Download Monoacylglycerols Are Components of Root Waxes

Monoacylglycerols Are Components of Root Waxes and
Can Be Produced in the Aerial Cuticle by Ectopic
Expression of a Suberin-Associated Acyltransferase1[W][OA]
Yonghua Li, Fred Beisson, John Ohlrogge, and Mike Pollard*
Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824
The interface between plants and the environment is provided for aerial organs by epicuticular waxes that have been extensively studied. By contrast, little is known about the nature, biosynthesis, and role of waxes at the root-rhizosphere interface.
Waxes isolated by rapid immersion of Arabidopsis (Arabidopsis thaliana) roots in organic solvents were rich in saturated C18C22 alkyl esters of p-hydroxycinnamic acids, but also contained significant amounts of both a- and b-isomers of monoacylglycerols with C22 and C24 saturated acyl groups and the corresponding free fatty acids. Production of these compounds in
root waxes was positively correlated to the expression of sn-glycerol-3-P acyltransferase5 (GPAT5), a gene encoding an acyltransferase previously shown to be involved in aliphatic suberin synthesis. This suggests a direct metabolic relationship
between suberin and some root waxes. Furthermore, when ectopically expressed in Arabidopsis, GPAT5 produced very-longchain saturated monoacylglycerols and free fatty acids as novel components of cuticular waxes. The crystal morphology of
stem waxes was altered and the load of total stem wax compounds was doubled, although the major components typical of the
waxes found on wild-type plants decreased. These results strongly suggest that GPAT5 functions in vivo as an acyltransferase
to a glycerol-containing acceptor and has access to the same pool of acyl intermediates and/or may be targeted to the same
membrane domain as that of wax synthesis in aerial organs.
Suberin and cutin are ubiquitous extracellular lipid
polymers found in plants. Each polymer is insoluble
in organic solvents, but is found in association with
solvent-extractable waxes. Cutin and its cuticular and
epicuticular waxes form the cuticle layer covering all
aerial organs of plants. The cuticle protects plants from
biotic and abiotic stresses, limits gas and water exchange, and is likely involved in developmental processes during plant growth (Kolattukudy, 2001;
Nawrath, 2006). Usually, cutin polyesters are composed largely of C16 and C18 v-hydroxy fatty acid
monomers, which often have midchain functionality,
such as epoxy, secondary hydroxyl, or vicinal diol
groups (Kolattukudy, 2001; Nawrath, 2002). Glycerol is
also a monomer and has been shown to be esterified to
v-hydroxy fatty acids (Gracxa et al., 2002). In Arabidopsis (Arabidopsis thaliana) and oilseed rape (Brassica
napus), the polyesters of leaf and stem epidermis and
isolated cuticles are unusual in that they contain high
1
This work was supported by the National Research Initiative of
the U.S. Department of Agriculture Cooperative State Research,
Education, and Extension Service (grant no. 2005–35318–15419).
* Corresponding author; e-mail [email protected]; fax 517–353–
1926.
The author responsible for distribution of materials integral to
the findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Mike Pollard ([email protected]).
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The online version of this article contains Web-only data.
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Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.107.099432
proportions of a,v-dicarboxylic acids and, particularly,
that derived from linoleic acid (Bonaventure et al., 2004;
Franke et al., 2006). Cuticular waxes are derived from
very-long-chain (C22-C34) saturated fatty acids. Alkanes are common, but a wide variety of neutral lipids
are found (Kolattukudy, 1980; Jetter et al., 2006). The
chemical genetics of plant cuticular waxes has been
extensively studied (Kunst and Samuels, 2003). Arabidopsis stem epicuticular waxes are dominated by nonacosane and its 14-hydroxy, 15-hydroxy, and 15-oxo
derivatives, but contain smaller amounts of free fatty
acids (FFAs), aldehydes, primary alcohols, and wax
esters (Rashotte et al., 2001). Thus, despite their cooccurrence, there are few structural similarities between cuticular waxes and cutin monomers.
Suberin and its associated waxes form the suberin
layer, which is often characterized by electron-translucent
and electron-dense lamellae observed by transmission
electron microscopy. Suberin is present in many external as well as internal tissues and has an important
role in controlling water and solute fluxes. Its deposition is often induced by wounding or stress stimuli,
thereby providing a barrier against pathogen invasion
(Lulai and Corsini, 1998; Kolattukudy, 2001; Bernards,
2002). Suberin has been proposed to comprise polyphenolic and polyaliphatic domains (Bernards, 2002).
The term aliphatic suberin is used in this article to describe the polyaliphatic domain of suberin. In contrast
to cutin, depolymerization of suberin or suberin-rich
tissues produces fatty acids, fatty alcohols, hydroxycinnamic acids, and a,v-dicarboxylic acids in addition to v-hydroxy fatty acid monomers. Midchain
functional groups are rare. Suberin often includes
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Li et al.
substantial amounts of saturated monomers with chain
length .C20 (Kolattukudy, 1980, 2001; Bernards,
2002; Schreiber et al., 2005). Analyses of Arabidopsis
tissues expected to be enriched in suberin, namely,
roots and seed coats, have shown typical suberin
monomer compositions (Franke et al., 2006; Molina
et al., 2006; Beisson et al., 2007). A substantial portion of the hydroxycinnamic acids of suberin are
believed to be cross-linked (Bernards et al., 1995;
Bernards, 2002). Besides aliphatic and aromatic components, suberins contain a substantial proportion of
glycerol (Holloway, 1982; Gracxa and Pereira, 1997;
Gracxa et al., 2002), the content of which is positively
correlated to the suberization process (Moire et al.,
1999). Partial chemical depolymerizations of bark and
potato (Solanum tuberosum) periderm suberins have
yielded fragments that include monoacylglycerols
(MAGs) of a,v-dicarboxylic acids, v-hydroxy fatty
acids and fatty acids, diglycerol esters of a,vdicarboxylic acids, and an v-ferulyloxy-acyl glycerol
(Gracxa and Pereira, 1997, 1999, 2000; Gracxa and
Santos, 2006; Santos and Gracxa, 2006). Unlike cuticular waxes and cutin, suberin waxes tend to reflect, in
part, suberin polymer compositions. Suberin-associated
waxes have been studied mostly in the native and
wound-healing periderm of plant subterranean storage organs, where the main constituents appear to
be alkanes, primary alcohols, fatty acids, and alkyl
ferulates (Espelie et al., 1980; Bernards and Lewis,
1992; Schreiber et al., 2005). Suberin-associated waxes
are considered major contributors to the barrier for
water diffusion across suberized cell walls (Soliday
et al., 1979), but other factors controlling permeability
remain to be identified (Schreiber et al., 2005). Green
cotton (Gossypium hirsutum) fibers contain suberin and
a significant amount of suberin-like waxes, including
1-(22-caffeyloxydocosanoyl)-glycerol as a major component (Schmutz et al., 1994).
A family of sn-glycerol-3-P acyltransferase (GPAT)
genes has been identified in Arabidopsis, five members of which gave detectable enzyme activity when
expressed heterologously in a gat1D strain of yeast
(Saccharomyces cerevisiae; Zheng et al., 2003). Our recent
characterization of a mutant gpat5, with a null mutation in the GPAT5 acyltransferase gene (At3g11430),
revealed its essential role in aliphatic suberin synthesis
(Beisson et al., 2007). The seeds of gpat5 plants have
50% of the wild-type polyester load with large reductions in C20-C24 suberin-like aliphatic monomers,
whereas roots from 1-week-old seedlings have reductions in C20-C24 monomers. These findings are consistent with glycerol as a suberin monomer and offer the
first clue as to how glycerol might be incorporated into
the polyester network. However, the exact biochemical
function of the GPAT5 enzyme remains unknown.
We report here on the characterization of root waxes
in the model plant Arabidopsis and show that they
have a distinct composition and contain MAGs. We
also demonstrate that the accumulation of these MAGs
is positively correlated with the expression of GPAT5,
suggesting a common pathway for the biosynthesis of
aliphatic suberin and some of the suberin-associated
waxes. Furthermore, we show that ectopic expression
of GPAT5 under the control of the cauliflower mosaic
virus (CaMV) 35S promoter leads to production of
a- and b-isomers of MAGs as novel components of leaf
and stem surface waxes, changing significantly the
composition and morphology of the aerial cuticle.
RESULTS
MAGs Are Components of Arabidopsis Root Waxes
Stem and leaf surface waxes are operationally defined as the lipid material extracted by quickly dipping
these organs into chloroform. Using the chloroformdipping procedure, we examined the presence of surface lipids in roots of 7-week-old Arabidopsis plants.
At this stage, the roots have undergone considerable
secondary growth and the epidermis, cortex, and endodermis have been cast off, leaving the newly formed
suberin-rich periderm as the root outer cell layers
(Dolan and Roberts, 1995; Franke et al., 2006). The presence of a suberized periderm at the periphery of the
roots used in this study was confirmed by staining the
root cross sections with the lipophilic dye Sudan Red
7B (Fig. 1). We found that a 10-s dip in chloroform,
sufficient to recover .98% (w/w) of total surface waxes
from Arabidopsis stems, also extracted significant
amounts of lipid material from roots (Fig. 2, A and B).
A 1-min dip of roots in chloroform was found to dissolve approximately 90% (w/w) of the lipid material
(Fig. 2A) extractable by this method without significant (,5% [w/w]) contamination from intracellular
membrane lipids (data not shown). This procedure
was therefore used for all subsequent root analyses
and the lipid material recovered by this procedure was
collectively termed root waxes. At 7 weeks, Arabidopsis roots contained 360 6 32 mg/g fresh weight of
Figure 1. Cross section of 7-week-old Arabidopsis roots stained by
Sudan Red 7B to reveal the suberized periderm.
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GPAT5 Expression Produces Extracellular Monoacylglycerols
Figure 2. Profiling of root waxes of 7-week-old wildtype Arabidopsis plants. A, Recovery of total waxes
by chloroform dipping of roots versus stems. B,
Extraction kinetics of individual root wax components. C, Composition and content of Arabidopsis
root waxes (mean with 95% CI; n 5 4). PA, Primary
alcohol; VLCFFA, very-long-chain FFA.
root waxes (mean with 95% confidence interval [CI];
n 5 4). This value compares with values of approximately 1,000 mg/g fresh weight for stem wax loads
and approximately 100 mg/g fresh weight for leaf wax
loads.
The composition of waxes of Arabidopsis roots (Fig.
2C) was very distinct from that of Arabidopsis aerial
parts (Rashotte et al., 2001) and significantly different
from that reported for the subterranean storage organs
of seven species, including Crucifers (Espelie et al.,
1980). In Arabidopsis roots, esters of p-coumaric,
caffeic, and ferulic acids with C18-C22 saturated fatty
alcohols were the major component (47% [w/w]). Primary alcohols were present in significant amounts
(10% [w/w]) with a chain length profile very similar to
those esterified to hydroxycinnamic acids. This profile
distinguishes them from the C26-C30 primary alcohols of Arabidopsis cuticular waxes in aerial organs
(Rashotte et al., 2001). Substantial amounts of sterols
and FFA were also observed (approximately 15% [w/w]
each). Components that dominate aerial waxes, namely,
nonacosane and its 15(14)-hydroxy and 15-oxo derivatives, were only minor contributors to root waxes
(approximately 5% [w/w]). We will collectively call
these major components found in the cuticular waxes
of wild-type plants standard cuticular waxes.
The most unusual feature of Arabidopsis root waxes
was the presence of both a- and b-isomers of MAGs
(approximately 7% [w/w]). The acyl groups were
C22 . C24 . C26-C30 saturates, with negligible C20
and, in this respect, MAG distribution was similar to the
longer chain FFAs. Identification of MAG isomers was
accomplished primarily by gas chromatography (GC)mass spectrometry (MS) of their bis-trimethylsilyl
(TMSi) derivatives (Murphy, 1993), as shown in Figure
3, A and B, for the tetracosanoyl species. Although
molecular ions are absent from the electron impact
mass spectra, the [M-15]1 ion at mass-to-charge ratio
(m/z) 5 571 gives Mr 5 586. For a-MAG isomers,
cleavage of the C(b)-C(g) bond of the glycerol backbone usually produces the base peak [M-CH2OTMSi]1.
The m/z 5 483 ion in Figure 3A corresponds to this
cleavage. This [M-103]1 ion is very weak in the mass
spectra of b-MAGs. Instead, their most diagnostic ions
are [M-RCOOH]1 and [M-RCOOH-OTMSi]1, which
correspond to m/z 5 218 and 129, respectively (Fig.
3B). Identification of these MAG isomers was consistent with their retention factor values on silica thinlayer chromatography and their retention time values
on GC analysis, when compared to standards. It is important to note that, although the a-MAG isomer is
thermodynamically more stable than the b-MAG isomer (Gunstone, 1967), the latter is more prevalent in
the root waxes.
Kinetics of chloroform-dipping extractions showed
that, after a 10-s dip sufficient to extract 100% of stem
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Li et al.
Figure 3. Identification of saturated MAGs present in Arabidopsis root
waxes by GC-MS of their bis-TMSi derivatives: mass spectra of C24
a-MAG (A) and C24 b-MAG (B).
et al., 2004). Ectopic expression of GPAT5 was confirmed via reverse transcription-PCR transcript analysis
of mRNA prepared from seven independent lines using leaves, an organ where GPAT5 is not expressed
in wild type (data not shown). Two lines (designated
OE-1 and OE-2) were used for subsequent analyses.
Plants ectopically expressing GPAT5 displayed no obvious difference in growth or morphology compared to
wild type. There was no significant change in the aliphatic suberin load and composition of 7-week-old
roots of 35STGPAT5-expressing plants (Supplemental
Fig. S1). This may indicate that enzyme activity of
GPAT5 by itself does not limit suberin biosynthesis in
roots at this stage of development.
However, analyses of the root waxes of wild type,
two 35STGPAT5-expressing lines (OE-1 and OE-2),
and two knockout mutant lines (gpat5-1 and gpat5-2)
revealed strong positive correlation between GPAT5
expression and the concentration of MAGs (Fig. 4). An
approximately 50% (w/w) reduction was observed in
the mutants compared to a 70% (w/w) increase in the
35STGPAT5-expressing lines. Both positional isomers
of MAG were affected to a similar degree (data not
shown). A positive correlation was also observed for
C22-C30 FFAs (Fig. 4). Other root wax components
were not significantly changed with altered GPAT5 expression (primary alcohols, sterols) or only weakly
reduced by GPAT5 knockout and not increased by
overexpression (alkyl ferulates; data not shown). The
simple explanation for these results is that GPAT5
waxes, the only root wax component completely recovered was alkanes (Fig. 2B). Other root wax components were extracted more slowly, most noticeably
primary alcohols. Also, these kinetics of extraction
showed that increasing dipping time did not allow
the recovery of additional amounts of C16-C20 FFAs
and sterols, although more of these lipids could be
recovered by grinding the roots (data not shown). This
indicated that there were distinct intracellular and wax
fractions for C16-C20 FFAs and sterols.
Accumulation of MAGs in Root Waxes Correlates with
GPAT5 Expression
Previously we noted that the T-DNA knockout
mutant gpat5 had large reductions in suberin-like aliphatic monomers released by transesterification of the
residual fraction of seeds and roots remaining after
extensive delipidation (Beisson et al., 2007). To better
understand the specific role of GPAT5 in the pathway
of aliphatic suberin synthesis, we expressed this gene
in Arabidopsis under the control of the CaMV 35S promoter. This constitutive promoter has been used in mutant complementation experiments with a number of
cuticular lipid synthesis genes. In particular, a number
of genes involved in cuticular lipid biosynthesis and
known to be differentially expressed in the epidermis
(Suh et al., 2005) were highly induced by 35S-driven
overexpression of the WIN1 transcription factor (Broun
Figure 4. Changes in the quantity of FFAs and MAGs (sum of both
isomers) in the root waxes prepared from the T-DNA insertional mutant
lines (gpat5-1 and gpat5-2) and the 35STGPAT5 overexpression lines
(OE-1 and OE-2) as compared to that of wild type (7-week-old soilgrown roots). Bars 5 mean with 95% CI (n 5 4). The difference
between the mean of the wild type and the mean of a transgenic line
was significantly different from zero in all cases (P , 0.05; two-tailed
t test with unequal variances).
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GPAT5 Expression Produces Extracellular Monoacylglycerols
GPAT5 Overexpression Produces MAGs as Novel
Components of Cuticular Waxes
Figure 5. SEM images of stem surface of wild type (A) and 35STGPAT5
overexpression line OE-2 (B). Scale bars in A and B are 20 mm; for insets
in A and B, 2 mm.
activity is limiting for the production of MAGs in
waxes from wild-type roots. Null mutations of GPAT5
may not completely deplete MAGs from the suberinassociated waxes because of gene redundancy within
the GPAT family. Indeed, all other GPATs, except one,
are expressed in roots (Beisson et al., 2007), although
their precise spatiotemporal expression patterns have
yet to be determined.
To further demonstrate the link between GPAT5 and
the production of MAGs, we characterized the stem
cuticular waxes of the 35STGPAT5-expressing plants.
Scanning electron microscopy (SEM) of the stem surface showed a large reduction in wax crystal density
compared to wild type (Fig. 5). Stems of wild-type plants
were covered primarily with columnar shaped crystals, although rods, tubes, vertical plates, and dendriticand umbrella-like structures were also visible (Rashotte
and Feldmann, 1998; Jetter et al., 2006). In the stems of
35STGPAT5 plants, crystals that were still visible are
mostly plate like. These observations suggested changes
of cuticular wax content and composition on the stem
surface of 35STGPAT5-expressing plants. Paradoxically, despite a reduction in wax crystal densities,
chemical analysis revealed 1.8- and 2.2-fold increases
in total cuticular wax load in OE-1 and OE-2 plants,
respectively.
Wax analysis identified both a- and b-isomers of
MAGs, with saturated C22-C30 acyl groups, as novel
components of the cuticular waxes from stems of 35ST
GPAT5-expressing plants, constituting up to 20% (w/w)
of the total wax load (Fig. 6, inset). Whereas FFAs are
only minor components of standard waxes (,5%),
greatly elevated levels of C22-C30 FFAs (approximately 50% [w/w]) were also noted in the transgenes.
The accumulated MAGs have a slightly altered acyl
chain length distribution when compared to those present in the root waxes of Arabidopsis plants (Figs. 2 and
6). Tetracosanoic acid was the major FFA and acyl
group in MAGs of stems. Another noticeable phenotype was a shift in distribution of the saturated wax
esters, which constitute about 7% to 9% (w/w) of total
wax load, from C40-C46 in wild type to C40-C54 in the
transgenes. In wild-type wax esters, the predominant
acyl group was C16, but in the transgene there was an
additional contribution largely from the C22 acyl
Figure 6. Composition and content of chloroformdipping fraction of epicuticular waxes from stems of
5-week-old wild-type and 35STGPAT5 overexpression lines OE-1 and OE-2 (mean with 95% CI; n 5 4).
ALK, Alkane; OH, secondary hydroxy; KET, ketone;
PA, primary alcohol. Inset, Amount of individual lipid
classes.
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Li et al.
Table I. Characteristics of the chloroform-dipping wax fraction from various organs of 35STGPAT5 overexpression lines (mean with 95% CI; n 5 4)
n.d., Not detected.
Lipid Phenotypes
Line
Wax Classes
Stems
Leaves
Siliques
Seeds
Total wax load (mg/g fresh weight)
Wild type
Standard waxes
MAG 1 FFA
Standard waxes
MAG 1 FFA
Standard waxes
MAG 1 FFA
860 6 200
n.d.
780 6 320
810 6 280
620 6 300
1,230 6 400
;2
3:1
1:2
C24
80 6 5
n.d.
68 6 10
310 6 12
80 6 10
380 6 5
;5
3:1
1:2
C26
1,500 6 100
n.d.
840 6 200
1,100 6 150
750 6 100
1,250 6 130
;1.5
2:1
1:2
C24
170 6 30
n.d.
150 6 50
n.d.
400 6 100
380 6 50
;5
1:2
2:1
C24
OE-1
OE-2
Fold increase in wax load
FFA:MAG (w/w)
a-MAG:b-MAG (w/w)
Dominant acyl-chain length
MAG 1 FFA
group (data not shown). The novel MAG and FFA
products observed in stem waxes for 35STGPAT5expressing plants were also noted for surface waxes
from leaves, siliques, and seeds (Table I).
By analyzing the lipids in both the 30-s chloroform
dip fraction and the remaining stem tissue, we demonstrated that two-thirds of very-long-chain fatty acids
(VLCFAs; total acyl groups from MAGs and FFAs)
were immediately extractable (Fig. 7A). This compares
with wild-type surface waxes, which are completely
extracted, and with intracellular lipids (i.e. C16-C20
fatty acids), which are not extracted. Furthermore, the
chain length distribution of FFA and MAG in these
two fractions was similar, although the longer chain
MAG species did appear to be preferentially found on
the surface (Fig. 7B). Epidermal peel experiments
confirmed that the MAGs plus FFAs remaining after
the short chloroform dip are largely epidermal (.90%
[w/w]), although whether their site of deposition is
extracellular, intracellular, or both remains to be determined. Taken together, these findings show that
ectopic expression of GPAT5 in the aerial parts of plants
results in novel MAG, wax ester, and FFA products and
that these products are largely secreted onto the plant
surface, where they radically alter the crystal morphology of the epicuticular wax layer. Although lysophosphatidic acid might be expected to be the first product
of the GPAT5 reaction (Zheng et al., 2003), analyses of
the short chloroform dip and lipids extracted from
residual stems failed to reveal any lysophosphatidic
acid containing VLCFAs. Also, an analysis of stem
cutin showed that no additional long-chain C22-C30
fatty acids were incorporated into this polyester (Supplemental Fig. S2).
GPAT5 Overexpression Reduces Accumulation of
Wild-Type Cuticular Wax Components
In addition to the appearance of novel components
in 35STGPAT5 overexpression lines, there was a reduction in the load of some standard wax components,
most dramatically for the C29 alkane component of
the decarboxylation/decarbonylation pathway, whereas
C26-C30 primary alcohols remained approximately
constant (Fig. 6). To confirm the reduction in standard
Roots
360 6 30
300 6 120
400 6 150
;1
1:1
1:2
C22
waxes, we analyzed the accumulation of C22-C30 fatty
acids (from MAGs plus FFAs) in stems from 6-weekold plants for 18 independent T2 35STGPAT5 lines. An
inverse relationship was observed between the amount
of standard waxes present in each line and the amount
of newly formed VLCFAs due to GPAT5 expression
(Fig. 8).
Surface MAGs Are Produced in 35STGPAT5-Expressing
Tobacco Plants
Although the main focus of this study was Arabidopsis, some experiments conducted with tobacco
Figure 7. Extractability of stem wax components from 35STGPAT5
overexpression Arabidopsis plants after rapid dipping in chloroform. A,
Chloroform-extracted lipid and the residual tissue were transmethylated to release total fatty acids as methyl esters prior to silylation and
GC analysis. C16-C20 fatty acids are derived mainly from polar membrane lipids, whereas C22-C30 fatty acids are derived almost exclusively from FFAs and MAGs. B, Chain length distribution of novel MAGs
and FFAs in the chloroform-dipping and residual fractions. These lipids
were analyzed without transmethylation (mean with 95% CI; n 5 4).
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GPAT5 Expression Produces Extracellular Monoacylglycerols
(Nicotiana tabacum) suggest that the results seen with
Arabidopsis may be more broadly applicable across
the plant kingdom. First, when wild-type tobacco roots
from 8-week-old plants were subjected to the rapid
chloroform dip protocol and the lipids analyzed by
GC-MS, trace amounts of docosanoyl and tetracosanoyl
glycerols were detected. These were largely the b-isomer.
Second, when tobacco plants were transformed with
the same 35STAtGPAT5 construct, significant amounts
of MAGs (15% [w/w] of total leaf wax load) were
produced as novel components of tobacco cuticular
waxes extracted from 4-week-old plants (Fig. 9). These
MAGs contained saturated C24-C32 acyl groups, with
C26 and C28 dominant. The b-MAG isomers were the
major species of MAGs ($90% [w/w] of total MAGs),
whereas FFAs accounted for ,5% of total MAG mass.
Both straight and branched-chain acyl groups were
present in the MAGs. This parallels the presence of
iso- and anteiso-C27-C33 branched-chain hydrocarbons reported for tobacco epicuticular waxes (Severson
et al., 1984). The molecular species distributions for the
alkane and MAG fractions for tobacco plants transformed with the 35STAtGPAT5 construct are given in
Supplemental Table S1. The alkane distribution is very
similar to that reported by Severson et al. (1984) and
is annotated accordingly. The bis-TMSi derivatives
of branched-chain b-MAG species have very similar
mass spectra to their straight-chain isomers, but elute
ahead at distinctive fractional equivalent carbon numbers. Using this type of retention time analysis that we
have previously used for the identification of branched-
Figure 9. 35STAtGPAT5 ectopic expression in transgenic tobacco
plants produces surface MAGs (mean with 95% CI; n 5 3).
chain components of Arabidopsis and Brassica seed
polyesters (Molina et al., 2006), and with the comparison with the tobacco cuticular alkane composition, we
tentatively identified the novel MAGs as containing
iso- and anteiso-branched-chain acyl groups synthesized from Val- or Ile-derived primers for fatty acid
synthesis.
DISCUSSION
Figure 8. Inverse relationship between the C22-C30 fatty acids (present
largely as MAGs and FFAs) and standard waxes released by transmethylation of intact stems from 18 independent 35STGPAT5 overexpression lines. OE-1 and OE-2 are highlighted in black and labeled on the
graph. The negative correlation between the amount of standard waxes
and VLCFAs is highly significant (Kendall rank correlation t-statistic 5
20.63; two-tailed P 5 0.0002 with normal approximation and correction for ties; n 5 18).
In this study, the composition of Arabidopsis root
waxes is reported and MAGs with C22-C24 saturated
acyl groups are identified as components of these
waxes. This phytochemical analysis is complemented
by characterization of Arabidopsis plants expressing
the acyltransferase GPAT5 of the aliphatic suberin biosynthesis pathway under the control of the 35S promoter. The results show that (1) GPAT5 is not only
involved in the synthesis of aliphatic suberin polymer
(Beisson et al., 2007), but also in the synthesis of the
MAGs present in the root waxes; (2) GPAT5 is an acyltransferase acting on a glycerol-based acceptor; and (3)
the ectopic expression of this gene can lead to the
production of MAGs in cuticular waxes of aerial organs. The significance of these results in terms of the
relationships of aliphatic suberin to root waxes, the
functional roles of GPAT5, and its utility in engineering cuticular waxes are discussed.
Relationships between Root Waxes and
Aliphatic Suberin
The presence in Arabidopsis root waxes of free and
esterified saturated fatty acids and alcohols with chain
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Li et al.
lengths of C18-C22 and of hydroxycinnamate and
glycerol building blocks (Fig. 2C) shows that, at least
in Arabidopsis, root waxes have characteristics common to the monomers released upon depolymerization of root suberin (Franke et al., 2006; Beisson et al.,
2007) and is thus suggestive of a metabolic relationship with aliphatic suberin. The fact that in green-lint
cotton fibers the wax composition reflects in part the
suberin composition (Schmutz et al., 1993, 1994, 1996)
and that the Arabidopsis root wax compounds, apart
from the simple MAGs, have also been found in isolated periderm of potato (Schreiber et al., 2005), clearly
supports this view.
Concerning the location of suberin-associated waxes,
it has been suggested that they are found within the
electron-translucent region of the suberin lamellae
(Soliday et al., 1979). However, this is still considered
hypothetical (Schmutz et al., 1994, 1996; Schreiber
et al., 2005). Because suberin is deposited outside the
plasma membrane, but under the primary cell wall, a
direct physical association between wax and suberin
implies an extracellular location. Our results using the
chloroform-dipping method show that Arabidopsis
root waxes are likely to be extracellular. Moreover,
from the difference in extraction kinetics (Fig. 2B), we
infer that most likely the alkanes are present at or near
the surface of the periderm, whereas most of the other
root waxes may be more deeply embedded in the
peridermal cell walls where suberization occurs.
The view that suberin-associated waxes are physically associated with and metabolically related to suberin is further substantiated genetically by the analysis
of MAGs in both knockout and ectopic expression
lines of GPAT5. The level of MAG and associated FFA
in root waxes is strongly dependent on GPAT5 expression. Furthermore, GPAT5 controls the level of suberin
monomers in young roots during development of
the specialization zone (Beisson et al., 2007). These
results show GPAT5 can control both aliphatic suberin
polyester and suberin-associated wax composition,
although not necessarily in the same stages of development. Because we know GPAT5 is expressed in older
roots largely in the peripheral zone (i.e. suberized
periderm; Beisson et al., 2007), we conclude that we
are indeed measuring suberin-associated waxes.
Although GPAT5 is clearly involved in deposition of
both waxes and polyesters, the metabolic relationships
between the two product classes is not understood.
In 7-week-old Arabidopsis roots, we observed about
350 mg/g fresh weight of suberin aliphatic monomers
released by depolymerization (transesterification), and
a similar amount of suberin-associated waxes (approximately 360 mg/g fresh weight). What is not
known is the relative timing of wax and suberin deposition in peridermal tissues. The MAG, FFA, primary
alcohols, and alkyl ferulates might be suberin precursors that have not been polymerized or they might
simply be synthesized on the same pathway as suberin; or, with the exception of the alkyl ferulates, they
might be derived by some postdeposition hydrolytic
events. A comparison can be made with the inhibition
of fatty acyl elongation in green-lint cotton fibers,
which differentially reduces waxes over suberin
(Schmutz et al., 1996). An explanation of this observation is that suberin formation may take priority over
suberin-associated wax formation. In the case of overexpression of GPAT5 in late-stage Arabidopsis roots,
where increases in MAGs and FFA root waxes, but not
suberin, are observed, one interpretation is that suberin synthesis has reached a set point and extra flux is
channeled into root waxes. This perspective is consistent with the cotton fiber inhibition experiment, although the experimental perturbation runs in the
opposite direction. Concerning function, we expect
the root waxes to augment the barrier properties of
suberin, but whether they collectively or individually
provide additional biological advantages is unknown.
Aspects of GPAT5 Function
Because we observed MAG in the cuticular waxes in
35STGPAT5 plants, whereas it is completely absent in
the wild type, a principal conclusion of this study is
that GPAT5 acts in vivo as an acyltransferase for a
glycerol-containing acceptor. GPAT5 is annotated as a
sn-glycerol-3-P acyl-CoA acyltransferase and has been
shown to have such activity in yeast (Zheng et al.,
2003). However, we cannot be certain yet that it acts
in vivo specifically as a sn-glycerol-3-P acyltransferase
because we have been unable to identify the expected
immediate product, 1-acyl-lysophosphatidic acid. If
GPAT5 is acting solely as a sn-glycerol-3-P sn-1 acyltransferase, then there must be both isomerase and
phosphatase activities present to produce b-MAG.
Furthermore, the high levels of FFA, which cooccur
with MAGs and which have acyl chain length distributions resembling that of MAGs, suggest a biosynthetic relationship between these two products. Genes
annotated as lysophospholipases and MAG lipases are
up-regulated in the epidermis of Arabidopsis stems
(Suh et al., 2005), so it is likely that enzyme activities to
convert lysophosphatidic acid to MAG and MAG to
FFA are present in tissues where polyester synthesis
occurs. The considerable variation in the ratios of FFA,
a-MAG, and b-MAG in the surface lipids of various
tissues with the ectopic expression of GPAT5 (Table I)
suggests that not all of these molecules are immediate
products of the enzyme. In particular, the relatively
low abundance of FFA products in the wax fraction of
35STGPAT5-expressing tobacco leaves compared to
Arabidopsis cuticular lipids implies derivation via
lipolysis.
Concerning acyl donor specificity of GPAT5, a preference for very long acyl chains could be inferred
based on strong reduction of such monomers in polyesters of the gpat5 mutants (Beisson et al., 2007). The
presence of only saturated C22-C30 acyl chains in
wild-type root waxes and in the products from ectopic
expression of GPAT5 supports this hypothesis.
Changes in the maxima within the acyl distributions,
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GPAT5 Expression Produces Extracellular Monoacylglycerols
from C22 to C24 and then C26 (Table I), probably
reflect the influence of acyl-CoA availability in the different tissues. In suberin-associated root waxes, there
are no unsaturated acyl groups or chain lengths shorter
than C22 in MAGs, although C18 and C20 acyl-CoAs
must be available to produce the observed primary alcohols. Also, putative 18-hydroxyoleate and octadecene-1,
18-dioate acyl-CoAs may be available to produce
suberin. The lack of C20 or shorter acyl groups in the
MAG products may result from low GPAT5 activity
for such chain lengths or from metabolite channeling,
or because these products, once synthesized, are rapidly metabolized. These speculations, and that in the
previous paragraph, define the scope for extensive
characterization of the in vitro activity of GPAT5,
which is now under way in our laboratory.
GPAT5 Appears to Intercept Pathways for
Wax Biosynthesis
A major biosynthetic pathway of epidermal cells is
the elongation of saturated fatty acyl-CoAs to produce
precursors for surface waxes (Kunst and Samuels,
2003). 35STGPAT5 overexpression lines with the strongest phenotypes show that the accumulation of MAGs
and FFAs occurs at the expense of the standard waxes
(Fig. 8), suggesting that GPAT5 competes for the same
pool of very-long-chain acyl-CoA substrates that are
usually devoted to cuticular wax synthesis. In 35ST
AtGPAT5 tobacco plants, the fact that iso- and anteisoacyl groups occur in MAGs alongside the endogenous
iso- and anteiso-alkanes confirms that GPAT5 indeed
intercepts the pool of elongating acyl intermediates
destined for cuticular wax biosynthesis. These results,
together with the observation that MAGs can be secreted
onto the plant surface, suggest that GPAT5 may be
targeted to the same subcellular domain as that involved
in cuticular wax synthesis. MAG might even be considered a precursor for suberin synthesis, whether that
polymerization occurs as an intracellular or an extracellular process. Arabidopsis leaf polyesters are dominated by a,v-dicarboxylic acids (Bonaventure et al.,
2004; Franke et al., 2006), which are likely esterified
with a glycerol as the polyol, so a MAG-based synthon
is not an unreasonable suggestion.
Ectopic expression of GPAT5 driven by the CaMV 35S
promoter causes a decrease in the levels of standard
cuticular waxes in leaf, stem, and siliques (Table I; Fig.
8). However, this reduction is not observed in seeds,
nor do we see a reduction in other long-chain aliphatics in roots. These differences may simply derive
from relative differences in promoter strength in different tissues, differences that may vary between lines.
However, a large number of other explanations may be
invoked because we know almost nothing about relative pool sizes of intermediates, feedback inhibition,
substrate channeling, temporal separation of accumulation of individual root wax components, and protein
stability.
Biotechnology of Plant Cuticles
Previously, alterations in the content and composition of surface waxes and polyesters have been made
largely through gene deletion (Kunst and Samuels,
2003). In addition, overexpression of AP2 domaincontaining transcription factors (Aharoni et al., 2004;
Broun et al., 2004; Zhang et al., 2005) has increased
cuticular wax load, but no novel components were reported. This study shows that, with the appropriate
biosynthetic gene, it is possible to produce novel cuticular wax components that are transported to the surface. The changes in surface chemistry are expected to
alter the functional properties of the cuticle. The design
and testing of efficient screens to measure changes
in functional properties of cuticles (gas and water
exchange, pathogen resistance, herbivore or predator
interactions, etc.) of plants transformed with such
genes will be an important next step in developing
feasible technology for cuticle engineering.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
All Arabidopsis (Arabidopsis thaliana) plants were in the Col-0 genetic
background. The gpat5-1 and gpat5-2 mutants were previously isolated and
characterized (Beisson et al., 2007). Arabidopsis plants were grown in a
controlled-growth chamber (at 21°C–22°C, 40%–60% relative humidity, and a
16/8-h photoperiod at 80–100 mmol m22 s21 provided by fluorescent bulbs).
Stem epidermal peels were collected as a thin transparent film under a dissecting microscope using a sharp forceps (Suh et al., 2005). Tobacco (Nicotiana
tabacum var. SR1) plants were grown in an air-conditioned greenhouse with
natural light.
Cloning of GPAT5 and Construction of 35STGPAT5
Overexpression Plants
Genomic DNA was prepared from Arabidopsis leaf tissue using a plant
mini DNA kit according to the manufacturer’s instructions (Qiagen). Genomic
DNA sequences encoding the GPAT5 gene (At3g11430) were amplified
by PCR using forward primer (5#-CACACTCTAGAATGGTTATGGAGCAAGC-3#) and reverse primer (5#-CACACGAGCTCTCAATGGAGACAAGG-3#).
The PCR product was initially cloned into pGEM-T Easy vector, and then
subcloned as an XbaI-SacI fragment into binary vector pBI121 to replace the
GUS gene. The construct (35STGPAT5) was introduced into Agrobacterium
tumefaciens strain C58C1 for Arabidopsis vacuum infiltration (Bechtold et al.,
1993) and strain LBA4404 for tobacco leaf disc transformation (Rogers et al.,
1986). Transgenic plants were then selected on Murashige and Skoog medium
containing 50 mg mL21 kanamycin (Murashige and Skoog, 1962).
SEM Analysis
To view epicuticular waxes, sections of stems were treated in 1% (w/v)
osmium tetroxide vapor for 24 h, air dried for 3 d, mounted onto standard
aluminum stubs for JEOL SEM, and then sputter coated with around 30 nm of
gold using an EMSCOPE SC-500 sputter coater. The images were taken with a
JEOL 6400V scanning electron microscope.
Sudan Red 7B Staining and Microscopy
Sudan Red 7B (Sigma) was prepared as a 0.05% (w/v) solution in
PEG400:glycerol (1:1 [v/v]; Brundrett et al., 1991). Soil-grown Arabidopsis
roots were stained in this solution for 1 h at room temperature, rinsed briefly
in distilled water, and free-hand sections were made at the base of the roots
with a razor blade. Images were taken with a Leica MZ 12.5 microscope.
Plant Physiol. Vol. 144, 2007
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Li et al.
Cuticular Wax Analysis
Stems were dipped in chloroform for 30 s, the solvent evaporated under a
stream of N2 gas, and tricosane, tricosanoic acid, monoheptadecanoin, and
tridodecanoin added as internal standards. The waxes were derivatized by
heating at 110°C for 10 min in pyridine:BSTFA (1:1 [v/v]). The silylated sample
was analyzed by GC using a 30-m DB5-ht capillary column temperature
programmed at 10°C min21 to 370°C. Eluting components were quantified
based on uncorrected peak areas from integrated flame ionization detector ion
current. For molecular identification, a Hewlett-Packard 5890 GC-coupled
MSD 5972 mass analyzer was used with the mass analyzer set in electron
impact mode (70 eV) and scanning from 40 to 700 atomic mass units. Tobacco
leaf epicuticular wax analysis was conducted as above, except that leaves were
dipped in dichloromethane instead of chloroform (Severson et al., 1984).
Root Wax Analysis
Arabidopsis roots were carefully and thoroughly washed in distilled
water, blotted, then air dried at 50°C for 30 min, and dipped in chloroform for
1 min, unless otherwise stated. The extracts were passed through a glass woolplugged column and evaporated to dryness under a stream of N2 gas. The
waxes were derivatized and analyzed as described above for cuticular waxes.
Due to the complex architecture of the roots, it is not practical to calculate the
root wax load based on surface area; therefore, we report root wax load as
micrograms per gram fresh weight. Inspection of root biomass of wild-type,
mutant, and 35STGPAT5 overexpression lines showed no obvious differences
in morphology, nor did the average peridermal root diameter measured close
to the crown vary significantly between lines, suggesting that per gram freshweight units are approximately proportional to surface area units.
Additional Lipid Analyses
Fatty acid content and composition of Arabidopsis tissues was analyzed
directly by acidic transmethylation according to Li et al. (2006). For polyester
analysis, the NaOMe depolymerization protocol from Bonaventure et al.
(2004) was used with slight modifications as described in Suh et al. (2005).
Polyester monomers were separated, identified, and quantified by GC-MS.
The mass spectrometer was run in scan mode (40–500 atomic mass units) with
peaks quantified on the basis of their total ion current. Polyester monomer
amounts were expressed per gram of solvent-extracted dry residue.
Statistics
Statistical tests were performed using Microsoft Excel and Analyze-it
(version 1.73) software.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Polyester analysis of 7-week-old roots of wild-type
and 35STGPAT5 overexpression line OE-2 (mean with 95% CI; n 5 3).
Supplemental Figure S2. Polyester analysis of 6-week-old stems of wildtype and 35STGPAT5 overexpression lines (mean with 95% CI; n 5 4).
Supplemental Table S1. Molecular distribution and identification of
alkanes and monoacylglycerols in the leaf waxes of tobacco plants
overexpressing 35STAtGPAT5 (6SD, n 5 3).
ACKNOWLEDGMENTS
We thank Katrin Weber (Department of Plant Biology, Michigan State
University) for assisting with plant transformations and Ewa Danielewics
(Center for Advanced Microscopy, Michigan State University) for SEM
analyses.
Received March 12, 2007; accepted May 4, 2007; published May 11, 2007.
LITERATURE CITED
Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A (2004) The
SHINE clade of AP2 domain transcription factors activates wax biosyn-
thesis, alters cuticle properties, and confers drought tolerance when
overexpressed in Arabidopsis. Plant Cell 16: 2463–2480
Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium-mediated
gene transfer by infiltration of adult Arabidopsis thaliana plants. C R
Acad Sci III 316: 1194–1199
Beisson F, Li Y, Bonaventure G, Pollard M, Ohlrogge J (2007) The
acyltransferase GPAT5 is required for synthesis of suberin in seed coat
and root of Arabidopsis. Plant Cell 19: 351–368
Bernards MA (2002) Demystifying suberin. Can J Bot 80: 227–240
Bernards MA, Lewis NG (1992) Alkyl ferulates in wound healing potato
tubers. Phytochemistry 31: 3409–3412
Bernards MA, Lopez ML, Zajicek J (1995) Hydroxycinnamic acid-derived
polymers constitute the polyaromatic domain of suberin. J Biol Chem
270: 7382–7386
Bonaventure G, Beisson F, Ohlrogge J, Pollard M (2004) Analysis of the
aliphatic monomer composition of polyesters associated with Arabidopsis epidermis: occurrence of octadeca-cis-6, cis-9-diene-1,18-dioate
as the major component. Plant J 40: 920–930
Broun P, Poindexter P, Osborne E, Jiang CZ, Reichmann J (2004) WIN1, a
transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci USA 101: 4706–4711
Brundrett MC, Kendrick B, Peterson CA (1991) Efficient lipid staining in
plant material with sudan red 7B or fluorol yellow 088 in polyethylene
glycol-glycerol. Biotech Histochem 66: 111–116
Dolan L, Roberts K (1995) Secondary thickening in roots of Arabidopsis
thaliana: anatomy and cell surface changes. New Phytol 131: 121–128
Espelie KE, Sadek NZ, Kolattukudy PE (1980) Composition of suberinassociated waxes from the subterranean storage organs of seven plants,
parsnip, carrot, rutabaga, turnip, red beet, sweet potato and potato.
Planta 148: 468–476
Franke R, Briesen I, Wojciechowski T, Faust A, Yephremov A, Nawrath C,
Schreiber L (2006) Apoplastic polyesters in Arabidopsis surface
tissues—a typical suberin and a particular cutin. Phytochemistry 66:
2643–2658
Grac
xa J, Pereira H (1997) Cork suberin: a glyceryl based polyester.
Holzforschung 51: 225–234
Grac
xa J, Pereira H (1999) Glyceryl-acyl and aryl-acyl dimers in Pseudotsuga
menziesii bark suberin. Holzforschung 53: 397–402
Grac
xa J, Pereira H (2000) Suberin structure in potato periderm: glycerol,
long-chain monomers, and glyceryl and feruroyl dimers. J Agric Food
Chem 48: 5476–5483
Grac
xa J, Santos S (2006) Linear aliphatic dimeric esters from cork suberin.
Biomacromolecules 7: 2003–2010
Grac
xa J, Schreiber L, Rodrigues J, Pereira H (2002) Glycerol and glyceryl
esters of omega-hydroxyacids in cutins. Phytochemistry 61: 205–215
Gunstone FD (1967) An Introduction to Chemistry and Biochemistry of
Fatty Acids and Their Glycerides. Chapman Hall, London, pp 141
Holloway PJ (1982) Structure and histochemistry of plant cuticular membranes: an overview. In DF Cutler, KF Alvin, CE Price, eds, The Plant
Cuticle. Academic Press, London, pp 1–32
Jetter R, Kunst L, Samuels L (2006) Composition of plant cuticular waxes.
In M Riederer, C Müller, eds, Biology of the Plant Cuticle. Annual Plant
Reviews, Vol 23. Blackwell Scientific Publishers, Oxford, pp 145–175
Kolattukudy PE (1980) Cutin, suberin and waxes. In PK Stumpf, ed, The
Biochemistry of Plants—A Comprehensive Treatise, Vol 4. Academic
Press, New York, pp 571–645
Kolattukudy PE (2001) Polyesters in higher plants. Adv Biochem Eng
Biotechnol 71: 1–49
Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular
wax. Prog Lipid Res 42: 51–80
Li Y, Beisson F, Pollard M, Ohlrogge J (2006) Oil content of Arabidopsis
seeds: the influence of seed anatomy, light and plant-to-plant variation.
Phytochemistry 67: 904–915
Lulai EC, Corsini DL (1998) Differential deposition of suberin phenolic and
aliphatic domains and their roles in resistance to infection during potato
tuber (Solanum tuberosum L.) wound-healing. Physiol Mol Plant Pathol
53: 209–222
Moire L, Schmutz A, Buchala A, Yan B, Stark RE, Ryser U (1999) Glycerol
is a suberin monomer: new experimental evidence for an old hypothesis. Plant Physiol 119: 1137–1146
Molina I, Bonaventure G, Ohlrogge J, Pollard M (2006) The lipid polyester
composition of Arabidopsis thaliana and Brassica napus seeds. Phytochemistry 67: 2597–2610
1276
Plant Physiol. Vol. 144, 2007
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2007 American Society of Plant Biologists. All rights reserved.
GPAT5 Expression Produces Extracellular Monoacylglycerols
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497
Murphy RC (1993) Mass Spectroscopy of Lipids—Handbook of Lipid
Research. Plenum Press, New York, pp 206
Nawrath C (2002) The biopolymers cutin and suberin. In CR Somerville,
EM Meyerowitz, eds, The Arabidopsis Book. American Society of Plant
Biologists, Rockville, MD, pp 1–14
Nawrath C (2006) Unraveling the complex network of cuticular structure
and function. Curr Opin Plant Biol 9: 281–287
Rashotte AM, Feldmann KA (1998) Correlations between epicuticular wax
structures and chemical composition in Arabidopsis thaliana. Int J Plant
Sci 159: 773–779
Rashotte AM, Jenks MA, Feldmann KA (2001) Cuticular waxes on
eceriferum mutants of Arabidopsis thaliana. Phytochemistry 57: 115–123
Rogers SG, Horsch RB, Fraley RT (1986) Gene transfer in plants: production of transformed plants using Ti plasmid vectors. Methods Enzymol
118: 627–640
Santos S, Gracxa J (2006) Glycerol-v-hydroxyacid-ferulic acid oligomers in
cork suberin structure. Holzforschung 60: 171–177
Schmutz A, Amrhein N, Ryser U (1993) Caffeic acid and glycerol are constituents of the suberin layers in green cotton fibres. Planta 189: 453–460
Schmutz A, Buchala A, Ryser U (1996) Changing the dimensions of suberin
lamellae of green cotton fibers with a specific inhibitor of the endoplasmic
reticulum-associated fatty acid elongases. Plant Physiol 110: 403–411
Schmutz A, Jenny T, Ryser U (1994) A caffeoyl-fatty acid glycerol ester
from wax associated with green cotton fiber suberin. Phytochemistry 36:
1343–1346
Schreiber L, Franke R, Hartmann K (2005) Wax and suberin development
of native and wound periderm of potato (Solanum tuberosum L.) and its
relation to peridermal transpiration. Planta 220: 520–530
Severson RF, Arrendale RF, Chortyk OT, Johnson AW, Jackson DM,
Gwynn GR, Chaplin JF, Stephenson MG (1984) Quantitation of the
major cuticular components from green leaf of different tobacco types.
J Agric Food Chem 32: 566–570
Soliday CL, Kolattukudy PE, Davis RW (1979) Chemical and ultrastructural evidence that waxes associated with the suberin polymer constitute the major diffusion barrier to water vapor in potato tuber (Solanum
tuberosum L.). Planta 146: 607–614
Suh MC, Samuels AL, Jetter R, Kunst L, Pollard M, Ohlrogge J, Beisson F
(2005) Cuticular lipid composition, surface structure, and gene expression in Arabidopsis stem epidermis. Plant Physiol 139: 1649–1665
Zhang JY, Broeckling CD, Blancaflor EB, Sledge MK, Sumner LW, Wang
ZY (2005) Overexpression of WXP1, a putative Medicago truncatula AP2
domain-containing transcription factor gene, increases cuticular wax
accumulation and enhances drought tolerance in transgenic alfalfa
(Medicago sativa). Plant J 42: 689–707
Zheng Z, Xia Q, Dauk M, Shen W, Selvaraj G, Zou J (2003) Arabidopsis
AtGPAT1, a member of the membrane-bound glycerol-3-phosphate
acyltransferase gene family, is essential for tapetum differentiation
and male fertility. Plant Cell 15: 1872–1887
Plant Physiol. Vol. 144, 2007
1277
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