Journal of Experimental Botany, Vol. 58, No. 5, pp. 1071–1082, 2007
doi:10.1093/jxb/erl268 Advance Access publication 11 January, 2007
RESEARCH PAPER
The outermost cuticle of soybean seeds: chemical
composition and function during imbibition
Suqin Shao1, Chris J. Meyer2, Fengshan Ma2, Carol A. Peterson2 and Mark A. Bernards1,*
1
Environmental Stress Biology Group, Department of Biology, University of Western Ontario, London, ON,
Canada N6A 5B7
2
Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1
Received 18 August 2006; Revised 2 November 2006; Accepted 15 November 2006
Abstract
Introduction
Seeds of different cultivars of Glycine max (L.) Merr.
(soybean) have strikingly different rates of water
imbibition. Seeds that readily imbibe water are termed
‘soft’ while those that remain non-permeable, even
after several days in water, are referred to as ‘hard’,
‘stone’, or ‘impermeable’ seeds. What prevents soybean hard seeds from taking up water? Previous work
established that the initial imbibition of soft soybean
seeds correlates with the presence of small cracks
in the outermost cuticle that covers the seed coat,
prompting a detailed analysis of soybean seed coat
cutin. In the present paper, it is shown that the
outermost cuticle of the seed coat has an unusual
chemical composition, lacking typical mid-chain hydroxylated fatty acids but being relatively rich in other
types of hydroxylated fatty acids. The cuticle of the
impermeable cultivar studied contained a disproportionately high amount of hydroxylated fatty acids
relative to that of the permeable ones. Moreover, a brief
treatment with hot alkali released the v-hydroxy fatty
acid component of the outermost cuticle and created
holes in it that caused the seeds to become permeable.
This demonstrates that the outermost cuticle of the
seed is the critical structure that prevents water uptake
by hard seeds.
Soybean (Glycine max L. Merr.) has enormous commercial value as it is the world’s largest oilseed and legume
crop, and is the primary source of protein feed supplement
for livestock. Many soybean cultivars have been selected
for seeds that imbibe water rapidly and uniformly but
even these varieties produce some hard seeds (also known
as ‘stone’ or ‘impermeable’ seeds). Such seeds do not
readily imbibe water when soaked, even for prolonged
periods. Other varieties, many of which possess otherwise
desirable agronomic traits, produce a large proportion of
hard seeds (Rolston, 1978). And while hard seeds may
benefit plants in nature by preserving a seed stock for
several years (Tyler, 1997), they constitute a major
problem during food processing where rapid and uniform
hydration is important for the production of quality
soybean foods such as soymilk, tofu, and miso. Understanding the basis for hard seed impermeability to water is
an essential first step in developing strategies to handle
such seeds on a large scale at the processing stage, and to
plan suitable genetic modifications.
Whether a legume seed will be soft (permeable) or hard
(impermeable) is controlled by the seed coat. This layer,
largely derived from the integuments of the maternal
tissue after fertilization (Esau, 1977; Boesewinkel and
Bouman, 1995), embodies a heritable form of physical or
seed coat-imposed dormancy (Bewley and Black, 1994;
Baskin et al., 2000). The seed coat of soybean (Fig. 1) is
typical of legumes, a group with a well-characterized
anatomy (Corner, 1951; Esau, 1977). The seed coat is
modified at the hilum and micropyle areas (Fig. 1A),
but these do not contribute to the initial uptake of water
in soybean (Ma et al., 2004) and, thus, will not be
considered here. In the remainder of the seed coat
Key words: Cutin, Glycine max, seed coat permeability,
soybean, stone seeds.
* To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1072 Shao et al.
Fig. 1. Soybean seed coat anatomy. (A) A seed of cv. Harosoy 63 under a dissecting microscope, viewed from the ventral side. M, Micropyle; H,
hilum; R, raphe; AB, abaxial side. Scale bar¼1 mm. (B) Cross-section (1.5 lm thickness) of a cv. OX-951 seed coat, dorsal side (opposite to the
ventral side), stained with toluidine blue O. PL, Palisade layer; HG, hourglass layer; PA, parenchyma tissue; AL, aleurone layer; EN, compressed
endosperm tissue. Scale bar¼50 lm. (C) Transmission electron micrograph of a cv. Harovinton palisade cell at the dorsal side of the seed coat. The
outer tangential wall is shown, with the cuticle (between the pair of arrows) and endocarp deposits (asterisks) denoted. Seed coat samples were fixed
with acrolein vapour in a sealed mini-chamber over a period of 2 weeks, then dehydrated with absolute ethanol and propylene oxide, and embedded
in Spurr’s resin. Sections (85–90 nm thick) were stained with uranyl acetate and lead citrate. Scale bar¼1 lm. (D) Surface view on the abaxial side of
a cv. Harovinton seed under a scanning electron microscope. There are endocarp deposits (arrowheads) on top of the cuticle (asterisks) of the palisade
layer. Scale bar¼20 lm.
(illustrated in Fig. 1B), the outermost layer consists of
closely packed macrosclereids (palisade cells), which are
dead cells with uneven wall thickenings. Within their
walls is the ‘light line’, a callose-rich wall area running
parallel to the edge of the seed coat (Bhalla and Slattery,
1984; Serrato-Valenti et al., 1993; Ma et al., 2004). The
next cell layer consists of osteosclereids (hourglass cells).
These cells are also dead and thick-walled, but their shape
is like an hourglass and large air spaces occur between
them. Next is a layer of crushed parenchyma. In addition
to the above tissues derived from the integuments, the
legume seed coat includes some cells of the endosperm.
There is one intact, closely packed, uniseriate layer
(herein termed aleurone) with many living cells (Yaklich
et al., 1992; Ma et al., 2004) as well as irregular, small
masses of crushed endosperm cells. Two thin (<0.2 lm)
and inconspicuous cuticles are associated with the seed
coat, one on the outside of the seed (covering the palisade
cells; Fig. 1C), and the other between the crushed
parenchyma and endosperm (Chamberlin et al., 1994;
Ma et al., 2004).
Various explanations have been advanced to account for
the strikingly different permeabilities of the coats to water
in hard and soft seeds. Some examples are: tightly bound
palisade cells (Corner, 1951; Ballard, 1973), thickened coat
(Wyatt, 1977; Miao et al., 2001), lack of pits (Yaklich
et al., 1986; Harris, 1987; Hahalis et al., 1996; Chachalis
and Smith, 2001), presence of endocarp deposits (Calero
et al., 1981; Yaklich et al., 1986), dark colour (Wyatt,
1977), closed hilum and/or micropyle (Hyde, 1954; Ballard,
1973; Rolston, 1978; Lush and Evans, 1980), outer
tangential walls of palisade cells (Werker et al., 1979), and
a prominent light line of the palisade cells (Bhalla and
Slattery, 1984; Harris, 1987; Serrato-Valenti et al., 1993).
The importance of the outer cuticle has been promoted
by some investigators (Arechavaleta-Medina and Snyder,
1981; Ragus, 1987), but opposed by others (Ballard, 1973;
Werker, 1980/1981; Chachalis and Smith, 2001). Generally,
the cuticles of the seed coat were overlooked, in part
because they are very thin and do not stain readily for lipids
as does a typical cuticle. However, a recent, exhaustive
anatomical study established that the only features consistently correlating with seed coat permeability to water were
small cuticular cracks, visible with SEM, on the surface of
the seed coat (Ma et al., 2004). These cracks were present
in soft but not hard seeds. The initial penetration of water
into soft seeds typically occurred on the dorsal side, the
location of the majority of the cracks (Ma et al., 2004). The
corollary of this observation is that the intact cuticle appears
to be an efficient barrier to water entry into the seed coat
in soft as well as hard seeds.
In the present study, the idea that the continuity of the
outermost cuticle governs water entry into soybean seeds
was confirmed experimentally. This brought up the
Soybean seed coat cutin chemistry and imbibition 1073
question of why the cuticles crack in some seeds (making
them soft) but not in others (making them hard).
According to Ma et al. (2004), isolated pieces of cuticle
from hard seeds were stronger and less prone to breaking
when handled than those isolated from soft seeds. Could
this property be related to the chemical composition of the
cuticle? To date, no detailed analysis of any seed coat
cuticle has been carried out. Accordingly, the monomer
composition of both the outer and inner cuticles of a
number of soybean cultivars, including those that produce
predominantly soft seeds and one that produces predominantly hard seeds, was characterized. These results
were further compared with the monomer compositions of
cuticles from pods and leaves of the same plants. The
effect of wax extraction from the outer cuticle on water
imbibition by hard seeds was also determined, as wax is
known to be the major contributor of hydrophobicity in
leaf cuticles (Schönherr, 1982).
Materials and methods
Plant material
Ripe seeds from six cultivars of soybean [Glycine max (L.) Merr.]
were hand-harvested during the 2000/2001 and 2001/2002 growing seasons from field-grown plants at Agriculture and Agri-Food
Canada, London, ON, Canada. Most seeds produced by five of the
cultivars (Tachanagaha, Harovinton, Harosoy 63, Bobcat, and AC
Onrei) were soft, whereas most produced by cv. OX-951 were hard.
[Hard seeds of the latter cultivar may comprise up to 76% of the
total (Mullin and Xu, 2001).] Such seeds were identified by their
lack of measurable water uptake after 24 h (see below). All seeds
were stored at room temperature until they were used. Fully
expanded soybean leaves and full-sized, green pods were collected
approximately 40 d post-anthesis.
Measurement of water uptake
Seeds were examined initially with a dissecting microscope, and
only those with no visible damage were chosen for imbibition trials.
Water uptake of at least five individual seeds from each cultivar or
experimental treatment was measured gravimetrically according to
Ma et al. (2004).
Disruption of the outer cuticle of intact stone seeds
It was not possible to devise a method to remove the entire outer
cuticle from a seed. However, it was possible to remove small patches
of this cuticle by boiling seeds individually in 1 M NaOH for 5 min
(modified from Beresniewicz et al., 1995). The alkali was removed
by rinsing three times with water. Control seeds were either untreated or boiled in water for 5 min. Seeds that had split during the
boiling procedures were discarded; the rest were tested for water
uptake or were observed for any changes in the seed coat surface.
Additionally, the NaOH solubles released from treated seeds were
pooled, acidified, and extracted with CHCl3. These solubles were
then dried, derivatized, and analysed by GC-FID/GC-MS as
described for surface waxes below.
Microscopic analyses
Tissue samples were prepared for light and scanning electron
microscopy as described elsewhere (Ma et al., 2004), but for
transmission electron microscopy the seed coat samples were
processed under anhydrous conditions. Changes in the seed coat
outer cuticle resulting from the hot NaOH treatment were observed
with a Zeiss Stemi SV 11 dissecting microscope (Carl Zeiss
Canada, Don Mills, ON, Canada) and a Zeiss Axiophot epifluorescence microscope (UV filter set: exciter filter G 365, dichroitic
mirror FT 395, and barrier filter LP 420; Carl Zeiss Canada).
Images were captured with a Q-Imaging digital camera system
(Retiga 2000R, Fast 1394, Cooled Mono, 12-bit; Quorum Technologies Inc., Guelph, ON, Canada). Details of the seed coat surface
structure of control and NaOH-treated seeds were observed with
a Hitachi S570 scanning electron microscope (SEM) at 15 kV and
photographed with black and white film (Ilford Pan F Plus ISO 50).
At least 10 seeds from each treatment were observed with each
microscope. Further details are provided in the figure legends.
Surface wax analysis
Surface waxes were isolated by dipping eight intact seeds, enclosed
in a tea ball, in 30 ml CHCl3 for 30 s. The CHCl3-soluble waxes
thus extracted were evaporated to dryness under vacuum at 40 C.
The residue was quantitatively transferred to a glass vial and the
solvent removed under a stream of nitrogen. After addition of a
known quantity of tetracosane (as an internal standard) the dried
samples were derivatized according to Riederer and Schönherr
(1986). The combined methyl ester and trimethylsilyl ether derivatives were analysed by GC on a Varian CP-3800 gas chromatograph equipped with a flame ionization detector (GC-FID) and an
ion trap mass spectrometer (GC-MS). The GC was equipped with
a pair of CP-Sil 5 CB low-bleed MS columns (WCOT silica 30 m3
0.25 mm ID), one in line with the FID and the other with the MS.
The injector ovens were set at 250 C, and the FID oven at
300 C. Samples (1 ll) were injected twice (once to each column)
in splitless mode and simultaneously eluted with the following oven
temperature programme: the initial temperature of 80 C was held
for 2 min, followed by a rapid increase (40 C min1) to 200 C,
a 2 min hold at 200 C, and a slower increase (3 C min1) to 300 C.
The final temperature was held for just over 9 min for a total run
time of 50 min. High purity He was used as a carrier gas at 1 ml
min1. Compounds were identified on the basis of their co-elution
with authentic standards and their EI-mass spectra (50–650 amu).
Quantification was based on FID peak areas and independently
derived calibration curves for each standard.
The total wax load on soybean leaves was measured by taking five
leaves from 13-d-old plants, dipping them in CHCl3 for 30 s, and
weighing the extract residue remaining after solvent evaporation
(n¼3).
Analysis of cutin from outer and inner seed coat cuticles
Seed coats were removed from hydrated seeds. While still wet, the
coats were partially digested with pectinase so that the outer cuticle
and its associated cells could be separated from the inner cuticle with
its associated cells. This paradermal separation was achieved by
modifying the method of Pacchiano et al. (1993). Briefly, seed coats
were first treated with 1.6 mg ml1 pectinase (from Aspergillus
niger, 25 U mg1; Sigma-Aldrich) in 50 mM acetate buffer (pH
4.0) for 2 d at 31 C. At this stage, a seed coat could be separated
with forceps into an outer layer (mainly palisade and hourglass
cells) and an inner layer (largely crushed parenchyma and aleurone).
Alternatively, the two layers of soybean seed coats were separated
after a 2 h treatment with boiling oxalic acid/oxalate solution
(4 g l1 oxalic acid, 16 g l1 ammonium oxalate; Kolattukudy and
Walton, 1972).
The two cuticles from the seed coats of selected cultivars were
isolated from their surrounding cells by treating the separated seed
coat layers sequentially with cellulase (Aspergillus niger, 1.92 U mg1;
1074 Shao et al.
30 mg ml1 solution, pH 5.0, at 44 C for 7 d), pectinase
(Aspergillus niger, 25 U mg1; 2.4 mg ml1 solution, pH 4.0, at
31 C for 2 d), and hemicellulase (Aspergillus niger, 1.5 U mg1;
2.8 mg ml1 solution, pH 5.5, 45 C for approximately 8 d).
The residues containing the cuticles were then dried at 50 C and
Soxhlet-extracted with a mixture of CHCl3:MeOH (2:1 v/v) for 7 h,
followed by CHCl3 alone for 12 h (Dean and Kolattukudy, 1977).
The amount and monomer composition of outer and inner seed coat
cutins were determined after BF3/MeOH depolymerization as
described by Riederer and Schönherr (1986). The depolymerizate
was derivatized and analysed by GC-FID/GC-MS as described for
waxes above.
The cuticles from both pods and leaves of soybean were isolated
enzymatically using the method of Pacchiano et al. (1993). Cuticle
monomers were separated and analysed as described above.
Sphingolipid analysis
Sphingolipids were isolated according to Cahoon and Lynch (1991)
and analysed by GC-FID and GC-MS according to Bonaventure
et al. (2003). Briefly, the soluble components isolated from fresh
tissue were hydrolysed under mild conditions with NaOH, followed
by TLC separation of compound classes (including free fatty acids
and sphingolipids) and hydrolysis with Ba(OH)2. After partitioning,
sphingoid bases and free fatty acids were analysed separately by
GC-FID/GC-MS.
Statistical analysis
Each chemical analysis was independently repeated at least three
times. Data were analysed by standard analysis of variance
(ANOVA) techniques, followed by a least squares difference (LSD)
comparison of mean data values at a P-value of 0.05, using Statistix
software (V2.0; Analytical Software, Tallahasse, FL, USA).
Results
Water uptake by soybean seeds
Imbibition curves for the seeds of the six soybean
cultivars used in this study (Fig. 2A) are in agreement
with those reported earlier (Ma et al., 2004). From these
curves it is clear that seeds from five cultivars (AC Onrei,
Bobcat, Harosoy 63, Harovinton, Tachanagaha) can be
categorized as soft (with varying degrees of permeability).
By contrast, hard seeds selected from cv. OX-951 failed to
imbibe any measurable amount of water.
Making hard seeds soft
Five procedures were used to try to make stone seeds
permeable to water. First, a brief (30 s) immersion in
CHCl3 was used to extract readily soluble waxes. However,
this failed to make the stone seeds imbibe (Fig. 2B). (The
extent of wax removal from the outer cuticle was judged to
be nearly complete based on subsequent exhaustive solvent
extraction of isolated seed coats; see below.) Next, endocarp deposits (material from the inner face of the pod that
can adhere to the seed coat surface; Gijzen et al., 1999;
Fig. 1D) were selectively removed from the surface of
intact seeds by a brief treatment with a cellulase–pectinase
mixture. Again, no change in permeability was observed
(data not shown). Then, intact seeds were treated with pancreatic lipase for 20 h at room temperature. This treatment
released predominantly 16:0 and 18:0 fatty acids (approximately 26% of that released from the outer cuticle by BF3/
MeOH depolymerization), albeit without any effect on
permeability (data not shown). Success was ultimately
achieved by boiling the seeds in 1 M NaOH for 5 min.
This alkaline treatment had a striking effect on the ability
of hard seeds to take up water, i.e. treated seeds imbibed
quickly and hydrated as efficiently as the soft seeds (Fig.
2). Finally, to test whether or not high temperature
contributed to the softening of the seeds, hard seeds were
boiled in water for 5 min but they remained true to their
nature and did not imbibe (Fig. 2B).
Hot alkali treatment caused small, brown patches to
appear on the light yellow seed coat (Fig. 3). This
browning was probably due to oxidation of cell wall
phenols exposed by de-esterification of some of the cuticle
monomer components. Patchy removal of the cuticle was
observed microscopically with ultraviolet light, under
which dark spots of various sizes, some merging with
Fig. 2. Soybean seed water uptake curves. Seeds of cv. OX-951 were pre-soaked for 24 h and only those that had not imbibed any water were used
for further water uptake analysis. (A) The water uptake of individual seeds (n¼5) from each of six soybean cultivars was determined gravimetrically
for the first 3 h of imbibition. (B) The water uptake of individual seeds (n¼5) from stone seeds of cv. OX-951 that had been de-waxed with
chloroform, boiled in 1 M NaOH for 5 min or boiled in water for 5 min was determined gravimetrically for the first 3 h of imbibition. The values are
averages 6SD.
Soybean seed coat cutin chemistry and imbibition 1075
each other, were evident against the natural blue autofluorescence of undamaged areas (Fig. 3, insets). Additional details of the seed coat surface were observed with
SEM. In control (untreated) seeds, the outer cuticle was
partially obscured by endocarp deposits. In any area where
it could be seen, the outer cuticle was smooth and continuous (Fig. 4A). Boiling a seed in water for 5 min did not
visibly affect either its endocarp deposits or its outer cuticle
(Fig. 4B). By contrast, after boiling in 1 M NaOH for
5 min, small areas of the cuticle (as well as some endocarp
deposits) were completely removed, revealing the outer
periclinal walls of the subtending palisade cells and
grooves where the individual cells abutted one another
(Fig. 4C). Thus, cell outlines, originally obscured by the
overlying cuticle, became visible. The surfaces of the
exposed walls were smooth, indicating that they had not
been substantially altered by the alkali treatment. [In the
example illustrated, cuticle removal had also occurred
around a pit in the seed coat (arrow). Despite their suggestive appearance, pits are not involved in imbibition of water
by soybean seeds (Ma et al., 2004).]
Chemical analyses of soybean seed coat
cutin and wax
Fig. 3. Light and fluorescence microscopy of soybean seed coats.
Surface views of seeds of cv. OX-951 after a 5 min boil in (A) water or
(B) 1 M NaOH. Seeds were viewed under white light. Scale bars¼1
mm. Insets: autofluorescence of the cuticle under ultraviolet light. Scale
bars¼100 lm.
Fig. 4. Scanning electron micrographs of soybean seed coats. (A)
Surface of untreated seed showing intact outer cuticle (asterisk) and
overlying endocarp material (arrowheads). (B) Surface of seeds after
boiling in water for 5 min. (C) Montage of scanning electron
micrographs of seed surface following NaOH treatment. A region
where the cuticle is still intact is indicated with asterisks. To the left of
this is an area where the cuticle has been removed, exposing the walls
of the underlying palisade cells (arrowheads). Cuticle was also removed
from a pit (arrow). Scale bars¼15 lm.
The apparent importance of the outer cuticle in controlling
imbibition led to the chemistry of the seed coat cuticles
being investigated more closely. Initial analysis of whole
seed coat cutin by BF3/MeOH depolymerization revealed
the presence of fatty acids, 2-hydroxy fatty acids, and xhydroxy fatty acids as the major components. No midchain hydroxy- or epoxy-derivatized fatty acids typical
of many plant cutins (Walton, 1990; Matzke and Riederer,
1991; Kolattukudy, 2000) were detected (Fig. 5A). This
unusual cutin chemistry was contrasted with that of cuticles isolated from either soybean leaves or green pods
which, after BF3/MeOH depolymerization, revealed a
typical profile of cutin monomers including substantial
amounts of 9(10),16-dihydroxyhexadecanoic acids (Fig.
5B, C). Importantly, depolymerization of seed coat cuticles
using sodium methoxide did not yield a different profile,
either quantitatively or qualitatively (data not shown).
Consequently, BF3/MeOH depolymerization was used for
all remaining cutin analyses.
To determine which components of the cuticle had been
removed by boiling in NaOH as described above (Figs 3,
4), the aliphatics present in the NaOH hydrolysate were
analysed. The aliphatic cutin monomer compounds consisted of fatty acids, 1-alkanols, n-alkanes, and x-hydroxy
fatty acids (Table 1). Interestingly, little of the 2-hydroxy
fatty acid component observed with whole seed coat
analyses (Fig. 5A) was released by NaOH treatment.
Analysis of the aliphatic monomers derived from the
remaining inner and outer seed coat cuticles (isolated from
NaOH-treated seeds) revealed additional fatty acids, 2hydroxy acids, and smaller amounts of x-hydroxy fatty
acids (Table 1).
Next, complete chemical analyses of the soluble wax
from the outer cuticle, and the cutins from both the inner
and outer cuticles (isolated and analysed separately) from
permeable and impermeable seeds were made (Tables 2–4).
From this information, it was possible to confirm that only
the outer cuticle had been affected by the NaOH treatment
(see below).
Outer cuticle wax: The surface wax of the seeds of all
cultivars tested was composed primarily of fatty acids,
1076 Shao et al.
Fig. 5. Gas chromatographic analysis of soybean cutins. Representative chromatograms of the cutin monomers released from soybean cutins of (A)
seed coats, (B) pod walls, and (C) leaves (cv. Harosoy 63). Tissues were extracted, depolymerized (BF3/MeOH), derivatized, and the cutin monomers
released analysed by gas chromatography (see Materials and methods). The main monomers detected in each sample are labelled. Samples are not
normalized. IS, Internal standard.
Table 1. Chemical analysis of NaOH-treated OX-951 seed coats
Intact seeds were boiled individually in 1 M NaOH for 5 min without prior removal of waxes, and the released aliphatic monomers recovered with
organic solvent after acidification. The cutin monomers of the inner and outer seed coats of NaOH-treated seeds were analysed after BF3/MeOH
depolymerization as described in Materials and methods. Values (ng mm2 seed surface area) represent the average of triplicate measurements 6SD.
nd, Not detected.
Extract/tissue
NaOH solubles
Inner seed coat
Outer seed coat
Aliphatic monomer class
Totals
Fatty acids
2-OH fatty acids
x-OH fatty acids
1-Alkanols
Alkanes
25.862.7
31.863.2
20.362.0
nd
15.261.9
27.564.7
11.161.0
0.360.1
1.160.1
5.060.1
nd
nd
7.861.6
nd
nd
1-alkanols, n-alkanes, and trace amounts of other hydroxylated fatty acids (Table 2). This profile is typical of the
cuticular waxes of many plant species and tissue types
(Jetter and Riederer, 1996; Riederer and Markstadter,
49.765.6
47.464.9
49.065.4
1996; Teusink et al., 2002). Essentially, the same amount
of total wax (i.e. 23.6–25.9 ng mm2), with the same
chemical profile was found for all six soybean cultivars,
regardless of their degree of permeability. Prolonged
Soybean seed coat cutin chemistry and imbibition 1077
Table 2. Analysis of soybean seed coat wax
Waxes were extracted from intact seeds by a brief (30 s) CHCl3 dip (except OX-951-S) and the monomer composition determined (as their methyl
esters/trimethylsilyl ethers) by GC-MS and comparison with authentic standards. For OX-951-S, the outer seed coat was removed, exhaustively
extracted with CHCl3 and CHCl3:MeOH, and the soluble components analysed as above. The amount of each component (ng mm2 seed surface
area) was determined by GC-FID. Values represent the mean of three replicate extractions 6SD. Within each column, values followed by the same
letter are not significantly different from each other by general ANOVA followed by an LSD comparison of means (P¼0.05). tr, Trace.
Cultivar
Harovinton
AC Onrei
Tachanagaha
Bobcat
Harosoy 63
OX-951
OX-951-S
Aliphatic monomer class
Totals
Fatty acids
2-OH fatty
acids
x-OH fatty
acids
1-Alkanols
Alkanes
11.760.3 b
11.862.2 b
12.662.0 b
10.360.1 b
12.661.2 b
13.361.8 b
44.963.8 a
tr
tr
tr
tr
tr
tr
tr
0.160.01 c
0.260.04 ab
0.260.02 ab
0.260.01 ab
0.160.01 bc
0.260.06 ab
0.360.04 a
4.660.1
5.560.6
4.660.3
5.360.8
5.160.5
4.760.1
5.060.6
6.960.6
6.760.9
7.360.8
7.560.6
6.561.1
6.460.2
6.960.5
a
a
a
a
a
a
a
a
a
a
a
a
a
a
23.660.7 b
24.263.6 b
24.660.9 b
23.360.2 b
25.963.2 b
25.662.2 b
57.164.9 a
Table 3. Analysis of soybean outer seed coat cutin
The seed coats from imbibed seeds were separated into inner and outer layers. The outer seed coat layers were extracted (to remove waxes and other
organic solubles), de-polymerized using BF3/MeOH, derivatized with BSTFA, and quantified by GC-FID. Individual compounds were identified by
GC-MS. The data are summarized by substance class. Values (ng mm2 seed surface area) represent the average of triplicate measurements 6SD.
Within each column, values followed by the same letter are not significantly different from each other by general ANOVA followed by an LSD
comparison of means (P¼0.05).
Cultivar
Harovinton
AC Onrei
Tachanagaha
Bobcat
Harosoy 63
OX-951
Aliphatic monomer class
Fatty acids
2-OH fatty acids
x-OH fatty acids
23.063.5 d
25.561.3 cd
27.361.8 bcd
26.362.2 bcd
30.661.1 a
28.860.9 abc
17.461.9 c
18.060.6 c
18.361.1 c
18.961.1 c
23.960.1 b
28.260.5 a
8.160.7 b
8.360.1 b
9.160.3 b
8.660.5 b
9.160.1 b
11.261.1 a
Totals
Fatty acids/
hydroxylated
fatty acids
48.466.0 c
51.762.7 bc
54.761.7 b
54.863.3 b
63.660.3 a
68.161.1 a
1:1.1
1:1.0
1:1.0
1:1.1
1:1.1
1:1.4
Table 4. Analysis of soybean inner seed coat cutin
The seed coats from imbibed seeds were separated into inner and outer layers. The inner layers were extracted and analysed as described in the
legend to Table 3 and in Materials and methods. Values (ng mm2 seed surface area) represent the average of triplicate measurements 6SD. Within
each column, values followed by the same letter are not significantly different from each other by general ANOVA followed by an LSD comparison
of means (P¼0.05).
Cultivars
Harovinton
Harosoy 63
OX-951
Aliphatic monomer class
Fatty acids
2-OH fatty acids
x-OH fatty acids
30.262.8 a
30.162.4 a
30.565.2 a
14.862.3 a
15.961.8 a
15.562.7 a
0.4160.1 a
0.4660.1 a
0.3560.1 a
Soxhlet extraction of outer seed coat cuticles with
CHCl3:MeOH and CHCl3 did not yield any greater amount
of n-alkanes or 1-alkanols (indicative of wax components)
than that obtained from a brief immersion in CHCl3
(Table 2), indicating that the latter treatment was sufficient
to remove both epicuticular and intracuticular waxes from
soybean seed coat cuticles.
Totals
Fatty acids/
hydroxylated
fatty acids
45.464.8 a
46.563.5 a
46.365.2 a
1:0.50
1:0.54
1:0.52
Outer cuticle: Depolymerization of the extractive-free (i.e.
previously exhaustively extracted with CHCl3:MeOH and
CHCl3 alone to remove soluble components) outer layer
of soybean seed coats using BF3/MeOH resulted in the
release of fatty acids, 2-hydroxy fatty acids, and xhydroxy fatty acids as the major components (Fig. 5A;
Table 3). No mid-chain hydroxy- or epoxy-derivatized
1078 Shao et al.
fatty acids typical of many plant cutins (Walton, 1990;
Matzke and Riederer, 1991; Kolattukudy, 2000) were
detected. Although the total amounts of cutin monomers
released were not directly correlated with imbibition, the
least permeable cultivar, OX-951, contained the largest
amount of outer cutin monomers among the six cultivars
tested. In addition, the monomer profile from seed coats of
cv. OX-951 was significantly different in amount with
respect to the hydroxylated fatty acid sub-classes from all
other soybean cultivars tested. This relationship is clearly evident in the calculated ratios of fatty acids to hydroxylated
fatty acids (Table 3), where all five permeable cultivars
had ratios at or near 1:1, while the OX-951 ratio was 1:1.4.
Inner cuticle: Depolymerization of the extractive-free inner layer of soybean seed coats using BF3/MeOH resulted
in the release of fatty acids and 2-hydroxy fatty acids as
the major components as well as small amounts of xhydroxy fatty acids (Table 4). No mid-chain hydroxy- or
epoxy-derivatized fatty acids, typical of many plant cutins
(Walton, 1990; Matzke and Riederer, 1991; Kolattukudy,
2000), were detected. By contrast to the data obtained for
outer seed coat cuticles, there were no statistical differences between any monomer class isolated from the inner
cuticle of the hard and soft seeds tested. Moreover, the
ratios of fatty acids to hydroxylated fatty acids were lower
(1:0.5) than that from the outer cuticle and did not differ
between cultivars (Table 4).
Origin of 2-hydroxy fatty acids: To determine whether
the 2-hydroxy fatty acids observed in soybean seed coat
cutin depolymerizates were sphingolipid- or cutin-derived,
soybean seed coats were subjected to standard sphingolipid analysis (Cahoon and Lynch, 1991; Bonaventure
et al., 2003). For comparison, living endosperm tissue
from soybean seeds was also analysed. Thus, when the
fatty acid components of the sphingolipid fraction isolated
from soybean seed endosperm were analysed by GC-MS,
the predominant (>95%) 2-hydroxy fatty acid observed
was 2-hydroxyhexadecanoic acid (Fig. 6), with minor
amounts of 2-hydroxylated 22:0 and 24:0 fatty acids.
These data are in agreement with those of Sullards et al.
(2000). Similarly, a sphingoid base peak was found in
the long chain base fraction isolated after sphingosine
hydrolysis (Fig. 6). By contrast, there was no phospholipid band apparent on the TLC plates of lipid extracts
prepared from soybean seed coats. Nevertheless, the
region of the TLC plate corresponding to phospholipids
band (i.e. that which would contain sphingolipids if they
were present) was isolated and subjected to hydrolysis and
GC analysis. Essentially no components consistent with
either 2-hydroxy fatty acids or sphingoid bases were
present in the sample (Fig. 6).
Chain length distribution: Detailed chain length distribution data for each aliphatic substance class were obtained
Fig. 6. Sphingolipid analysis of soybean seed tissues. Representative
chromatograms of the fatty acids (A, C) and sphingoid bases (B, D)
released by Ba(OH)2 hydrolysis of sphingolipids isolated from (A, B)
soybean seed coats and (C, D) soybean endosperm. Sphingolipids were
extracted, purified by TLC, hydrolysed with Ba(OH)2, and the fatty
acids recovered by partitioning, derivatized, and analysed by gas
chromatography. The main fatty acids are labelled.
for each extract and tissue analysed. Regardless of their
source (i.e. wax, inner or outer seed coat) all six cultivars
studied had essentially the same distribution of monomers
(data not shown) and these are summarized for cultivar
OX-951 (Table 5). Consequently, the differences in cutin
composition between cultivars lay primarily in the
quantity of different aliphatic substance classes and not
the presence or absence of specific components.
Discussion
The results presented herein provide direct experimental
evidence that the inconspicuous, outermost cuticle of a
soybean seed governs its initial ability to imbibe water, a
necessary prerequisite for full hydration and seed germination. Selective removal of patches of the outer cuticle
with hot alkali did render the seeds capable of imbibition. In
accordance with this premise, when areas of the outer cuticle
were removed by hot alkali treatment, seeds were changed
from hard to soft. This experimental result is consistent
with an earlier anatomical study showing that soft seeds of
soybean have naturally occurring, minute cracks in their
outer cuticle whereas hard seeds do not (Ma et al., 2004).
Knowing that it is the integrity of the outer cuticle that is
responsible for hard-seededness in soybean will enable
steps to be taken in the food processing industry to solve
this problem, for example, scarifying seeds prior to soaking.
What components of the intact outer cuticle are
responsible for its impermeability to water?
In comparing the profile of wax monomers collected by
CHCl3 dip (Table 2) to that of monomers released by brief
NaOH treatment (Table 1), it becomes clear that the
NaOH treatment also removed waxes, since there are
roughly equal amounts of 1-alkanols and n-alkanes in
Soybean seed coat cutin chemistry and imbibition 1079
Table 5. Chain length distribution of soybean seed coat cutin monomers
The seed coats from imbibed seeds were separated into inner and outer layers. The layers were extracted and analysed separately as described in the
legends to Tables 2 and 3 and in Materials and methods. Values represent mol % within each substance class. Nd, Not detected.
Substance class
Fatty acids
x-OH-fatty acids
2-OH-fatty acids
1-Alcohols
n-Alkanes
Extract/tissue
Wax
Inner
Outer
Major (>10%): 26:0, 30:0
Minor (<10%): 16:0, 18:0, 20:0,
22:0, 24:0, 25:0, 27:0, 28:0,
29:0, 31:0, 32:0
Traces:
17:0, 18:1, 18:2, 19:0,
Major (>10%): 18:0
nd
Major (>10%): 16:0, 18:0
Minor (<10%): 20:0, 22:0, 23:0,
24:0, 25:0, 26:0
Major (>10%): 16:0, 18:0
Minor (<10%): 19:0, 20:0, 22:0,
23:0, 24:0, 25:0, 26:0
Major (>10%): 18:0
Major (>10%): 23:0, 24:0, 25:0
Minor (<10%): 18:0, 22:0, 26:0
nd
Major (>10%): 18:0
Major (>10%): 24:0, 25:0, 26:0
Minor (<10%): 21:0, 22:0, 23:0
nd
nd
nd
Major (>10%): 26:0, 28:0, 30:0,
32:0
Minor (<10%): 22:0, 24:0, 27:0,
29:0, 31:0
Major (>10%): 29:0, 30:0, 31:0
Minor (<10%): 27:0, 28:0, 32:0,
33:0
both samples. The main differences between these two
preparations lie in the greater amount of fatty acids
(approximately double in the NaOH extractives) and the
presence of x-hydroxy fatty acids in the NaOH extractives.
The origin of the additional fatty acids and x-hydroxy fatty
acids in the NaOH solubles appears to be exclusively from
the outer cuticle since the chemical composition of the
inner cuticle remained essentially unchanged by the alkali
treatment (compare inner seed coat data for OX-951 seeds
in Tables 1 and 4). In support of this, the sum of monomers
measured in the NaOH extractives and BF3/MeOHdepolymerized outer seed coats of NaOH-treated OX-951
seeds (Table 1) equals that of the CHCl3-extracted waxes
(Table 2) and BF3/MeOH-depolymerized outer seed coats
of solvent-extracted OX-951 seeds (Table 3). These data
confirm that the brief surface treatment with NaOH acted
only on the outer cuticle as expected. The (apparent)
selective removal of x-hydroxy fatty acids by the NaOH
treatment may be due, in part, to the more labile nature of
alkyl esters of primary alcohols compared with either alkyl
esters of secondary alcohols or alkyl esters in which there
is a neighbouring a-hydroxyl group (whether it is involved
in an ester or not), as may be the case for 2-OH-fatty acids.
Since removal of waxes from hard seeds by brief
immersion in CHCl3 alone did not result in imbibition
(Fig. 2B), the impermeability of the soybean seed cuticle
to water does not appear to depend on its wax component,
but rather on its cutin components. That both epicuticular
and intracuticular waxes were removed by this brief
exposure to solvent is supported by the observation that
exhaustive solvent extraction of isolated seed coats did not
yield more wax-associated components (i.e. alkanes and
1-alkanols) than brief immersion in CHCl3 (Table 2),
although a 3-fold higher yield of fatty acids was obtained.
The latter are likely to have originated from the exhaustive
solvent extraction of isolated seed coats, in which
underlying cells, that would not have been accessible to
the solvent used for surface wax dissolution, were
exposed. This result is in contrast to those obtained from
leaf cuticles, for which the intracuticular wax component
has been shown to be responsible for its impermeable
nature (Schönherr, 1982), albeit to varying degrees. The
amount of wax associated with soybean seed coats
(approximately 25 ng mm2) is relatively low compared
with that of soybean leaf cuticles (e.g. 99.663.2 ng mm2
extracted from 13-d-old leaves by CHCl3 dip) and the
cuticle from which they were extracted much thinner. It
is plausible, therefore, to assume that brief solvent extraction of the thin outer seed coat cuticle was sufficient to
remove the bulk of the soluble waxes (i.e. both epiand intra-cuticular), even though this would not be possible with thicker leaf cuticles.
Soybean seed coat cutin differs from that of
leaves and pods
The present findings show that both the inner and outer
cuticles of soybean seed coats are unique in chemical
composition and that they differ significantly from typical
cuticles, including those from leaves and pods of the same
plant. This is especially evident in (i) the predominance of
2-hydroxy- and x-hydroxy-fatty acids, (ii) the absence of
mid-chain hydroxylated fatty acids (Fig. 5; Walton, 1990;
Kolattukudy, 2000), and (iii) the high proportion of long
chain (non-wax) monomers in the monomer profile of the
seed cuticle (Table 5).
The presence of substantial amounts of 2-hydroxy fatty
acids in the present cutin depolymerizates was surprising.
1080 Shao et al.
While this class of compounds has recently been reported
as a component of Arabidopsis leaf cuticle (Franke et al.,
2005), its origin remains uncertain. That is, the 2-hydroxy
fatty acids reported for Arabidopsis cutins may have arisen
from sphingolipids of the plasma membranes of underlying
tissue. However, several lines of evidence support the conclusion that the 2-hydroxy fatty acids identified in soybean
seed coat cutin depolymerizates are derived from cutin: (i)
the preparation from which the outer cuticle aliphatic
monomer profile was obtained consisted of dead cells
(mainly palisade and hourglass cells), which are unlikely to
contain plasma membrane-derived lipids (Kolodziejek
et al., 2003), (ii) typical soybean sphingolipids contain predominantly 2-hydroxyhexadecanoic acids (Sullards et al.,
2000) whereas predominantly 2-hydroxytetracosanoic
(24:0) acid (and no 2-hydroxyhexadecanoic acid) was observed in cutin depolymerizates, (iii) no evidence for
sphingoid bases in soybean outer seed coats was found
using standard sphingolipid analytical techniques (Cahoon
and Lynch, 1991; Bonaventure, 2003; Fig. 6), (iv) typical
soybean sphingolipid-derived 2-hydroxy fatty acids (predominantly 2-hydroxyhexadecanoic acid) were observed
in soybean endosperm tissue (Fig. 6), and (v) BF3/MeOH
hydrolysates of cutins from exhaustively extracted whole
leaf and pod tissues (both of which contain live cells and
therefore intact plasma membranes) contained no 2hydroxy fatty acids (Fig. 5B, C; data not shown). These
data confirm that the 2-hydroxy fatty acids found in
soybean seed coat cutin depolymerizates are derived from
the cutin polymer. (This conclusion does not preclude the
possibility that the 2-hydroxy fatty acids isolated from
soybean seed coat cutin are of sphingolipid origin, being
incorporated into the cutin polymer during palisade or
hourglass cell scenescence.)
Why should the cuticles of some seeds crack while
those of others do not?
According to the results of this study and a previous paper
(Ma et al., 2004), the difference between hard and soft
soybean seeds is the continuity of the outermost seed
cuticle in the former and the presence of small cracks in the
cuticles of the latter. In addition, Ma et al. (2004) reported
that isolated pieces of cuticle from soft seeds are more
delicate and prone to breakage than those from hard seeds.
Thus, the strength of the cutin polymer may be critical to
forming one or the other type of seed. There are chemical
differences between the components of the outermost
cuticles of hard and soft seeds, and these differences may
have a bearing on the tendency of cuticles to crack. In other
words, the softness of soybean seed coats is a result of
cuticular ‘crack-ability’ rather than cuticular permeability.
Although a direct study of the cutin polymer was not
possible, the monomer compositions of the two seed types
showed intriguing differences. The most notable of these was
the relatively high amount of hydroxylated components
(especially 2-hydroxy fatty acids) found in the BF3/MeOH
depolymerizate of hard seeds. The presence of a greater
proportion of bi-functional (hydroxylated) fatty acids may
provide a greater inter-connectivity between monomers in
the cutin of hard seeds. And while bi-functional monomers themselves are not sufficient for the assembly of an
integrated, three-dimensional polymer, recent evidence
suggests that glycerol plays a significant role in the
polyester structure of cutins (Gracxa et al., 2002) and these
may provide the necessary bridging between bi-functional
monomers.
The relevance of the hydroxylated monomers is also
underscored by the results of the NaOH treatment of
entire hard seeds, in which the x-hydroxy fatty acid component of the outer cuticle (along with part of the fatty
acid component) was extracted (compare Tables 1 and 3).
This resulted in the disruption of the outer cuticle structure
in discrete areas, allowing the seed to imbibe. Although
the NaOH treatment resulted in removal of only patches
of cuticle (Figs 3, 4), it extracted essentially all of the xhydroxy fatty acids (compare Tables 1 and 3); it seems
that the outer cuticle may be rich in x-hydroxy fatty acids
in localized regions. In any case, it is clear that x-hydroxy
fatty acids play an integral role in establishing or
stabilizing the overall cutin structure, possibly making it
resistant to cracking in the case of hard seeds. It seems
less likely that the NaOH treatment modified the underlying carbohydrate cell walls, and thereby making hard
seeds soft, since in order to affect the cell walls the cuticle
must first be breached; as soon as this occurs, imbibition
can also occur.
Nevertheless, the interaction between cutin monomers
and the underlying carbohydrate cell wall matrix cannot
be overlooked in establishing the differences between hard
and soft soybean seeds. Indeed, preliminary evidence
suggests that the structural organization of outer seed
coats differs between hard- and soft-seeded cultivars (MA
Bernards, S Shao, unpublished results). For example, after
exhaustive enzymatic digestion with cellulase, pectinase,
and hemicellulase, the amount of cutin in the outer seed
coats of both Harosoy 63 and OX-951 remains <1% dry
weight (data not shown). The remaining >99% is likely
to be residual carbohydrates not digested by the enzymes.
However, when subjected to neutral sugar analysis
(Blakeney et al., 1983), approximately 90% of the mass
of Harosoy 63 outer seed coats could be accounted for as
carbohydrates, while only 67% of the outer seed coat of
OX-951 could be recovered as free carbohydrates. The
remaining 10–33% of the digested seed coats are likely to
contain cutin monomers (approximately 1%), galacturonic
acid (not detected in the present analysis), and possibly
carbohydrate-aliphatic adducts. The profile of monosaccharides released by acid hydrolysis was similar between
the two cultivars, consisting primarily of glucose and
Soybean seed coat cutin chemistry and imbibition 1081
xylose; i.e. hemicellulose components (data not shown),
in agreement with Mullin and Xu (2001). Together these
data suggest the possibility that a more extensive integration (possibly due to covalent linkages) exists between
cutin and carbohydrate monomers in the OX-951 cultivar,
which may give the surface material either more structural
integrity or greater flexibility, making it less susceptible to
cracking and thereby less permeable. This idea requires
further testing.
In summary, evidence from the present study, coupled
with that of Ma et al. (2004) demonstrates that hardseededness in soybean can be ascribed to the presence of an
unbroken, outer cuticle covering the seed. The outer seed
cuticle differs from the inner cuticle in chemical composition, and both are strikingly different from cuticles isolated
from pods and leaves of the same plant. The feature of the
outer cuticle that correlates with a lack of cuticular cracks
is a relatively high level of hydroxylated fatty acids. It is
postulated that these chemicals are critical in modifying
either the degree of cross-linking of the components of the
cuticle or its integration with the underlying cell wall
components, thereby preventing the cuticle from cracking
during development.
Acknowledgements
We thank Dale Weber (University of Waterloo) for technical
assistance with SEM and Dr Mark Gijzen (Agriculture and AgriFood Canada, London, ON) for providing the seeds. This research
was funded by a Strategic Grant from the Natural Sciences and
Engineering Research Council of Canada.
Note added in proof
The impermeability of intact cuticle areas of soft seeds has been
indicated by applying water only to their abaxial sides. Meyer CJ,
Steudle E, Peterson CA, 2007. Patterns and kinetics of water uptake
by soybean seeds. Journal of Experimental Botany 58, 717–732.
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