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Biochemical Systematics and Ecology 63 (2015) 198e200

Contents lists available at ScienceDirect

Biochemical Systematics and Ecology


journal homepage: www.elsevier.com/locate/biochemsyseco

Esters and other constituents of the foliar cuticular wax of a


soybean variety
C.E.J. Palacios a, G. Negri b, A. Salatino a, *
a ~o Paulo, Rua do Mata
Department of Botany, Institute of Biosciences, University of Sa ~o 277, 05508-090, Sa
~o Paulo, Brazil
b
Department of Psychobiology, Federal University of Sa~o Paulo, CEBRID-UNIFESP, Sa~o Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: The constituents of the foliar cuticular wax of the variety ‘MG/BR 46 Conquista’ were
Received 11 September 2015 isolated and chemically characterized. N-Alkanes, esters and triterpenoids are major
Received in revised form 14 October 2015 constituents of the cuticular wax. Esters, taraxerol and steroids seem to be valuable
Accepted 17 October 2015
cuticular wax constituents for characterization and distinction among soybean varieties.
Available online xxx
© 2015 Published by Elsevier Ltd.

Keywords:
Cuticular wax
Fatty esters
Glycine max
Soybean
Sterols
Triterpenoids

1. Subject and source

The cuticular wax is a crucial factor in the resistance against water loss by plant leaves. Evidence has been raised that cuticular
wax from arid habitats may be more efficient as protection against water loss than their counterparts from other habitats
(Oliveira et al., 2003). Drought affects severely soybean production (Seversike et al., 2014). The composition of the foliar cuticular
wax is probably relevant to search varieties with higher drought resistance. Cuticular wax composition is also useful for char-
acterization and distinction among crop species and varieties (Halinski et al., 2011; Kitagami et al., 2013). The present work deals
with the composition of the foliar cuticular wax of ‘MG/BR 46 Conquista’, currently a soybean variety widely cultivated in Brazil.

2. Previous work

To our knowledge, so far the paper by Kim et al. (2007) has been the only report addressing the composition of soybean
foliar cuticular wax.

3. Present study

Pesticide-free seeds of the variety ‘MG/BR Conquista’ of soybean (Glycine max (L.) Merril) were provided by EMBRAPA
Soybean Division. Germinated seedlings were placed on trays with vermiculite and grown at 30  C during 4 days under

* Corresponding author.
E-mail address: asalatin@ib.usp.br (A. Salatino).

http://dx.doi.org/10.1016/j.bse.2015.10.012
0305-1978/© 2015 Published by Elsevier Ltd.
C.E.J. Palacios et al. / Biochemical Systematics and Ecology 63 (2015) 198e200 199

photoperiod of 12 h. The plantlets were maintained in greenhouse for 10 days. The young plants were transferred to open air
plots containing soil fertilized with vegetal ash in the experimental garden of the Department of Botany of the University of
Sa~o Paulo (Brazil). The cultivation took place in JuneAug 2011 (winter season in southeast Brazil).
Total areas of five samples, each comprising 10 leaflets from 60 day old plants, were determined with the software ImageJ
1.44p. The cuticular wax was extracted by three consecutive immersions (30, 20 and 10 s) of the leaflets in dichloromethane.
The solvent was evaporated and the wax weighed. Five fractions of wax constituents were isolated by preparative thin layer
chromatography (TLC), using silicagel impregnated with 0.02% sodium fluoresceine (Oliveira et al., 2003), dichlorometane as
mobile phase and visualization under long wave UV. The fractions of alkanes and fatty esters were analyzed directly by GC/
MS. Fractions containing fatty acids, triterpenes and sterols were derivatized with N,O-bis-(trimethylsilyl)-trifluoroacetamide
(Jetter et al., 2000) prior to GC/MS analysis.
Analysis of 1 mL of n-hexane solutions of n-alkane and derivatized fractions were performed with a GC/MS system Agilent
GC/MS 6859/5975B, equipped with capillary column HP-5MS (crosslinked 5% MePh siloxane, 30 m  0.25 mm) according to
Kitagami et al. (2013). The esters were analyzed starting at 250  C during 3 min, increasing 5  C min1 to 320  C and isotherm
condition for 23 min. The n-alkane and fatty ester fractions were also analyzed in a HP 5890 series II gas chromatograph with
FID detector. The relative percents of n-alkanes and esters were estimated by the area under the corresponding peaks.
Compounds identification followed mass spectroscopy, comparison with NIST 05 Library and authentic samples. The esters
were identified by the base peak (corresponding to the protonated acid moiety) and the molecular ion (Moldovan et al., 2002).
Identification of TMSi-derivatized triterpenoids was based on Ogunkoya (1981) and Carvalho et al. (2010), and the derivatized
sterols on Dumazer et al. (1986), Buckley et al. (1999) and Moreau et al. (2002).
The leaves analyzed contained 15.2 ± 1.1 mg cm2 of cuticular wax. Contents in the range 10e30 mg cm2 are common on
leaves of cultivated plants (Baker, 1982). An average of 8.26 ± 1.67 mg cm2 of cuticular wax was reported for 18 soybean
varieties (Kim et al., 2007). Five fractions were obtained from the wax: n-alkanes (10.8% of the products recovered from TLC),
esters (25.7%), triterpenoids plus primary alcohols plus unknowns (16.2%), and sterols plus unknowns (37.5%). The n-alkane
homologues ranged from C23 to C33, with predominance of C31 (27.5%). Acid moieties of the esters were C16, C18 and C20 and of
alcohols were C18, C20, C26 and C28. The predominant ester was octacosanoyl-eicosanoate (C28eC20, 46.7%). The primary al-
cohols detected were C16, C18, C20, C22, C26 and C28, with high predominance (47%) of the latter homologue. Amyrins (a and b),
lupeol and taraxerol were the triterpenoids detected. Taraxerol was characterized by the molecular ion (Mþ, m/z 498) and the
base peak m/z 204, and eluted before a-amyrin (Bauer et al., 2004). The intensity of the taraxerol peak in the chromatogram,
relative the total triterpenoids, was 9.6%. Sterols (sitosterol, stigmasterol and campesterol) were also detected as minor
constituents.

4. Chemotaxonomic significance

Cuticular wax constituents have been widely used as chemotaxonomic markers, particularly the distribution of n-alkanes
 ski et al., 2011; Jingjing et al., 2013). Little has been done regarding the chemotaxonomic potential of esters of cuticular
(Halin
wax. The distribution of wax esters can be analyzed by GC/MS without derivatization (Furuhasi et al., 2015; present work).
Wax esters seem to be relevant chemotaxonomic markers for characterization of soybean varieties and distinction among
them. In fact, they are major constituents of the foliar cuticular wax of the variety ‘MG/BR 46 Conquista’ and have not been
detected in the cuticular wax of 18 other varieties of soybean (Kim et al., 2007). Not only presence/absence, but also the
homologue distribution of cuticular esters may be chemotaxonomically relevant, as has been observed regarding n-alkanes;
for example, the cuticular waxes of Coffea arabica and C. canephora are characterized by the predominance of the C29, and C.
racemosa by the C31 n-alkane (Kitagami et al., 2013). Caution must be taken, however, regarding presence/absence of esters as
soybean chemotaxonomic markers, since these constituents may have been overlooked or were not detected by Kim et al.
(2007), due to the methodology they employed, which was quite distinct from the one used in the present work. Wax tri-
terpenes also seem to be useful chemotaxonomic markers for distinction among soybean varieties: taraxerol was detected in
the cuticular wax of ‘MG/BR 46 Conquista’ and not in the varieties analyzed by Kim et al. (2007), while the reverse holds for
lupenone and 3-keto-olean-12-ene. Sterols are rare foliar wax constituents, except in Solanaceae (Halin  ski et al., 2012). They
were detected in the foliar wax of ‘MG/BR 46 Conquista’, but were not mentioned in the work by Kim et al. (2007).

Acknowledgments

~o de Aperfeiçoamento do Pessoal de Ensino Superior) is


Provision of MSc scholarship to CPJ by CAPES (Coordenaça
acknowledged. AS is a research fellow of CNPq.

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