F.M. Razeq et al. / Phytochemistry 106 (2014) 188–196
189
attributes of C. sativa, and whether they can be further improved, are
currently unclear.
Scanning electron microscopy studies of leaf surfaces revealed
an absence of epicuticular wax crystal structures (Fig. 1H). Similarly,
smooth surfaces without crystals were observed on leaves of
A. thaliana ecotypes Wassilewskija (WS), Landsberg erecta (Ler)
(Jenks et al., 1995), and C24 (Teusink et al., 2002). Wax loads in these
All land plants have aliphatic and phenolic-based barriers pres-
ent at environmental interfaces that serve to protect them against
water loss and various stresses. Their aerial surfaces are coated
with a cuticle, which is comprised of cutin and waxes (Samuels
et al., 2008). Cutin is a polymer mostly made up of glycerol and
long-chain (C16 and C18) fatty acid derivatives overlaying the cell
wall and serves as the structural backbone of the cuticle. Cuticular
waxes are mixtures of low polarity compounds, including very-
long-chain (PC20) straight-chain aliphatics (e.g. fatty acids, fatty
aldehydes, alkanes, primary and secondary fatty alcohols, wax
esters, and b-diketones), triterpenoids, and phenolic-lipids (e.g.
alkylrescorcinols) (von Wettstein-Knowles, 2012). Intracuticular
waxes are embedded in the cutin matrix and epicuticular waxes,
often in the form of crystals, cover the outer surface. Cuticular
waxes serve the essential function of limiting non-stomatal water
loss (Riederer and Schreiber, 2001; Vogg et al., 2004). Epicuticular
waxes being in direct contact with the environment play key roles
in plant–insect and plant–pathogen interactions as well as in the
shedding of dust, spores, and water droplets (Kerstiens, 1996).
Cuticular wax composition varies substantially between species,
organs of the same plant, developmental stages and environmental
conditions (Jetter et al., 2006). Another hydrophobic surface barrier
is suberin. Similar to cutin, suberin is a polyester of glycerol, phen-
olics and fatty acid derivatives (long-chain and very-long-chain)
(Franke and Schreiber, 2007). Suberin is found on the inner face
of cell walls of certain tissue layers, for example the root endoder-
mis and peridermis. There are also waxes associated with suberin
and, when in roots, termed root waxes (Espelie et al., 1980; Kosma
et al., 2012; Li et al., 2007). Suberin-associated or root wax com-
pounds variably include long-chain and very-long-chain fatty
acids, primary fatty alcohols, alkanes, monoacyglycerols, sterols,
and alkyl hydroxycinnamates (fatty alcohols esterified with cou-
maric, caffeic, or ferulic acids) (Kosma et al., 2012).
A. thaliana ecotypes (1.6, 1.0, and 1.1 l
g cmꢀ2, respectively) were
comparable to total waxes accumulated on C. sativa leaves. It is pos-
sible that the leaves on these species are covered solely by an amor-
phous wax layer, because a critical amount of lipids is necessary to
form crystals, although it is well documented that crystal formation
is also influenced by wax chemical composition (Baker, 1982;
Rowland et al., 2006). The wax loads on leaves of Brassica oleracea
and B. napus are much higher (25–50 l
g cmꢀ2) than C. sativa leaves
and their leaf surfaces are characterized by densely packed dendritic
tubes of wax crystals (Baker, 1974; Holloway et al., 1977). The leaf
wax compositions of B. oleracea and B. napus are also very different,
being dominated by alkanes, ketones, and secondary alcohols.
2.2. Composition of wax esters found in leaf cuticular wax
Alkyl esters of long-chain fatty acids and long-chain fatty alco-
hols (wax esters), with carbon chain lengths ranging from C38 to
C50, were present in the waxes extracted from leaves but not in
those extracted from other organs. Wax esters were quantified
by GC-FID, and equaled 4.7 0.6
4.6 0.5
g cmꢀ2 in bottom leaves (Fig. 2, inset). By comparison,
wax ester content in Ler and WS ecotypes of A. thaliana stem cutic-
ular wax is 0.17 and 0.2
g cmꢀ2, respectively, with only traces
l
g cmꢀ2 in top leaves and
l
l
present in the rosette leaf cuticular wax fractions (0.002
l )
g cmꢀ2
(Jenks et al., 1995).
Saturated wax ester homologues were identified according to
their molecular ions (M+Å). Like wax esters found in cuticular waxes
of other species, each C. sativa wax ester peak was composed of a
mixture of constitutional isomers. To characterize these isomeric
mixtures, the relative amounts of the acyl moieties were compared
within each ester group present in the mass spectrum. The alcohol
(R0OH) and acid (RCOOH) moieties of the isomeric mixtures were
identified according to their most abundant diagnostic fragments,
[R0–H]+Å and [RCOOH2]+, respectively, which are products of cleav-
age near the ester bond (Table 1) (Aasen et al., 1971; Lai et al.,
2007; Sümmchen et al., 1995; Urbanova et al., 2012). For each
even-numbered wax ester (C38–C48), the protonated acyl frag-
ments [RCOOH2]+ had higher intensities and were used to calculate
the abundance of each wax ester isomer (n = 4). The most abun-
dant ions representing the alcohol moiety of the esters were radi-
cal cations [R0ꢀH]+Å, produced by elimination of the acyl moiety;
these were identified to confirm isomer structure.
The surface waxes of C. sativa may confer, in part, its relative
tolerance to various abiotic and biotic stresses. These extracellular
lipids may also represent an additional source of high-value com-
ponents that can be extracted from the plant. Herein a detailed
chemical description of the extracellular waxes of C. sativa aerial
and root organs is reported.
2. Results and discussion
Wax mixtures from stems, leaves, flowers, seeds and roots of C.
sativa were extracted by rapid immersion in chloroform, silylated,
and analyzed by gas chromatography (GC). Molecular components
were identified from both their GC-retention behavior and charac-
teristic mass spectra (MS), and quantified by flame ionization
detection (FID).
Table 1 details the relative amounts of different isomers in the
major wax ester homologues (C38, C40, C42, C44, C46, C48 and C50).
Small amounts of odd-carbon number ester homologues (0.06–
0.16 l
g cmꢀ2 each) with carbon chain lengths C41, C43, C45, C47
and C49, were also identified (data not shown); their isomer com-
positions are not reported here because the method is reliable only
when substantial concentrations of components are present in the
mixture (Aasen et al., 1971). The most abundant wax esters were
2.1. Leaf cuticular wax
Leaf samples were analyzed at two different growth stages along
the vegetative or primary stem, identified as ‘‘top’’ (youngest) and
‘‘bottom’’ (oldest) (Fig. 1A). Total leaf wax loads were not signifi-
cantly different between the top and bottom portions, which accu-
C42, C44 and C46, which comprised 18, 26 and 13 mol% of the total
wax esters of bottom leaves, respectively, and 19, 25 and 18 mol%
of the total wax esters in top leaves, respectively (Fig. 2). Substan-
tial amounts of C40 (10–11 mol%), and C48 (8–11 mol%) were also
found in both young and old leaves. The predominant wax ester
isomers were composed of eicosanoic acid (20:0) and 1-tetracosa-
nol (24:0-OH), constituting 68% of the isomeric molecular species
in the C44 wax esters, eicosanoic acid (20:0) and 1-docosanol
(22:0-OH), constituting 52% of the C42 wax esters, and eicosanoic
acid (20:0) and 1-hexacosanol (26:0-OH), constituting 63% of the
C46 wax esters (Table 1).
mulated 6.3 1.1 and 5.8 0.8 l
g cmꢀ2 (mean SD., n = 4; Fig. 2,
inset), respectively. However, they differed in their relative compo-
sitions of wax esters and alkanes (T-test, two-sided P < 0.05). Wax
esters were the dominant compounds in both top and bottom
leaves (64 and 69 mol%, respectively), followed by very-long-chain
primary fatty alcohols, ranging from C22 to C34 (26–27 mol%), fatty
acids (4–5 mol%) and odd-carbon number alkanes (C29–C33) (3 and
1 mol% in top and bottom leaves, respectively) (Fig. 2).
Razeq, Fakhria M.
