Biochemical Systematics and Ecology 46 (2013) 116–119

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Biochemical Systematics and Ecology

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Foliar cuticular waxes of cultivated species and varieties of Coffea

Julia Toshie Kitagami a, Antonio Salatino a, Oliveiro Guerreiro-Filho b,Maria Luiza Faria Salatino a,*

aDepartment of Botany, Institute of Biosciences, University of São Paulo, Rua do Matão 277, Cidade Universitária, 05508-09 São Paulo, SP, BrazilbAgronomic Institute of Campinas, Rua Barão de Itapura 1481, CP 28, 13012-970 Campinas, SP, Brazil

a r t i c l e i n f o

Article history:Received 18 April 2012Accepted 8 September 2012Available online 16 October 2012

Keywords:Foliar cuticular waxn-AlkanesFatty alcoholsTriterpenoidsChemotaxonomy

* Corresponding author. Tel./fax: þ55 11 3091 754E-mail address: mlfsalat@usp.br (M.L. Faria Salat

0305-1978/$ – see front matter � 2012 Elsevier Ltdhttp://dx.doi.org/10.1016/j.bse.2012.09.012

a b s t r a c t

The cuticular waxes of leaves of Coffea arabica cv. ‘Catuaí Vermelho’, C. arabica cv. ‘Obatã’,Coffea canephora cv. ‘Apoatã’, Coffea racemosa and two hybrids between C. arabica and C.racemosawere extracted by rapid washing of the surface with chloroform. The waxes werefractionated by thin layer chromatography over silicagel. The fractions of the constituentclasses were characterized by infrared spectroscopy and the distribution of the homologsof the n-alkanes and n-primary alcohols was determined by GC/MS and GC/FID. Amongthe samples analyzed, leaves of C. racemosa have the highest content of foliar wax(22.9 mg cm�2). Most samples contain either n-alkanes (C. canephora and C. racemosa) orn-primary alcohols (C. arabica) as predominant wax constituents. The distribution ofn-alkanes allowed the distinction of C. racemosa from the other samples; the distributionof alcohols allowed the distinction of the three species. The two hybrids have waxes similarto the wax of C. arabica.

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1. Introduction

A cuticle covering the leaf surface is a characteristic of vascular plants. The cuticle contains a biopolymer (cutin) and twotypes of waxes: the intracuticular and the epicuticular waxes (Stark and Tian, 2006). The former embeds the cutin matrix,while the latter is deposited on the cuticle surface (Guhling et al., 2005). Washing of the foliar surface with low polaritysolvents removes both the intracuticular and epicuticular waxes. The product obtained is then referred to as cuticular wax(Jetter et al., 2006). The amount of cuticular wax varies widely among plant species. For example, leaves of varieties ofsoybean contain 8 mg cm�2 (Kim et al., 2007), while leaves of wild plants often contain thick deposits of cuticular wax;examples are leaves of Tocoyena formosa (82 mg cm�2) and Ziziphus joazeiro (72 mg cm�2), both species native to Brazil(Oliveira and Salatino, 2000).

The cuticular waxes are composed by long chain aliphatic substances, such as hydrocarbons (chiefly n-alkanes), esters offatty acids and fatty alcohols, aldehydes, primary and secondary alcohols and free fatty acids (Bianchi, 1995). Other commonwax constituents are triterpenes, while flavonoids occur more rarely in cuticular wax (Hamilton, 2004; Gao et al., 2012).Waxes play several roles in the plant biology, such as maintenance of an impermeable foliar surface (and thus contributing toavoid the growth of pathogens), restriction of the loss of water and protection against the attack of herbivore insects and UVirradiation (Baker, 1982; Hamilton, 2004; Gao et al., 2012).

7.ino).

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J.T. Kitagami et al. / Biochemical Systematics and Ecology 46 (2013) 116–119 117

The composition of the cuticular wax is a characteristic of the plant species or variety. Hence it may be used for taxonomicpurposes and to distinguish among taxa (Medina et al., 2006). The distribution of wax alkanes in particular has been oftenused with taxonomic purposes (Kolattukudy, 1976; Salatino et al., 1989; Mimura et al., 1998; Sonibare et al., 2005; Rodriguesand Salatino, 2006; Motta et al., 2009; de Souza et al., 2010; Huang et al., 2011) and to determine hybrid parentage (Chadwicket al., 2000).

Coffee is among themost consumed drinks in theworld (FAO, 2006). Several species of Coffea are cultivated, such as Coffeaarabica and Coffea canephora. The former contributes with 70% of the coffee consumed worldwide. Other species are culti-vated to meet specific purposes. For example, Coffea racemosa is cultivated because it is more resistant to water deficiencythan other species of Coffea (Guerreiro-Filho, 1992).

Stocker and Wanner (1977) reported that the chemistry of the foliar wax is useful for characterization of species of Coffea.The present work aims to compare the composition of the foliar cuticular wax of species and varieties of Coffea.

2. Material and methods

2.1. Plant material

Fully expanded and undamaged leaves were collected from specimens cultivated at the Agronomic Institute of Campinas,in the state of São Paulo (Brazil, Southeast). The accessions collected were: 1) C. arabica L. cv. ‘Catuaí Vermelho’; 2) C. arabica L.cv. ‘Obatã’; 3) C. canephora Pierre cv. ‘Apoatã’; 4) C. racemosa Lour.; 5) hybrid H 13685-1, obtained from crosses between C.arabica and C. racemosa, followed by backcrosses with C. arabica; 6) hybrid H 13685-1-27, obtained from endocrosses amongindividuals of H 13685-1. The leaves were dried in ventilated oven at 35 �C.

2.2. Chemical analyses

Thewaxes were extracted by three consecutive immersions in chloroform, the first for 30 s, the second for 20 s and the lastone for 15 s. The pooled extracts were evaporated to dryness in a rotatory evaporator. The waxes were dissolved in a smallvolume of chloroform; the solvent was evaporated on a steam bath and the flasks maintained in dessicator until constantmass. The total leaf areas were determined using the software “Leaf Measurement System”, version 2.0 (Skye InstrumentsLtd., Llandrindod Wells, UK.). The wax contents are expressed by the ratio between wax mass and leaf area (mg cm�2), takinginto account both adaxial and abaxial surfaces.

The classes of constituents of the waxes were isolated by thin layer chromatography over silicagel 60, impregnated withsodium fluorescein, using the mobile phase n-hexane: chloroform (7:3) (Salatino and Silva, 1988). For characterization of therespective organic functions, the isolated fractions were analyzed by infrared (IR) spectroscopy, using a spectrophotometerBomem MB 100 FTIR. Ketones and diketones were distinguished by the lower Rf of the diketones and the absorption of thecarbonyl bands at 1732 and 1725 cm�1, respectively.

The primary alcohols were converted to the corresponding acetyl esters (Kanya et al., 2007) prior to analysis by GC/MS.Quantitative analyses of the homolog n-alkanes and acetyl esters of n-primary alcohols were carried out in a chromato-graph HP 5890 series II coupled to a mass spectrometer HP 5989 B Engine Chem Station System, using a capillary columnHP-5MS (Crosslinked 5% MePh Siloxane, 30 m � 0.25 mm) and He as carrier gas at a flux of 1 cm3.s�1. A columntemperature program was used, starting at 150 �C in the beginning of a ramp rate of 5 �C.s�1 until 300 �C and holding thistemperature for 10 min. The temperatures of the injector and detector were 250 �C and 300 �C, respectively. The massspectra were obtained at 70 eV and the temperatures of the quadrupole and ion source were 100 �C and 150 �C,respectively. The alkane and alcohol homologs were identified by comparison of retention times and respective massspectra with standards and data from the NIST library. Analysis of the n-alkanes and acetyl esters of n-primary alcoholswere also performed by gas chromatography and detection by flame ionization (GC/FID), using the chromatographicconditions described above.

3. Results

The contents of wax and classes of constituents of the samples of Coffea analyzed are given in Table 1. C. racemosa standsout due to a wax content that is almost twofold the contents of the other genotypes. C. canephora and the two varieties of C.arabica have practically the same wax content. The contents of the two hybrids are very similar (Table 1) and practically thesame as C. arabica.

The predominant classes of wax constituents of most Coffea samples analyzedwere either n-alkanes or n-primary alcohols(Table 1). Other constituents may be abundant in waxes of some samples and appear as minor components in the waxes ofother genotypes. Fatty acids and ketones, for example, are relatively abundant in the foliar wax of both hybrids, but the formerare minor constituents in the foliar wax of the other samples. Diketones and esters are relevant constituents in the foliar waxof C. arabica cv. ‘Obatã.’ Esters are also present as major constituents in the foliar wax of the hybrid H 13686-1. C. canephorastands out for the nearly complete absence of diketones (Table 1). The content of n-alkanes is a characteristic that seeminglydistinguishes the Coffea accessions analyzed: it is highest in C. canephora, intermediate in C. racemosa and lower in C. arabicaand in both hybrids (Table 1).

Table 2Distribution of n-alkanes of the foliar cuticular waxes of genotypes of Coffea. See caption of Table 1 for explanation about the hybrids H 13685-1 and H 13685-1-27.

Genotypes of Coffea Homologs of n-alkanes (%)a

C27 C28 C29 C30 C31 C32 C33

C. arabica cv ‘Catuaí Vermelho’ – – 57.8 1.3 40.7 – –

C. arabica cv ‘Obatã’ – – 63.3 2.1 34.3 – –

C. canephora cv ‘Apoatã’ 1.0 – 61.0 2.0 35.0 – –

C. racemosa – – 11.3 1.1 84.8 – 2.7Hybrid H 13685-1 – – 61.7 2.0 35.3 – –

Hybrid H 13685-1-27 – – 55.3 2.8 41.0 – –

a Dashes: undetected or trace amounts (below 1%).

Table 1Contents of foliar cuticular waxes (mg cm�2) and percentage of classes of constituents of genotypes of Coffea. CarC: C. arabica cv. ‘Catuaí Vermelho’; CarO: C.arabica cv ‘Obatã’; Ccan: C. canephora cv ‘Apoatã’; Crac: C. racemosa. Hybrid 13685-1 was obtained by crosses between C. arabica and C. racemosa, followed bybackcrosses with C. arabica; hybrid 13685-1-27 is the result of endocrosses among individuals of H 13685-1.

Content of wax/percent ofclasses of constituentsa

Genotypes of Coffea

CarC CarO Ccan Crac H 13685-1 H 13685-1-27

Content of wax 12.3 � 2.5 11.5 � 0.4 12.3 � 2.1 22.9 � 3.1 13.5 � 3.3 13.2 � 3.4Acids 6.2 4.7 5.0 4.2 13.7 16.1Alcohols 25.6 19.3 15.0 14.0 14.2 23.1Ketones 12.8 8.2 14.0 12.0 18.1 18.5Diketones 11.9 17.2 trb 3.6 10.8 4.0Esters 3.1 13.6 7.1 9.0 12.8 3.0n-Alkanes 16.2 16.2 49.8 29.9 16.6 14.2

a Acids: monocarboxylic fatty acids; Alcohols: n-primary fatty alcohols; Esters: monesters of fatty acids and alcohols.b Trace amounts.

J.T. Kitagami et al. / Biochemical Systematics and Ecology 46 (2013) 116–119118

Among the samples analyzed, the foliar wax of C. racemosa is unique for bearing hentriacontane (C31) as main n-alkanehomolog, in addition to detectable amounts of tritriacontane (C33). In the foliar wax of all other genotypes nonacosane (C29) isthe main homolog (Table 2). The distribution of n-alkanes of both hybrids is closer to the distribution of the C. arabica parentalthan to C. racemosa. It is not possible to distinguish C. canephora from C. arabica by the distribution of n-alkanes (Table 2).

However, the distribution of n-primary alcohols allows the distinction of C. canephora from the other genotypes: its foliarwax is exclusive for having detectable amounts of homologs with odd numbers of carbon atoms (Table 3). C. racemosa is alsodistinct from the other samples, due to its higher content of octacosanol (C28) and a lower content of triacontanol (C30). Againthe hybrids are closer to the C. arabica parental rather than to C. racemosa: their content of C30 alcohol is similar to bothvarieties of C. arabica (Table 3).

4. Discussion

The contents of wax reported in this study are much lower than values 66–90 mg cm�2 reported by Lichston and Godoy(2006) for foliar waxes of the varieties ‘Catuaí Vermelho’ and ‘Obatã’. These differences may be accounted for by the fact thatLichston and Godoy (2006) analyzed leaves from young plants, which were cultivated in greenhouse, while our data refer toadult plants cultivated outside.

The predominance of alkanes and alcohols in the waxes of most genotypes analyzed corroborates the observation ofStocker and Wanner (1975) regarding C. arabica cv. ‘Bourbon’.

Table 3Distribution of n-primary alcohols of the foliar cuticular waxes of genotypes of Coffea. See caption of Table 1 for explanation about the hybrids H 13685-1 andH 13685-1-27.

Genotypes of Coffea Homologs of n-primary alcohols (%)a

C28 C29 C30 C31 C32

C. arabica cv ‘Catuaí Vermelho’ 2.8 – 73.1 – 24.1C. arabica cv ‘Obatã’ 3.9 – 64.1 – 32.0C. canephora cv ‘Apoatã’ 9.9 5.3 57.5 4.5 22.8C. racemosa 30.3 – 39.5 – 30.2Hybrid H 13685-1 – – 68.6 – 31.4Hybrid H 13685-1-27 – – 58.3 – 41.7

a Dashes: undetected or trace amounts (below 1%).

J.T. Kitagami et al. / Biochemical Systematics and Ecology 46 (2013) 116–119 119

Among the genotypes dealt with in the present work, C. racemosa is the most resistant to water deficits (Guerreiro-Filho,1992). Such hardiness may be linked with the higher content of leaf wax, when compared with the levels of the othergenotypes. The chemical composition of foliar wax may also be important to provide resistance to water vapor diffusion(Juniper and Jeffree, 1983). In this regard, the predominance of alkanes over other classes of wax constituents may be anadditional factor accounting for the hardiness by C. racemosa. Among the common classes of foliar wax components, alkanesmay be the most efficient as a barrier to cuticular transpiration (Oliveira et al., 2003). The distributions of n-alkanes andn-primary alcohols have been used to characterize and distinguish among species and varieties of Coffea (Stocker andWanner,1977). The results presented in this study show that the hybrids H 13685-1 and H 13685-1-27 are close to the parental C.arabica.

Acknowledgments

The authors are grateful to CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).

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