postharvest Laboratory at INIA-La Platina in Santiago (Chile). On each harvest, twenty

109

fruit were used for the assessment of quality parameters and five fruits were used for

110

volatile analysis.

111


Page 6 of 36

Page 7 of 36


112

Volatile extraction. In 2015, flesh with peel from pepino samples were analyzed. In 2016,

113

whole intact fruit, and flesh with and without peel were analyzed. Placentary tissue and

114

seeds were removed from flesh. For the flesh and peel samples, a small-scale liquid/liquid

115

extraction (LLE) method 28 was used for the volatile analysis. Amounts of approximately

116

16 g each of flesh with and without peel were cut from the fruit with a knife, placed into a

117

50 mL screw-capped falcon tube and homogenized with Ultra-Turrax T18 (IKB, Staufen,

118

Germany). Then, 50 µL of 1-heptanol (8.22 µg/mL) (internal standard) was added to the

119

homogenate and centrifuged (13,500 rpm, 10 min, room temperature); the supernatant was

120

transferred to a new 50 µL Falcon tube and centrifuged again (13,500 rpm, 15 min, room

121

temperature). The clear supernatant was transferred to a 50 mL pear-shaped distillation

122

flask and 20 mL of diethyl ether was added. After gentle agitation, the upper phase (organic

123

solvent) of the extract was transferred to a new 50 mL pear-shaped distillation glass. The

124

solvent was removed using a Vigreux column at 42 °C, concentrated to ~1 mL, and

125

immediately injected (1 µL) in a GC-MS. The volatile compounds were expressed as 1-

126

heptanol equivalents (assuming all of the response factors were 1).

127

For the intact fruit analysis, a whole fruit was placed in a 1 L glass jar closed with an

128

aluminum lid following 5 min incubation period at room temperature. Headspace

129

compounds were trapped by SPME (65 µm polydimethylsiloxane-divinylbenzene coated

130

fiber, Supelco, Steinheim, Germany) at room temperature for 20 min, and immediately

131

desorbed for 5 min in the GC-MS injector.

132 133

Analysis of volatiles by GC-MS. The volatile compounds collected by liquid/liquid

134

extraction or by SPME, were analyzed on a Thermo Finnigan Trace DSQ mass



135

spectrometer coupled to a 0.25 µm BPX5 20 M fused silica capillary column with a 30 m x

136

0.25 mm inner diameter. Helium (1.1 mL min-1) was used as carrier gas. Alternatively, a

137

Zebron ZB-Wax 60 m column with 0.32 mm ID and 0.25 µm FD was used. The injector

138

temperature was 250 °C, set for splitless injection. The temperature program for the

139

liquid/liquid extraction samples was 40 °C for 2 min, ramped to 140 °C at a rate of 4 °C

140

min-1, at 140 °C for 2 min, ramped to 230 °C at 10 °C min-1, at 230 °C for 10 min.

141

Likewise, the temperature program for the headspace volatiles was 40 °C for 8 min, ramped

142

to 100 °C at a rate of 4 °C min-1, ramped to 190 °C at a rate of 30 °C min-1, at 190 °C for 5

143

min, ramped at 30 °C min-1 to 230 °C, at 230 °C for 15 min. The ion source temperature

144

was 250 °C. Mass range was recorded from m/z 50 to 300, and the spectra were evaluated

145

with the Xcalibur Software version 1.4 supplied with the device. Identification of all

146

quantified compounds was made by the comparison of the mass spectrum with the

147

authenticated reference standards and with spectra in the National Institute for Standard and

148

Technology (NIST) mass spectral library (Search Version 1.5). The volatiles of extracted

149

pepino samples were both identified and quantified, whereas in the intact fruit, the volatiles

150

were only identified.

151

152

RNA extraction and cDNA synthesis. Total RNA, from 1-3 g of frozen tissue of flesh and

153

peel from each developmental stage (2015 and 2016 years), was extracted using the

154

modified hot borate method.29 The RNA quantity was calculated using a Qubit® 2.0

155

fluorometer (Invitrogen™, Eugene, OR, USA). The RNA quality was assessed by

156

spectrophotometry (Picodrop) measuring the A260/280 and A260/230 ratios and by

157

electrophoresis through a 1.5% formaldehyde-agarose gel. Two micrograms of total RNA


Page 8 of 36

Page 9 of 36


158

were pretreated with RNase-free DNase I (Epicentre, Madison, WI, USA) to remove

159

contaminating genomic DNA. First-strand cDNA was synthesized from 2 µg of total RNA

160

treated, using MMLV-RT reverse transcriptase (Promega, Madison, WI, USA) and oligo

161

dT primers according to a standard procedure. Each cDNA sample was diluted to 50 ng µL-

162

1

163

leaf of pepino were also extracted and cDNA synthesized as described above. This cDNA

164

was used to perform RT-PCR gene expression of the different plant tissues.

before use in qPCR assays. Additionally, 1-3 g of RNA of frozen tissue of root, shoot, and

165

166

Identification and isolation of partial cDNAs for lipoxygenase genes. Degenerate

167

primers were designed from conserved sequences of the closest Solanaceae members

168

known for pepino: tomato (Solanum lycopersicum) and potato (Solanum tuberosum)

169

(Supporting Information Table S1). Additionally, LOX degenerate primers described in the

170

literature for potato 5-LOX (GenBank Accession CAA64765)30 were also tested. The PCR

171

programs involved an initial denaturation step at 94 °C for 1 min, followed by 35 cycles of

172

94 °C for 30 s, 50–60 °C (depending on the primer characteristics) for 30 s, and 72 °C for

173

60 s, and a final extension step at 72 °C for 10 min. All PCR reactions were performed in a

174

MyCycler thermal cycler (Bio-Rad). The PCR products were verified by electrophoresis on

175

a 1.5% (w/v) agarose gel containing ethidium bromide and cloned into the pGEM-T Easy

176

vector (Promega) followed by sequencing (Macrogen Corp., Seoul, Korea). All primary

177

sequences were compared to sequences from National Center for Biotechnology

178

Information (NCBI) using BLAST alignment programs. Later, specific qPCR primers for

179

pepino were designed on the cloned sequences. PCR program and the cloning and

180

sequencing procedures were the same as described above.



181

182

Real-Time Quantitative PCR (qPCR) assays of flesh and peel.

183

The transcript abundance of the eight lipoxygenase genes identified in this study was

184

analyzed by qPCR with a LightCycler® 96 Real-Time PCR System (Roche Diagnostics,

185

Mannheim, Germany) using LC-FastStart DNA Master SYBR Green I to measure

186

amplified RNA-derived DNA products and gene-specific primers (Supporting Information

187

Table S1), as described by Gonzalez-Agüero et al.31 qPCR was performed on four

188

replicates, and the gene expression values were normalized to the SmTCPB gene (GenBank

189

KY761980).

190

191

RESULTS AND DISCUSSION

192

LOX-derived volatile compounds. Ten compounds presumably derived from the

193

lipoxygenase pathway were identified and quantified by GC-MS in both years of study

194

(Figure 1). Of these, half were alcohols, four aldehydes and one ester. C5, C6 and C9

195

compounds were found in pepino.

196

Only two C5 LOX-derived compounds were detected: 1-penten-3-ol and (Z)-2-penten-ol

197

(Figure 1). The highest emission for both volatiles occurred at stage 5 (commercial harvest;

198

70 days after fruit set) and stage 6 (90 days after fruit set) in 2015 and 2016, respectively.

199

Previous studies in pepino cultivars such as El Camino, Kawi, Suma,7 Sweet Round, Sweet

200

Long 32 and six other parent clones and their hybrids 1,33 did not report these two

201

compounds or any other C5 volatiles. In a close relative such as tomato, one of the most

202

abundant C5 volatiles produced is 1-penten-3-one,25 and 1-penten-3-ol and (Z)-2-penten-ol,


Page 10 of 36

Page 11 of 36


203

among other C5 volatiles. These three C5 compounds are the most important for consumer

204

liking of tomato flavor.17

205

Regarding the C6 compounds, the major compound produced in pepino was (E)-2-hexenal,

206

followed by (Z)-3-hexenol, hexanal and 1-hexanol. Shiota et al.7 also found these

207

compounds in the three pepino cultivars they studied; however, the highest C6 volatile

208

produced was hexyl acetate, a compound almost undetectable in our study. Rodríguez-

209

Burruezo et al. 34 also found that (E)-2-hexenal was the main C6 volatile produced,

210

followed by hexanal and hexyl acetate in their pepino breeding study of three parent clones

211

and their hybrids. Moreover, Rodríguez-Burruezo et al. 34 reported (E)-2-hexenal as the

212

major volatile produced followed by hexanal in the cv. Valencia. Compared to the pepino’s

213

closest cultivated relative, volatile aroma studies in several tomato varieties have shown

214

that the largest C6 volatile production corresponded to (Z)-3-hexenal, followed by its

215

isomer (E)-2-hexenal.25 Hexanal and hexanol are also abundant in tomato fruit.22

216

Two C9 compounds were also found in pepino: (E)-2-nonenal and (E,Z)-2,6-nonadienal.

217

These C9 compounds were produced in small amounts (100-200 µg.kg-1 equivalent of 1-

218

heptanol) and in an opposite pattern compared to the C5 and C6 volatiles, showing higher

219

volatile production during early stages of development and then decreasing as the fruit

220

ripened. These two compounds give cucumber its ‘fresh green’ odor; 12 thus, they may also

221

be conferring the ‘green’ notes at the immature stages of pepino fruit. (E)-2-nonenal and

222

(E,Z)-2,6-nonadienal have also been found in pepino accessions, although the highest C9

223

produced was (Z)-2-nonenal.33 Others authors have also reported (E)-2-nonenal in pepino

224

cvs. Suma and Kawi, but it was not detected in the cv. El Camino.7 Shiota et al.7 in the

225

same study reported the production of different C9 compounds, such as (Z)-6-nonenal, and



226

its alcohol and ester, which were all present in mature stages of pepino fruit. According to

227

Shiota et al.7 all C9 compounds except, (Z)-6-nonenol and (Z)-6-nonenyl acetate which give

228

a cucumber and melon-like note to pepino fruit, do not contribute to a pleasant aroma in

229

pepino. None of the compounds derived from (Z)-6-nonenal were detected in the present

230

study.

231

Regarding intact fruit analysis, no LOX-derived volatiles were found in pepino, except for

232

a small amount of hexyl acetate at stage 5 (Supporting Information Figure S1) consistent

233

with the fact that LOX volatiles are immediately and mainly produced after cell

234

disruption.35 A small production of a C6 volatile in intact fruit is not uncommon, as the

235

precursors for hexyl acetate can also be formed from β-oxidation of fatty acids rather than

236

the LOX pathway.36 It has also been suggested, that the LOX pathway might contribute to

237

the straight-chain ester production in intact tissues by channeling aldehydes derived from

238

the linoleic acid, which would yield hexanal, hexanol, and hexyl acetate, to the metabolic

239

pathway.21 However, it is unknown whether the LOX pathway contributes in a meaningful

240

way to the aroma compounds production in the intact fruit. Additionally, several esters

241

(mostly terpenes) were found and showed higher peak heights relative to hexyl acetate.

242

243

Peel versus flesh volatile production.

244

There were slight differences between the amount of volatiles produced by the peel+flesh

245

during 2016 (Figure 1) and the flesh (Figure 2). The flesh always produced less volatiles

246

than the peel+flesh. Interestingly, other authors 32 have reported that the peel of the pepino

247

fruits had low percentages of acetates and alcohols. Ruiz-Beviá et al. 32 found that only one


Page 12 of 36

Page 13 of 36


248

peel sample (with a part of the pulp) had higher acetate content. In other fresh produce,

249

such as tomato, apple and kiwifruit, it has been previously shown, that the volatile

250

production in the peel is several times greater than in the flesh or whole fruit.23,24,26,27 For

251

instance, apple peel contains nine times higher levels of ethyl 2-methylbutyrate compared

252

with the flesh.23

253

An intriguing finding in our study was that there was no production of (E)-2-hexenal

254

(Figure 2, middle) in the flesh samples; therefore, hexanal was the major C6 volatile

255

produced in the flesh instead of (E)-2-hexenal. As a result, the current study investigated in

256

more detail the production of LOX-derived products in different tissues in one sample fruit

257

(Figure 3).

258

Figure 3 shows that there was no production of (E)-2-hexenal and its alcohol (E)-2-hexenol

259

in the flesh. This would suggest that the C6 volatiles derived from the 18:3 fatty acid

260

(linolenic acid) were probably not synthesized in the flesh, or were synthesized in low,

261

undetectable amounts. On the other hand, the products derived from the 18:2 fatty acid

262

(linoleic acid) were found in all tissues. Similar results were reported by Defilippi et al. 26

263

in apple fruit, where (E)-2-hexenal accumulated in higher amounts in the peel than in the

264

flesh, and hexanal levels remained unchanged in both tissues. These authors observed that

265

(E)-2-hexenal accumulation occurred during ripening; therefore, they concluded that the

266

accumulation pattern was ethylene-independent since the authors used transgenic apple

267

lines (Greensleeves) in which ethylene biosynthesis was suppressed. Likely, the higher (E)-

268

2-hexenal production in the peel of pepino might be explained by a higher accumulation of

269

18:3 fatty acid. Perhaps, 18:3 fatty acid might have a biological function in the wax

270

synthesis of the fruit, which is unrelated to volatile production. Guadagni et al. 23 removed



271

the wax from the apple peel surface and did not observe any volatile change in the aroma

272

profile.

273

Another plausible biological explanation for a higher (E)-2-hexenal accumulation in the

274

fruit peel may lie in angiosperm evolution since it has been suggested that the origin of fruit

275

fleshiness is related to defense against pathogens rather than to seed dispersal.37 It is well

276

documented that many secondary chemicals are mostly concentrated in the fruit peel with

277

specific biological functions such as stress protection, deterrents and fruit defense.38 Thus,

278

it is not surprising that (E)-2-hexenal is produced mostly in the pepino skin, especially

279

during fruit ripening. Some of these defense compounds are present in high concentrations

280

in immature fruits, and others only when the fruit ripens;38 thus, the fruits respond to

281

different ecological requirements at different stages of the plant. (E)-2-hexenal has been

282

shown to be toxic to microorganisms particularly fungi and bacteria 39 and also inhibits

283

seed germination and seedling growth in several plant species.40 Gardner et al. 40 estimated

284

the relative molar toxicity and concluded that (E)-2-nonenal and (E)-2-hexenal were highly

285

toxic aldehydes with the former being more toxic. Moreover, in assays, the growth of seeds

286

and seedlings was reduced the most by (E)-2-hexenal.

287

The production of C5 volatiles decreased nearly 6-fold in the flesh compared with the peel

288

(Figure 3, top). This might be the reason why Shiota et al.7 did not detect or report any C5

289

compounds in pepino, since they used peeled fruit and removed the seeds, using only flesh

290

in their studies.

291

If the proposed LOX pathway for the C5 synthesis is accurate with the same lipoxygenase

292

as the enzyme essential for the production of C5 and C6 compounds,16 then the C5 volatiles

293

should also be deficient or decreased in the flesh. Our study confirmed this result. Notably,


Page 14 of 36

Page 15 of 36


294

the reduction of C6 volatiles derived from 18:3 was greater than C5 because of the low

295

production of C5 in the flesh, suggesting that: (i) the pathway favors the production of C5

296

over the C6 volatiles when the availability of 18:3 fatty acid substrate is scarce, i.e., the

297

HPL enzyme does not metabolize the 13-HPOT as normally occurs, instead, the LOX

298

enzyme metabolizes 13-HPOT; (ii) if there is no 18:3 fatty acid available, then the C5

299

compounds are produced from another substrate: 13-HPOD, a hydroperoxide generated by

300

LOX from the 18:2 fatty acid present in the flesh (this is assumed since hexanal and

301

hexanol, 18:2 fatty acid-derived volatiles, were present in the flesh in slightly higher

302

amounts than in the peel). The low quantities of C5 volatiles can be attributed to 13-HPOD

303

being a poor substrate for lipoxygenases to generate C5 compounds;14 and (iii) based on the

304

fact that LOXs act upon free fatty acids rather than esterified fatty acids,9 probably the de

305

novo production of 18:2 fatty acids accumulate in the free fatty acid pool at a greater extent

306

than the 18:3 fatty acid. The differential pattern of fatty acid accumulation is not

307

uncommon. It has been recently reported in apple, that the 18:1 and 18:2 accumulates in the

308

free fatty acid fraction, whereas 18:3 was not present, however, these results were found in

309

peel. Therefore, the mechanism allowing this accumulation pattern was unclear.41

310

It is known that enzyme products depend upon substrate availability.42 In other words, the

311

quantity and quality of aroma profiles are determined by the substrate supply. For example,

312

ester formation in strawberry and banana, depend more on the supply of specific substrates

313

rather than enzyme specificity.42 Likewise, LOX-derived aldehydes and ester production

314

parallels the fatty acid availability in apple.41

315

316

Lipoxygenase gene family in pepino fruit.



317

Relative gene expression analysis by qPCR revealed that eight putative LOX genes were

318

expressed in the flesh and peel of pepino fruit (Table 1). In most angiosperms, LOX genes

319

are encoded by a multigene family. For instance, there are 23 LOX genes in apple,43 23 in

320

cucumber,44 18 in melon,45 18 in grape 46 and many other LOX gene families of similar

321

size. Interestingly, in the Solanaceae, the LOX gene family seems to be smaller: six genes

322

are expressed in tomato 47 and 5 in potato.48 Of the eight LOX genes found in pepino, two

323

genes corresponded to putative 13-LOX genes and the rest to 9-LOX genes. The

324

predicted LOX protein sequence alignments revealed as much as 87 to 96% similarity to

325

tomato and potato characterized full-length LOX sequences (Table 1). Additionally, these

326

eight LOX sequences were aligned using Blastn with the 75,832 unigenes of the pepino

327

transcriptome reported by Herraiz et al.49 Five out of eight sequences showed homology

328

with the pepino sequences assembled by these authors 49 (Table S2, Supporting

329

Information). These five sequences presented high degree of identity (95-99%), and in all

330

cases the full transcript of the respective LOX genes were obtained. The three remaining

331

sequences did not show homology with the pepino transcriptome database, which might be

332

explained by the use of different genetic material (Herraiz et al.49 used the cultivar Sweet

333

Long, whereas the present study used the most common ecotype found in Chile), and

334

different phenological stages (Herraiz et al.49 used a ripe stage whereas we studied six

335

phenological stages from immature to senescent fruit). Finally, four new sequences were

336

found in the pepino transcriptome, which may be encoding other LOX genes not included

337

in this study (Table S2, Supporting Information).

338

To determine which LOX genes might be involved in the volatile production we analyzed

339

LOX gene expression during fruit development. Different expression patterns were


Page 16 of 36

Page 17 of 36


340

observed among the eight candidate LOX genes, but each gene showed a similar expression

341

pattern between the peel and flesh tissues during fruit development in 2015 (Supporting

342

Information Figure S2) and 2016 (Figure 4). Additionally, the LOX transcripts were

343

present at roughly equivalent levels in the peel and flesh during fruit development, in both

344

years of study (Supporting Information Figure S3).

345

Of the two genes annotated as 13-LOXs, SmLOXC and SmLOXD, only SmLOXD had an

346

expression profile with pattern matching the production of C6 and C5 compounds (Figure

347

4) (Figure 1). Pepino genes SmLOXC and SmLOXD have 90% and 94% identity with

348

tomato homologue genes TomloxC and TomLoxD, respectively (Table 1). Both tomato

349

LOX genes are 13-LOXs and chloroplast localized. TomloxC is mostly expressed in

350

ripening fruit and encodes a LOX enzyme essential for aroma production,22 whereas

351

TomLoxD is expressed in fruit and leaves and is possibly involved in the jasmonic acid

352

pathway as its expression is stimulated by wounding.47 Unlike TomloxC, the pepino

353

SmLOXC expression decreased during fruit ripening (Figure 4) and showed an expression

354

pattern different from the C6 or C5 volatiles produced in the peel and flesh (Figure 1). On

355

the other hand, SmLOXD showed an increase in expression at stage 5 (harvest) (Figure 4)

356

and continued increasing at stage 6 paralleling the C5 and C6 volatile production (Figure

357

1). SmLOXD might be related to aroma production, although we do not overlook the

358

possibility that this LOX probably acts with a separate LOX simultaneously in the

359

production of volatiles. For instance, Shen et al.16 demonstrated that the C5 and C6 volatile

360

synthesis was catalyzed in part by TomloxC (13-LOX), suggesting that another 13-LOX

361

might play a role in the pathway for the production of these volatiles. TomLoxF, which is

362

78% identical to TomloxC, might share the same gene function with TomLoxC. In their



363

work, Shen et al.16 observed reductions in leaf-produced C5 and C6 volatiles, and in both

364

gene trancripts (TomloxC and TomloxF) in LoxC-antisense lines; therefore, the authors

365

suggested that TomloxC and posibly TomloxF were the main 13-LOX(s) enzymes

366

responsable for the C5 and C6 volatile synthesis. However, this was not proven.

367

Interestingly, two putative 9-LOX genes, SmLOXB and SmLOX5-like1, were ripening-

368

dependent and showed a sharp increase in the expression at phenological stages 5 and 6

369

(Figure 4). The 9-LOXs are the most abundant forms found in plants, but they are not the

370

major contributors to the flavor volatiles, except in cucumber and melon.12,13 Other

371

functions that have been attributed to the 9-LOXs involve resistance against pathogens,

372

especially in Solanaceae, tuber development, plant-pathogen interactions, and protein

373

storage.9 However, several cases of LOXs with a predicted 9-LOX regiospecificity have

374

been shown to have dual reaction specificity (possessing both 9- and 13- LOX activity).50,51

375

Recently, a LOX with 13/9-LOX activity was found to be linked to aldehyde and ester

376

production in ripening apples.22 Thus, if one of these genes, SmLOXB or SmLOX5-like1,

377

would have a dual role specificity, it might be involved in the production of C5 and C6

378

LOX-derived volatiles in pepino fruit and responsible for the sharp increase in the

379

production of these compounds in ripe fruit.

380

As for the C9 volatiles, the SmLOXA, a putative 9-LOX gene, showed the same pattern of

381

expression as the C9 volatile emissions (Figure 4). Another possible candidate might be

382

SmLOX5-like3; however, its pattern was rather inconsistent although its expression

383

decreased throughout ripening. In other species that also produce C9 volatiles, such as

384

cucumber, only one lipoxygenase, CsLOX2, a predicted 9-LOX, was reported to be

385

involved in C9 aroma synthesis in cucumber.52 Interestingly, the expression pattern of


Page 18 of 36

Page 19 of 36


386

CsLOX2 was also high in early stages of development after anthesis and was suggested to

387

be involved in fruit quality.

388

Finally, RT-PCR was used to examine the expression pattern of the eight LOX genes in

389

root, shoot, leaf, and fruit flesh and peel of pepino (Figure 5). SmLOXC and SmLOXD

390

(putative 13-LOXs) had different expression patterns. SmLOXC showed a rather low

391

transcript level in all tissues, whereas SmLOXD appeared to have a higher expression in the

392

shoots. Compared to SmLOXC, SmLOXD showed higher transcript abundance in the flesh

393

and peel. Interestingly, SmLOXB and SmLOX5-like1 showed same expression patterns and

394

had highest transcript levels in flesh and peel tissues. Other LOXs such as SmLOX5-like2

395

showed a tissue-specific expression in leaf, while SmLOX5-like3 showed a homogeneous

396

level of expression across the different pepino tissues.

397

398

In this study, we reported the production of C5 volatiles for pepino, which to the best of our

399

knowledge have not been previously found for this species. Moreover, we presented

400

evidence that the (E)-2-hexenal and its alcohol accumulate in the peel, but they are not

401

detected in the flesh of pepino fruit. This tissue specificity for aroma production is likely

402

due to substrate supply, although this remains to be further proven. Finally, we identified

403

eight members of the LOX gene family and suggested three candidate genes (SmLOXD,

404

SmLOXB and SmLOX5-like1) are putatively involved in the aroma production of C6 and C5

405

volatiles, and one candidate (SmLOXA) for C9 compounds. Further studies of LOX enzyme

406

activity are needed to test all proposed candidates.

407



408

Supporting Information

409

Primers for LOX genes (pdf)

410

Putative lipoxygenase genes aligned against pepino transcriptome (pdf)

411

GC-MS chromatogram of a ripe intact pepino fruit (pdf)

412

Relative LOX gene expression in peel and flesh 2015 season (pdf)

413

LOX transcript levels in pepino peel and flesh tissue during fruit ripening for 2015 and

414

2016 seasons (pdf)

415

416

ACKNOWLEDGMENTS

417

We thank the funding support from CONICYT-Chile (FONDECYT project 3150082), and

418

Thomas Hoffmann from the Technical University of Munich for technical support.

419

420

421

422

423

424

425

426


Page 20 of 36

Page 21 of 36

427

428


REFERENCES (1)Rodríguez-Burruezo, A.; Kollmannsberger, H.; Prohens, J.; Nitz, S.; Nuez, F.

429

Analysis of the volatile aroma constituents of parental and hybrid clones of pepino

430

(Solanum muricatum). J. Agr. Food Chem. 2004, 52, 5663–5669.

431

(2)Anderson, G.; Jansen, R.; Kim, Y. The origin and relationships of the pepino,

432

Solanum muricatum (Solanaceae): DNA restriction fragment evidence. Econ. Bot.

433

1996, 50, 369–380.

434 435 436 437

(3)Prohens, J.; Ruiz, J.; Nuez, F. The pepino (Solanum muricatum, Solanaceae). A ‘‘new’’crop with a history. Econ. Bot. 1996, 50, 355–368. (4)Weese, T.; Bohs, L. A three-gene phylogeny of the genus Solanum (Solanaceae). Syst. Bot. 2007, 32, 445–463.

438

(5)Contreras, C.; González-Agüero, M.; Defilippi, B. A review of pepino (Solanum

439

muricatum Aiton) fruit: a quality perspective. HortScience 2016, 5, 1127-1133.

440

(6)Dudareva, N.; Klempien, A.; Muhlemann, J.; Kaplan, I. Biosynthesis, function and

441

metabolic engineering of plant volatile organic compounds. New Phytologyst 2013,

442

198, 16-32.

443 444

(7)Shiota, H.; Young, H.; Paterson, V.; Irie, M. Volatile aroma constituents of pepino fruit. J. Sci. Food Agr. 1988, 43, 343–354.

445

(8)Rodríguez-Burruezo, A.; Prohens, J.; Nuez, F. Genetic analysis of quantitative traits

446

in pepino (Solanum muricatum) in two growing seasons. J. Amer. Soc. Hort. Sci.

447

2002, 127, 271–278.

448 449

(9)Feussner, I.; Wasternack, C. The lipoxygenase pathway. Annu. Rev. Plant Biol. 2002, 53, 275-297.



450

(10)Baldwin, E.; Scott, J.; Shewmaker, C.; Schuch, W. Flavor trivia and tomato aroma:

451

Biochemistry and possible mechanisms for control of important aroma

452

components. HortScience 2000, 35, 1013-1022.

453

(11)Mehinagic, E.; Royer, G.; Symoneaux, R.; Jourjon, F.; Prost, C. Characterization of

454

odor-active volatiles in apples: Influence of cultivars and maturity stage. J. Agric.

455

Food Chem. 2006, 54, 2678-2687.

456 457

(12)Buescher, R.W.; Buescher, R.H. Production and stability of (E,Z)-2,6-nonadienal, the major flavor volatile of cucumbers. J. Food Sci. 2001, 66, 357-361.

458

(13)Homatidou, V.; Karvouni, S.; Dourtoglou, V.; Poulos, C. Determination of total

459

volatile components of Cucumis melo L. variety Cantaloupensis. J. Agric. Food

460

Chem. 1992, 40, 1385-1388.

461

(14)Salch, Y.; Grove, M.; Takamura, H.; Gardner, H. Characterization of a C-5, 13-

462

cleaving enzyme of 13(S)-hydroperoxide of linolenic acid by soybean seed. Plant

463

Physiol. 1995, 108, 1211-1218.

464

(15)Fall, R.; Karl, T.; Jordan, A.; Lindinger, W. Biogenic C5 VOCs: release from

465

leaves after freeze–thaw wounding and occurrence in air at a high mountain

466

observatory. Atmos. Environ. 2001, 35, 3905–3916.

467

(16)Shen, J.; Tieman, D.; Jeffrey, J.; Taylor, M.; Schmelz, E.; Huffaker, A.; Bies, D.;

468

Chen, K.; Klee, H. A 13-lipoxygenase, TomloxC, is essential for synthesis of C5

469

flavour volatiles in tomato. J. Exp. Bot. 2014, 65, 419-428.

509 510

(17)Tieman, D.; Bliss, P.; McIntyre, L. The chemical interactions underlying tomato ﬂavor preferences. Curr. Biol. 2012, 22, 1035–1039.


Page 22 of 36

Page 23 of 36

511


(18)Vuorinen, T.; Nerg, A.; Holopainen, J. Ozone exposure triggers the emission of

512

herbivore-induced plant volatiles, but does not disturb tritrophic signalling.

513

Environ. Pollut. 2004, 131, 305-311.

514

(19)León, J.; Royo, J.; Vancanneyt, G.; Sanz, C.; Silkowski, H.; Griffiths, G.; Sanchez-

515

Serrano, J. Lipoxygenase H1 gene silencing reveals a specific role in supplying

516

fatty acid hydroperoxides for aliphatic aldehyde production. J. Biol. Chem. 2002,

517

277, 416–423.

518

(20)Salas, J.; García-González, D.; Aparicio, R. Volatile compound biosynthesis by

519

green leaves from an Arabidopsis thaliana hydroperoxide lyase knockout mutant.

520

J. Agric. Food Chem. 2006, 54, 8199-8205.

521

(21)Schiller, D.; Contreras, C.; Vogt, J.; Dunemann, F.; Defilippi, B.; Beaudry, R.;

522

Schwab, W. A dual positional specific lipoxygenase functions in the generation of

523

flavor compounds during climacteric ripening of apple. Hortic. Res. 2015, 2, 1-13.

524

(22)Chen, G.; Hackett, R.; Walker, D.; Taylor, A.; Lin, Z.; Grierson, D. Identification

525

of a specific isoform of tomato lipoxygenase (TomloxC) involved in the generation

526

of fatty acid-derived flavor compounds. Plant Physiol. 2004, 136, 2641-2651.

527 528 529

(23)Guadagni, D.; Bomben, J.; Hudson, J. Factors influencing the development of aroma in apple peels. J. Sci. Food Agric. 1971, 22, 110–115. (24)Ties, P.; Barringer, S. Influence of lipid content and lipoxygenase on flavor

530

volatiles in the tomato peel and flesh. J. Food Sci. 2012, 77, C830-C837.

531

(25)Buttery, R.; Teranishi, R.; Ling, L.; Flath, R.; Stern, D. Quantitative studies on

532

origins of fresh tomato aroma volatiles. J. Agric. Food Chem. 1988, 36, 1247-

533

1250.



534

(26)Defilippi, B.; Dandekar, A.; Kader, A. Relationship of ethylene biosynthesis to

535

volatile production, related enzymes, and precursor availability in apple peel and

536

flesh tissues. J. Agric. Food Chem. 2005, 53, 3133–3141.

537

(27)Zhang, B.; Yin, X.; Shen, Y.; Chen, K.; Ferguson, I. Volatiles production and

538

lipoxygenase gene expression in kiwifruit peel and flesh during fruit ripening. J.

539

Amer. Soc. Hort. Sci. 2009, 134, 472–477.

540

(28)Aubert, C.; Baumann, S.; Arguel, H. Optimization of the analysis of flavor volatile

541

compounds by liquid-liquid microextraction (LLME). Application to the aroma

542

analysis of melons, peaches, grapes, strawberries, and tomatoes. J. Agric. Food

543

Chem. 2005, 53, 8881-8885.

544

(29)Gudenschwager, O.; González-Agüero, M.; Defilippi, B. A general method for

545

high-quality RNA isolation from metabolite-rich fruits. S. Afr. J. Bot. 2012, 83,

546

186-192.

547

(30)Chen, X.; Reddanna, P.; Reddy, R.; Kidd, R.; Hildenbrandt, G.; Reddy, C.

548

Expression, purification, and characterization of a recombinant 5-lipoxygenase

549

from potato tuber. Biochem. Biophys. Res. Commun. 1998, 243, 438-443.

550

(31)González-Agüero, M.; Pavez, L.; Ibañez, F.; Pacheco, I.; Campos-Vargas, R.;

551

Meisel, L. Orellana, A.; Retamales, J.; Silva, H.; González, M.; Cambiazo, V.

552

Identification of woolliness response genes in peach fruit after post-harvest

553

treatments. J. Exp. Bot. 2008, 59, 1973–1986.

554

(32)Ruiz-Beviá, F.; Font, A.; García, A.; Blasco, P.; Ruiz, J. Quantitative analysis of

555

the volatile aroma components of pepino fruit by purge-and-trap and gas

556

chromatography. J. Sci. Food Agr. 2002, 82, 1182–1188.


Page 24 of 36

Page 25 of 36


557

(33)Rodríguez-Burruezo, A.; Prohens, J.; Fita, A. Breeding strategies for improving the

558

performance and fruit quality of the pepino (Solanum muricatum): A model for the

559

enhancement of underutilized exotic fruits. Food Res. Int. 2011, 44, 1927–1935.

560

(34)Rodríguez-Burruezo, A.; Prohens, J.; Nuez, F. ‘Valencia’: A new pepino (Solanum

561

muricatum) cultivar with improved fruit quality. HortScience, 2004, 39, 1500–

562

1502.

563 564 565

(35)Hatanaka, A. The biogeneration of green odour by green leaves. Phytochemistry 1993, 34, 1201–1218. (36)Rowan, D.; Allen, J.; Fielder, S.; Hunt, M. Biosynthesis of straight-chain ester

566

volatiles in Red Delicious and Granny Smith apples using deuterium-labeled

567

precursors. J. Agric. Food Chem. 1999, 47, 2553-2562.

568 569

(37)Knapp, S. Tobacco to tomatoes: a phylogenetic perspective on fruit diversity in the Solanaceae. J. Exp. Bot. 2002, 53, 2001-2022.

570

(38)Cipollini, M.; Stiles, E. Relative risks of microbial rot for fleshy fruits: Significance

571

with respect to dispersal and selection for secondary defense. Adv. Ecol. Res.

572

1992, 23, 35-91.

573

(39)Matsui, K.; Minami, A.; Hornung, E.; Shibata, H.; Kishimoto, K.; Ahnert, V.;

574

Kindl, H.; Kajiwara, T.; Feussner, I. Biosynthesis of fatty acid derived aldehydes

575

is induced upon mechanical wounding and its products show fungicidal activities

576

in cucumber. Phytochemistry 2006, 67, 649-657.

577

(40)Gardner, H.; Dornbos, D.; Desjardins, A. Hexanal, trans-2-hexenal, and trans-2-

578

nonenal inhibit soybean, Glycine max, seed germination. J. Agric. Food Chem.

579

1990, 38, 1316-1320.



580

(41)Contreras, C.; Tjellström, H.; Beaudry, R. Relationships between free and esterified

581

fatty acids and LOX-derived volatiles during ripening in apple. Postharvest Biol.

582

Technol. 2016, 112, 105-113.

583 584 585

(42)Schwab, W. Review: Metabolome diversity: too few genes, too many metabolites?. Phytochemistry, 2003, 62, 837–849. (43)Vogt, J.; Schiller, D.; Ulrich, D.; Schwab, W.; Dunemann, F. Identification of

586

lipoxygenase (LOX) genes putatively involved in fruit flavour formation in apple

587

(Malus x domestica). Tree Genet. Genomes 2013, 9, 1493-1511.

588

(44)Liu, S.; Liu, X.; Jiang, L. Genome-wide identification, phylogeny and expression

589

analysis of the lipoxygenase gene family in cucumber. Genet. Mol. Res. 2011, 10,

590

2613–2636.

591

(45)Zhang, C.; Jin, Y.; Liu, J.; Tang, Y.; Cao, S.; Qi, H. The phylogeny and expression

592

profiles of the lipoxygenase (LOX) family genes in the melon (Cucumis melo L.)

593

genome. Sci. Hortic. 2014, 170, 94-102.

594

(46)Podolyan, A.; White, J.; Jordan, B.; Winefield, C. Identification of the lipoxygenase

595

gene family from Vitis vinifera and biochemical characterization of two 13-

596

lipoxygenases expressed in grape berries of Sauvignon Blanc. Funct. Plant Biol.

597

2010, 37, 767–784.

598

(47)Mariutto, M.; Duby, F.; Adam, A.; Bureau, C.; Fauconnier, M.; Ongena, M.;

599

Thonart, P.; Dommes, J. The elicitation of a systemic resistance by Pseudomonas

600

putida BTP1 in tomato involves the stimulation of two lipoxygenases isoforms.

601

BMC Plant Biol. 2011, 11, 29.


Page 26 of 36

Page 27 of 36

602


(48)Kolomiets, M.; Hannapel, D.; Chen, H.; Tymeson, M.; Gladon, R. Lipoxygenase is

603

involved in the control of potato tuber development. The Plant Cell 2001, 13, 613–

604

626.

605

(49)Herraiz, F.; Blanca, J.; Ziarsolo, P.; Gramazio, P.; Plazas, M.; Anderson, G.;

606

Prohens, J.; Volanova, S. 2016. The first de novo transcriptome of pepino

607

(Solanum muricatum): assembly, comprehensive analysis and comparison with the

608

closely related species S. caripense, potato and tomato. BMC Genomics 2016, 17,

609

321.

610

(50)Palmieri-Thiers, C.; Canaan, S.; Brunini, V.; Lorenzi, V.; Tomi, F.; Desseyn, J.;

611

Garscha, U.; Oliw, E.; Berti, L.; Maury, J. A lipoxygenase with dual positional

612

specificity is expressed in olives (Olea europaea L.) during ripening. Biochim.

613

Biophys. Acta, Mol. Cell Biol. Lipids 2009, 1791, 339–346.

614

(51)Acosta, I.; Laparra, H.; Romero, S.; Schmelz, E.; Hamberg, M.; Mottinger, J.;

615

Moreno, M.; Dellaporta, S. tasselseed1 is a lipoxygenase affecting jasmonic acid

616

signaling in sex determination of maize. Science 2009, 323, 262-265.

617

(52)Yang, X.; Jiang, W.; Yu, H. The expression profiling of the lipoxygenase (LOX)

618

family genes during fruit development, abiotic stress and hormonal

619

treatments in cucumber (Cucumis sativus L.). Int. J. Mol. Sci. 2012, 13, 2481-

620

2500.

621

622

623



624

FIGURE CAPTIONS

625

Figure 1. LOX-derived volatiles emitted by the flesh + peel of the pepino fruit during

626

development: (top) C5 volatiles, (middle) C6 volatiles, and (bottom) C9 volatiles. Values

627

are mean ± standard error of five replicates.

628

629

Figure 2. Volatiles emitted by the flesh of pepino fruit and derived from the LOX pathway

630

during development: (top) C5 volatiles, (middle) C6 volatiles, and (bottom) C9 volatiles.

631

Values are mean ± standard error of three biological replicates.

632

633

Figure 3. Volatile production of different tissue compartments in a fully ripe (stage 5)

634

pepino fruit: (top) C5 and (bottom) C6 volatiles.

635

636

Table 1. Putative lipoxygenase genes found in pepino fruit.

637

638

Figure 4. Relative expression of lipoxygenase (LOX) genes during pepino development in

639

the 2016 season. Error bars indicate SE from three biological replicates. The expression

640

levels of each gene are expressed as a ratio relative to the phenological stage 1 (10 days

641

after fruit set), which was set at 1.

642 643

Figure 5. Expression analysis of the members of the pepino LOX gene family in different

644

plant tissues by RT-PCR.


Page 28 of 36

Page 29 of 36


TABLES

Putative gene

SmLOXA

GenBank access S. muricatum

Fragment size (bp)a

KY783402

864

Predicted lipoxygenase type 9-LOX

Solanum species

GenBank access b

S. lycopersicum

AAA53184

Table 1. a

Length of sequences identified and sequenced to annotation date.

b

Access code obtained from BLASTx sequence alignment.

c

Comparison of the S. muricatum sequences with Solanum species orthologs.


Identity (%)c


Page 30 of 36

87

SmLOXB

KY783403

1,558

9-LOX

S. lycopersicum

AAA74393

87

SmLOXC

KY783404

1,535

13-LOX

S. lycopersicum

AAB65766

90

SmLOXD

KY783405

351

13-LOX

S. lycopersicum

AAB65767

94

SmLOX5

KY783406

447

9-LOX

S. tuberosum

XP_006344836

95

SmLOX5-like1

KY783407

326

9-LOX

S. tuberosum

XP_006344623

96

SmLOX5-like2

KY783408

867

9-LOX

S. lycopersicum

AAG21691

93

SmLOX5-like3

KY783409

1,492

9-LOX

S. tuberosum

XP_006359922

92

FIGURES


Page 31 of 36


Figure 1.



Figure 2.


Page 32 of 36

Page 33 of 36


Figure 3.



Figure 4.


Page 34 of 36

Page 35 of 36


Figure 5.



TOC GRAPHICS


Page 36 of 36

Volatile Compound and Gene Expression Analyses Reveal Temporal

Recommend Documents