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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

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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


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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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possible roles of individual LOX genes in pepino.

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

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INTRODUCTION

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

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

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

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

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

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potato (Solanum tuberosum).4

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

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

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

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

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

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

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

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the lipoxygenase (LOX) pathway.

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

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

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

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

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

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

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

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fruit.



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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apple MdLOX1a.21

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

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

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

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

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

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

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

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

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



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

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aroma volatiles.

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MATERIALS AND METHODS

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

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

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(Taufkirchen, Germany).

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

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

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

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

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

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

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

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

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

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

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

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

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

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volatile analysis.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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desorbed for 5 min in the GC-MS injector.

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

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



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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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were only identified.

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

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

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

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

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

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


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

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

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

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

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1

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

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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

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Identification and isolation of partial cDNAs for lipoxygenase genes. Degenerate

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primers were designed from conserved sequences of the closest Solanaceae members

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

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

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

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

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

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

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

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

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

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sequences were compared to sequences from National Center for Biotechnology

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

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

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sequencing procedures were the same as described above.



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

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

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

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

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

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

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

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KY761980).

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RESULTS AND DISCUSSION

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

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

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

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compounds were found in pepino.

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

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

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

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

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

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

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


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

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liking of tomato flavor.17

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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study.

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

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

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

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

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

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

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

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

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

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

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

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Peel versus flesh volatile production.

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

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

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

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


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

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

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

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

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with the flesh.23

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

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

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

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

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(Figure 3).

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

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

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

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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

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

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

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

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

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

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

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

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



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

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profile.

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

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

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

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

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

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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

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

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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

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

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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.

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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

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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,


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

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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

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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

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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

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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


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427

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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


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Volatile Compound and Gene Expression ... - ACS Publications

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