L. Busta et al. / Phytochemistry 121 (2016) 38–49
39
distinguished: elongation and diversification. First, even-
carbon-numbered long-chain fatty acyl-CoAs originating from
plastidial FA de novo biosynthesis are elongated by the addition
of C2 units derived from malonyl-CoA. The fatty acid elongase
(FAE) multi-enzyme complex accomplishes the transformation by
on the fern Osmunda regalis (Jetter and Riederer, 2000), and also
in the form of b-diketones in the Poaceae (Barthlott et al., 1998).
Based on their isomer patterns, these wax constituents with sec-
ondary functionalities have long been suspected to originate from
processes other than P450 hydroxylation, possibly through the
activity of polyketide synthase (PKS) enzymes (von Wettstein-
Knowles, 1995). In essence, their secondary functional groups were
hypothesized to originate during chain elongation, as remnants of
b-functionalities introduced through Claisen condensation reac-
tions, thus explaining their exclusive presence on particular car-
bons. Diverse biochemical experiments have since confirmed this
hypothesis for b-diketones, but have not provided information
about the enzymes and genes involved (von Wettstein-Knowles,
1993). Similar biochemical and molecular genetic evidence is also
lacking for other specialty compounds.
catalyzing four sequential reactions. Initially
a ketoacyl-CoA
synthase (KCS) condenses the fatty acyl-CoA and malonyl-CoA sub-
strates to form a b-ketoacyl intermediate (Joubès et al., 2008), then
a ketoacyl reductase (KCR) reduces the secondary oxo group to a
hydroxyl function (Beaudoin et al., 2009). Next, the b-hydroxyl
group is eliminated by a dehydratase (HCD), and the resulting
a,
b-double bond is finally saturated by an enoyl-CoA reductase
(ECR) to form a fatty acyl-CoA two carbons longer than the original
KCS substrate (Bach et al., 2008; Zheng et al., 2005). Repeated FAE
cycles lead to acyl-CoAs with chain lengths typically ranging from
C24 to C34
.
In the absence of enzymological or genetic information, the
biosynthetic machinery behind the formation of specialty wax con-
stituents is best assessed by further detailed analyses of diverse
compounds with secondary functionalities. In particular, those
with oxygen functionalities on both a primary and a secondary car-
bon are informative, for example 5-hydroxy aldehydes and 1,5-
alkanediols in Taxus baccata and 5-hydroxy acids in Cerinthe minor
(Jetter and Riederer, 1999a; Wen and Jetter, 2007), 11-keto alco-
hols and aldehydes in O. regalis (Jetter and Riederer, 1999b), 1,7-,
1,9-, and 1,11-diols in Papaver alpinum (Jetter et al., 1996), and 3-
hydroxy fatty acids in Aloe arborescens (Racovita et al., 2014). The
primary functionalities therein added substantial chemical diversity
in the form of compound classes with terminal carboxylic acid, ester,
aldehyde, or alcohol group. Comparisons between the homolog
and isomer patterns of these bifunctional compound classes, where
they were co-occurring, thus proved to be particularly informative
for narrowing down possible, novel biosynthetic pathways.
In order to further our understanding of wax biosynthesis, novel
compounds with both primary and secondary functional groups
have to be identified, which necessitates analyses of waxes from
diverse plant lineages. It is important that both vascular and
non-vascular plants are included in this survey, especially since
specialty compounds have been found as prominent members of
the waxes of angiosperms, gymnosperms, ferns, and mosses alike.
Most notably, waxes from several Polytrichales mosses comprised
large percentages of compounds with secondary functional groups,
including 10-nonacosanol (Neinhuis and Jetter, 1995). Waxes of
Andreaea, Pogonatum, Syntrichia, and Physcomitrella moss species
contained ubiquitous wax compounds including fatty acids, alka-
nols, alkyl esters, aldehydes, and alkanes (Buda et al., 2013; Haas,
1982; Xu et al., 2009). However, bifunctional compound classes
from moss waxes have yet to be identified. The goal of the present
study was to provide further moss wax analyses and to identify
bifunctional compounds if possible.
Recently, microscopic examination of multiple structures of the
moss Funaria hygrometrica revealed that the calyptra, a maternal
protective structure of mosses, has a cuticle (Budke et al., 2011).
Interest in the wax of a cuticle that had not been previously ana-
lyzed prompted a preliminary analysis, which indicated the pres-
ence of unknown compounds that potentially contained two
functional groups. As candidates for compounds that could expand
our understanding of the diversity and potentially the biosynthesis
of specialty wax compounds, structural elucidation of these com-
pounds was the focus of this work. Accordingly, waxes were
extracted from the surfaces of the three main structures of F. hygro-
metrica, the maternal leafy gametophyte, the offspring sporophyte
capsule, and the maternal calyptra. The wax mixtures were sepa-
rated with thin layer chromatography (TLC) where necessary,
and analyzed as trimethylsilyl (TMS) derivatives with GC–MS.
Authentic standards were then synthesized to confirm the struc-
tures of the unknown compounds.
In the second stage of ubiquitous wax compound biosynthesis,
elongated VLC acyl-CoAs are diversified into various derivatives
through head group modifications. These take place in a chain-
length-specific manner such that the full range or only a selection
of the chain lengths in the acyl-CoA precursor pool is found in each
of the final wax products. Matching chain length profiles have been
reported for the free and esterified n-alkanols in various plant spe-
cies (Lai et al., 2007; Razeq et al., 2014), indicating that the esters
and free alkanols are biosynthetically related. Indeed, it has been
shown that in Arabidopsis a fatty acyl-CoA reductase (FAR) gener-
ates VLC n-alkanols that are then linked with fatty acyl-CoAs by a
wax ester synthase (Li et al., 2008), and that both the alcohol inter-
mediates and the alkyl ester end products are exported to the cuticle.
Further chain length comparisons across species suggested that a
separate biosynthetic pathway leads to aldehydes and alkanes.
Molecular genetic evidence, again for Arabidopsis, has confirmed that
reduction of VLC acyl-CoAs truly leads to even-carbon-numbered
aldehydes and further decarbonylation to corresponding odd-
carbon-numbered alkanes (Bernard et al., 2012; Chen et al., 2003).
While the biosynthesis of the ubiquitous wax compounds is well
understood, the processes leading to specialty compounds have
received far less attention. Exceptions to this are the secondary
alcohols, diols, ketones, and ketols found as major constituents of
Brassicaceae waxes (Lee et al., 2015; Zhang et al., 2013). Again,
homolog profiles suggest a biosynthetic relationship: that all these
specialty compound classes are derived from the ubiquitous alka-
nes. Further detailed isomer analysis of Arabidopsis stem wax con-
stituents with secondary functional groups indicates that
hydroxylation on one or more of three carbon atoms near the center
of alkane precursor molecules likely leads to the secondary alco-
hols, and repeat hydroxylation or oxidation to the ketones, diols,
and ketols (Wen and Jetter, 2009). Molecular genetic investigations
confirmed this hypothesis, and a P450 monooxygenase with mid-
chain alkane hydroxylase (MAH) activity was shown to be involved
in the process by oxidizing any carbon from C-13 to C-15 of the C29
alkane chain to produce a mixture of isomers whose secondary
functional groups are on adjacent carbons (Greer et al., 2007).
Many other wax compounds with one or more secondary oxy-
gen-containing functional groups that do not seem to be biosyn-
thetically related to the secondary functional compounds of the
Brassicaceae have been described as major wax constituents in
several plant taxa. For example, the secondary alcohol 10-nona-
cosanol is a very prominent wax component of various gym-
nosperm and angiosperm taxa, as well as multiple Pogonatum
moss species (Barthlott et al., 1996; Jetter et al., 1996; Neinhuis
and Jetter, 1995). In contrast to the isomer mixtures of Arabidopsis
secondary alcohols, this compound usually appears as a single iso-
mer, and while some species also contained trace amounts of 8- or
12-nonacosanol, the 9- or 11-isomers were not detected. Similarly,
ketones have been found in some plant waxes, most prominently