Article pubs.acs.org/JAFC
Epicuticular Wax in Developing Olives (Olea europaea) Is Highly Dependent upon Cultivar and Fruit Ripeness Stefania Vichi,*,† Nuria Cortés-Francisco,‡ Josep Caixach,§ Gonçal Barrios,∥ Jordi Mateu,∥ Antonia Ninot,⊥ and Agustí Romero⊥ †
Nutrition, Food Science and Gastronomy Department, Catalonian Reference Network on Food Technology (XaRTA), Institute for Research on Nutrition and Food Safety (INSA), University of Barcelona, Food and Nutrition Torribera Campus, Avenida Prat de la Riba 171, 08921 Santa Coloma de Gramenet, Spain ‡ Laboratory of the Public Health Agency of Barcelona, Avenida de les Drassanes 13, 08001 Barcelona, Spain § Mass Spectrometry Laboratory/Organic Pollutants, Institute of Environmental Assessment and Water Research (IDAEA), Spanish Council for Scientific Research (CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain ∥ Section of Agriculture and Plant Health, Territorial Service of Tarragona, Ministry of Agriculture, Livestock, Fisheries and Food, Catalonian Government, Avenida Catalunya 50, 3a, 43002 Tarragona, Spain ⊥ Olive Production, Oil Processing and Nut Trees, Institute for Food and Agricultural Research and Technology (IRTA), Mas de Bover, Carretera de Reus El Morell, 43120 Constantí, Tarragona, Spain S Supporting Information *
ABSTRACT: The epicuticular wax (EW) layer is located on the surface of most plant organs. It provides the cuticle with most of its properties and is the primary barrier against biotic and abiotic stress. Despite the importance of Olea europaea cultivation, few studies have characterized the EW covering leaves and olives, which could be involved in resistance to both infection and environmental conditions. In the present study, wide-ranging screening was carried out using direct-injection electrospray ionization coupled to high-resolution mass spectrometry to analyze EW in developing olives of nine varieties. The proportions of EW fractions [wax esters (WEs), diacylglycerols, triacylglycerols (TAGs), triterpenic acids, and aldehydes] strongly depended upon the olive cultivar and, in only a few cases, were influenced by the sampling date. The specific compositions of the major fractions, WEs and TAGs, were strictly related to the cultivar, while the degree of unsaturation and chain length of the WEs evolved throughout the 4 weeks prior to the olive turning color. KEYWORDS: epicuticular wax, olive, cultivar, ripening, high-resolution mass spectrometry
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INTRODUCTION Epicuticular wax (EW) is the complex monomeric mixture that forms the highly hydrophobic layer, which covers the polymeric cutin structure on the surface of most plant organs.1,2 Because this layer is located at the interface between the plant and the environment, it provides the cuticle with most of its properties. EW is the primary barrier against biotic and abiotic stress, and for this reason, it is particularly important during fruit development.3 EW contributes to the prevention of water loss and determines the wettability of the plant surface,4,5 protects against incident radiation by favoring light reflection,5−7 shields from bacterial and fungal pathogens,8,9 and plays a significant role in host−plant recognition by insects.5,10,11 It has been further shown that wax constituents can influence insect behavior regarding oviposition.12−14 The chemical composition of EW greatly affects the physical properties of plant surfaces,3 and our understanding of the effect of EW composition on different biological functions deserves to be extended. In turn, EW can be influenced by environmental conditions, such as ambient temperature, irradiation, and moisture,15,16 as well as genetic factors, as evidenced by the diversity of EW composition in different plant species and varieties.17,18 © XXXX American Chemical Society
Olea europaea cultivation is of great importance in the Mediterranean Basin, but relatively few studies have characterized the EW covering olive leaves19,20 and olives;17,20−22 these studies would be useful to elucidate the role of EW in the adaptation of olive trees to Mediterranean agro-climatic conditions or its possible function in resistance toward plagues and pathogens. The EW of ripe, healthy olive is composed of alkanes, alcohols, aldehydes (ALDs), alkyl esters (AEs), benzyl esters (BEs), triacylglycerols (TAGs), fatty acids (FAs), and triterpenic acids (TAs), among others.17,20−23 At present, little is known of EW compositional differences in olives from distinct varieties19 and at different stages of maturity.17 Most of the information available is related to the amount of wax esters (WEs) in the oils obtained from olives21,24−26 and little about EW composition at early stages of olive development. At the stage of olives turning color, the barrier between the plant and the environment becomes much more important, because this period corresponds to high levels of temperature and ultraviolet Received: June 7, 2016 Revised: July 12, 2016 Accepted: July 12, 2016
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DOI: 10.1021/acs.jafc.6b02494 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. (a) Positive and (b) negative ESI−UHRMS spectra of ‘Sevillenca’ olive EW extract. Elemental formulas, RDBE, and mass error are shown. R = 100 000 (m/z 200, fwhm).
(UV) irradiance, together with summer storms that increase humidity in the olive canopy, resulting in very appropriate conditions for the development of pests and diseases. Because EW chemical composition can significantly influence the properties of the olive surface, characterization of EW according to the olive cultivar and the stage of maturity could help us to better understand differences among olive cultivars in tolerance or resistance to biotic and abiotic factors. With the aim of documenting the diversity of EW composition on olive fruits and to provide a starting point for further understanding of the mechanisms that determine olive adaptation and resistance to environmental stresses during olive development, a wide-ranging screening was carried out using a rapid and efficient analytical method developed recently.23
The EW and olive physical characteristics of nine olive varieties grown in the same geographical area were screened and monitored for 4 weeks, corresponding to the developmental stage prior to the olives changing color.
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MATERIALS AND METHODS
Chemicals. Reagents were of mass spectrometry grade. Dichloromethane, methanol, and hexane (MS SupraSolv) were purchased from Merck (Darmstadt, Germany). Ammonium formate was from SigmaAldrich (St. Louis, MO). Nitrogen (Alphagaz, 99.999%, Air Liquide) was used in the Orbitrap Exactive as the nebulization gas. Plant Material. Healthy olive fruits of nine varieties pertaining to the collection of IRTA, Mas de Bover (Constanti,́ Spain), grown in the same parcel were studied. Cultivars were selected to cover the full range of ripening from very early [majority of fruits reach maturity B
DOI: 10.1021/acs.jafc.6b02494 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
C
% phenols
HCs ALDs FAs WEs TAs DFAs DAGs TAGs
38.1 cd
7.2 1.6 cde 0.9 44.1 a 1.7 ab 0.4 2.5 b 41.6 c
41.8 a −12.6 d 25.8 a 15.7 e 13.4 bc 1.8 b
25.0 d
4.0 1.9 abc 1.0 23.6 b 1.0 b 0.4 2.8 b 65.3 a
41.0 ab −12.5 cd 26.3 a 19.8 bc 12.7 cd 1.9 b
2
73.7 ab
6.5 1.1 e 0.9 21.2 b 0.8 b 0.3 2.4 b 66.8 a
31.2 e −9.7 a 16.8 f 15.9 e 13.3 bc 2.0 b
3 cd d bcd cd d bc
41.6 cd
5.7 1.8 bcd 1.1 40.7 a 2.8 a 0.4 3.0 b 44.5 bc
36.4 −12.9 21.9 18.0 12.0 1.5
4 bcd b cd d f d
28.7 d
8.8 1.1 de 1.3 39.7 a 2.0 ab 0.6 3.2 b 43.4 bc
37.0 −11.2 20.7 17.8 9.6 1.1
5
7
8
Fruit Parameters 38.4 bc 34.7 c 35.9 cd −12.1 bcd −11.5 bc −11.4 b 23.0 abc 17.7 ef 19.8 ed 14.1 f 22.3 a 20.9 ab 11.6 de 14.3 ab 15.1 a 1.2 cd 2.6 a 2.5 a Proportions of EW Classes (ESI+ Mode) 6.9 7.3 6.5 1.9 abc 2.4 ab 2.6 a 1.3 0.8 1.3 17.9 b 22.6 b 40.2 a 1.6 ab 2.8 a 0.7 b 0.3 0.3 0.4 4.1 a 2.3 b 3.1 b 65.9 a 61.5 ba 45.3 bc Proportions of EW Classes (ESI− Mode) 53.1 bc 92.3 a 74.0 ab
6
b abc
ab b
bcd
bc d ab e ef d
74.1 ab
6.7 1.8 1.2 27.9 1.4 0.3 2.3 58.3
38.6 −12.8 24.2 15.9 10.6 1.1
9
