Annals of Botany 84 : 297–304, 1999
Article No. anbo.1999.0915, available online at http:\\www.idealibrary.com on
Early Development of the Seed Coat of Soybean (Glycine max)
S. S H E A M I L L ER*†, L U-A N N A. B O W M A N*, M A R K G I J Z E N‡ and B R I A N L. A. M I K I*
* Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Centre, Ottawa, Ontario, Canada
K1A 0C6 and ‡ Southern Crop Protection and Food Research Centre, London, Ontario N5V 4T3
Received : 24 February 1999
Returned for revision : 15 April 1998
Accepted : 4 May 1999
Although the development of the soybean ovule has been fairly well studied, knowledge of the sequence of events in
the seed coat during the first 3 weeks after flowering is incomplete. The goal of the present study was to document,
using light microscopy, the early development of the soybean seed coat with respect to changes in structure and
histochemistry. At anthesis, the seed coat consists of an outer layer of cuboidal epidermal cells surrounding several
layers of undifferentiated parenchyma (which together constitute the outer integument), and an inner layer of
cuboidal endothelial cells (the inner integument). At 3 d post anthesis (dpa), the inner integument has expanded to
include three to five layers of relatively large cells with thick, heavily-staining cell walls immediately adjacent to the
endothelium. By 18 dpa, the outer integument has developed into a complex of tissues comprised of an inner layer
of thick-walled parenchyma, an outer layer of thin-walled parenchyma containing vascular tissue which has grown
down from the lateral vascular bundles in the hilum region, a hypodermis of hourglass cells, and palisade layer
(epidermis). The thick-walled parenchyma of the inner integument has become completely stretched and compressed,
leaving a single, deeply staining wall layer directly above the endothelium. At 21 dpa, the outermost cells of the
endosperm have begun to compress the endothelium. At 45 dpa (physiological maturity) the seed coat retains only
the palisade layer, hourglass cells, and a few layers of thin-walled parenchyma. The innermost layer of the endosperm,
the aleurone layer, adheres to the inside of the seed coat. This knowledge will be invaluable in future studies of
manipulation of gene expression in the seed coat to modify seed or seed coat characteristics.
# 1999 Annals of Botany Company
Key words : Soybean, Glycine max, seed coat, development, aleurone.
INTRODUCTION
The seed coat, or testa, of the mature soybean has been well
characterized, and contains features in common with the
majority of the legumes : an epidermal layer of palisade
cells, or macrosclereids, a sub-epidermal layer of hourglass
cells, or osteosclereids, a few layers of parenchyma, and an
aleurone layer (Williams, 1950 ; Corner, 1951). The seed
coat is developmentally transitory, and there are a number
of tissues present during early and mid-development which
do not persist in the mature seed coat. Despite a number of
studies examining different stages in the development of the
ovule and the seed coat (Pamplin, 1963 ; Thorne, 1981 ;
Yaklich et al., 1986, 1992, 1995 ; Baker, Minor and Cumbie,
1987 ; Carlson and Lersten, 1987), information on the very
early stages of seed coat development (i.e. the first 3 weeks
after anthesis) is incomplete, and the origin of the aleurone
layer remains in question. Many consider the aleurone layer
in soybean to be derived from the endosperm (Winton and
Winton, 1932 ; Williams, 1950 ; Carlson and Lersten, 1987 ;
Yaklich et al., 1992), hence the use of the term aleurone,
while others maintain that this layer is an endothelium of
maternal origin (Thorne, 1981 ; Baker et al., 1987).
Because our goal was to investigate gene expression in the
soybean seed coat during early development, we found it
† For correspondence.
Fax j1 613 759 1701, e-mail millers!em.agr.ca
0305-7364\99\090297j08 $30.00\0
necessary to first clarify events at these early stages. Thus,
our objective was to document the occurrence, and
replacement or modification of tissues during development,
with particular emphasis on the first 3 weeks after anthesis
(flowering). If we are to manipulate pigmentation, or
change constituents in the seed coat, or in the seed itself, it
is important to understand the developmental sequence,
and to try to elucidate the functions of the different tissues.
Using light microscopy, we have studied the development of
the soybean seed coat at 3 d intervals during the 3 week
period following anthesis, as well as a brief look at some
later stages of development. In addition to structure, the
carbohydrate, protein and lipid status of the tissues has also
been examined. In this study, we focused on cross sections
through the hilum region, as that is where the developing
seed attaches to the pod, and the area through which
assimilate enters the seed.
MATERIALS AND METHODS
Plant growth
Seeds of Glycine max (L.) Merrill, ‘ Maple Presto ’ were
germinated in vermiculite. Seedlings were grown in a
controlled environment in growth cabinets (25 mC day\20 mC
night with a 12 h photoperiod, relative humidity 80 %).
After approx. 1 week, seedlings were transplanted into soil
in 127 mm peat pots. At each node, flowers were tagged on
# 1999 Annals of Botany Company
298
Miller et al.—Soybean Seed Coat DeŠelopment
the days of full anthesis (banner petal fully extended :
Peterson et al., 1992). This was monitored daily throughout
the duration of flowering due to the number of flowers per
node. The relative humidity of the cabinets was changed to
70 % at approx. 21 d post anthesis (dpa) to allow for seed
drying.
Seed collection and fixation
Tagged pods were harvested at 3 d intervals from 1 to
30 dpa and at 45 dpa. Seeds were fixed at 4 mC overnight in
4 % phosphate buffered (25 m, pH 6n8) glutaraldehyde.
Small seeds (1–6 dpa) were fixed whole, with a portion of
the pod still attached, and larger seeds ( 9 dpa) were cut
into slices approx. 1n5 mm thick. After fixation, the
dehydration and embedding regime of O’Brien and McCully
(1981) was followed. Briefly, samples were dehydrated
through a solvent series (methylcellosolve, 95 % ethanol, npropanol and n-butanol) and infiltrated dropwise with
glycol methacrylate (GMA) over 2 d. The tissue was
polymerized overnight, under UV light, in an O -free
#
atmosphere.
was obtained by staining similar sections with iodinepotassium iodide (0n2 % iodine in 2 % aqueous potassium
iodide). Slides were either mounted wet in glycerol, or air
dried and mounted in immersion oil.
For observation of lipids (triacylglycerols), frozen sections
(approx. 10 µm) were used, as the dehydration series for
GMA embedding extracted the lipids from the tissue. Fixed
seeds were infiltrated with 1 sucrose overnight as a
cryoprotectant, then sectioned on a Reichert-Jung Cryocut
E microtome (Reichert, Vienna). Sections were transferred
to slides that were pre-treated with a Fro-Tissuer Pen
(Electron Microscopy Sciences, Fort Washington, PA) and
allowed to dry overnight. Sections were stained with 0n02 %
aqueous Nile Blue A, rinsed briefly in water, and aqueous
mounts were viewed using fluorescence optics at 450\ 520
excitation\emission.
For bright field optics, micrographs were recorded on
Kodak E100S colour slide film. Fluorescence micrographs
were recorded on Kodak EPL400X colour slide film. For
reproduction, 35 mm slides were scanned using a Kodak
Professional RFS 3570 Film Scanner. Images obtained were
imported into Adobe Photoshop for formatting and
labelling, then saved on CD or printed using a Kodak 8650
printer.
Histochemistry and microscopy
Sections (approx. 2 µm) were cut with a glass knife on
a Porter-Blum Ultra-microtome MT-1 (Sorvall, Newtown,
CT), and mounted on acid-alcohol washed slides. For
general structural observations, sections were stained with
either Toluidine Blue O (0n05 % in 50 m acetate buffer,
pH 4n4) or the fluorescent dye Coriphosphine O (0n3 %
aqueous). Slides were stained for 2 min, rinsed in distilled
water and mounted in glycerol, or air dried and mounted in
immersion oil and then viewed using a Zeiss Universal
Research microscope (Carl Zeiss, Germany). The microscope was equipped with a tungsten illuminator and substage condenser for bright field observations, and a 100W
mercury arc lamp and epi-illuminating condenser for
fluorescence analysis. Toluidine Blue stained sections were
observed using standard bright-field conditions ; for observation of Coriphosphine O, fluorescence optics were used
with excitation\emission wavelengths 365\ 420 nm.
For observation of protein and carbohydrates, a stepwise
staining procedure was used. Slides were first stained for
carbohydrates using the Periodic Acid Schiff’s (PAS)
procedure (O’Brien and McCully, 1981). Briefly, sections
were first subjected to an aldehyde blockade using 2,4dinitrophenylhydrazine (saturated, in 15 % acetic acid) for
10 min. After washing in running tap water (10 min), slides
were oxidized in 1 % aqueous periodic acid (10 min), and
washed again. The aldehydes created by periodate oxidation
were detected using Schiff’s reagent for 30 min, after which
sections were transferred directly into three successive baths
of freshly prepared 0n5 % sodium metabisulfite in a 1 %
dilution of concentrated HCl (2 min each), then rinsed in
running water for 10 min. The slides were then stained for
protein with Light Green (0n1 % in 1 % acetic acid, pH 2n8)
for 5 min, rinsed briefly in 1 % acetic acid, then washed for
1 min with running water. Specific identification of starch
RESULTS
In order to sequence the development of the seed coat, it is
valuable to refer to a cross section of the mature seed coat
(Fig. 1). The characteristic features of the mature seed coat
have long been recognized (Winton and Winton, 1932 ;
Williams, 1950 ; Corner, 1951). The outermost, epidermal
layer of the mature soybean seed coat is the palisade layer :
a single cell layer of thick-walled macrosclereids that are
elongated perpendicular to the surface of the seed. Inside
the palisade layer is a single-celled hypodermal layer of
thick-walled osterosclereids, or hourglass cells. The innermost portion of the seed coat proper is a multicellular
layer of partially flattened parenchyma. Immediately inside
the inner parenchyma is the aleurone layer : the outermost
layer of the endosperm, which has been tightly compressed
against the seed coat by the expansion of the cotyledons.
Anthesis
At anthesis (Fig. 2), the seed coat is bitegmic, and
contains none of the characteristic features of the mature
tissue (Fig. 1). The outer integument consists of a cuboidal
epidermis surrounding several layers of undifferentiated
thin-walled parenchyma ; the inner integument consists of a
cuboidal endothelium which provides the inner boundary of
the seed coat. The seed coat is proportionately the largest
part of the seed : about 90 % (as compared to less than 8 %
in the mature seed ; Saio, Arai and Watanabe, 1973). The
seed is attached to the pod by the funiculus. Although the
cells stain more intensely where the funiculus meets the seed
coat (not shown for anthesis, but see Fig. 3 A for an
example), the characteristic double palisade layer and
tracheid bar of the mature hilum have not yet developed.
Although there are no distinct protein bodies in the seed
Miller et al.—Soybean Seed Coat DeŠelopment
299
F. 1. Cross section of physiologically mature soybean seed coat
(45 dpa), stained with PAS and Light Green. Carbohydrates are
fuschia, proteins are green. p, Palisade layer ; h, hourglass cells ; pa,
partially crushed parenchyma (aerenchyma) ; c, crushed remnants of
parenchyma and endothelium ; a, aleurone ; em, crushed remnants of
endosperm. Bar l 100 µm.
F. 2. Cross section of seed coat at anthesis, stained with PAS and
Light Green. Carbohydrates are fuschia, proteins are green. ep ;
Epidermis ; pa, undifferentiated parenchyma ; en, endothelium ; s,
starch granules. Note darker green nuclei, with darkly staining nucleoli,
particularly in the parenchyma cells. The large, loose cells of the
embryo sac are visible inside the seed coat. Bar l 20 µm.
F. 3. Cross sections of seed coat 3 dpa. A, Stained with Toluidine
Blue. Note densely staining cells at base of funiculus (*) ; vb, vascular
bundles ; en, endothelium ; ii, inner integument ; oi, outer integument ;
ep, epidermis. Bar l 100 µm. B, Stained with PAS and Light Green.
Carbohydrates are fuschia, proteins are green. s, Starch granules. Bar
l 50 µm.
F. 4. 6 dpa. A, Cross section of whole soybean seed coat, stained with
Toluidine Blue. Part of the pod is visible in the upper portion of the
micrograph, showing sclerenchyma (sc) and inner epidermis (ie) of the
pod endocarp. vb, Vascular bundles ; en, endothelium ; ii, inner
integument ; oi, outer integument ; ep, epidermis. Bar l 100 µm. B,
Cross section at developing hilum region, stained with PAS and Light
Green. Carbohydrates are fuschia, proteins are green. f, Funiculus ; cp,
counter-palisade ; p, palisade ; s, starch granules. Bar l 50 µm.
coat, the cytoplasm stains a light green for protein. In fact,
at no point during development were discrete protein bodies
observed in the seed coat. The nuclei stain quite distinctly
green for protein due to the presence of chromosomal
proteins in addition to the nucleic acids. There are starch
granules in the undifferentiated parenchyma, but no lipid
bodies were observed in the seed coat at this stage.
Three days post anthesis
During the first 3 dpa, significant changes occur in the
structure of the seed coat (Fig. 3). The inner integument
expands to include three to five layers of relatively large cells
300
Miller et al.—Soybean Seed Coat DeŠelopment
F. 5. 9 dpa. A, Cross section through the hilum region of soybean seed coat, stained with Toluidine Blue. Note well developed tracheid bar (tb),
counter-palisade (cp) and palisade layer (p). Also visible are the endothelium (en), crushed walls of the inner integument (ii), thick-walled
parenchyma of the outer integument (k), thin-walled parenchyma of the outer integument (pa) with embedded vascular bundles (vb), and modified
parenchyma in the sub-hilum region (mp). Bar l 50 µm. B, Cross section of soybean seed coat at the bottom of the seed (opposite the hilum),
stained with Toluidine Blue. Note the distinct hypodermis (hp) beginning to differentiate into hourglass cells. ep, Epidermis ; pa, thin-walled
parenchyma of outer integument ; k, thick-walled parenchyma of outer integument ; ii, inner integument ; en, endothelium. The endosperm is
visible inside the seed coat (em). Bar l 100 µm. C, Cryo-section of hilum area, stained with Nile Blue. Note presence of bright yellow lipid droplets
in the funiculus (f) and counterpalisade (cp), but not in the palisade (p). Bar l 50 µm. D, Cryo-section of outer seed coat stained with Nile Blue.
Note presence of bright yellow lipid droplets in the epidermis (ep), but not in the thin-walled parenchyma of the outer integument (pa). Also visible
is the inner epidermis of the pod endocarp (ie). Bar l 50 µm.
with thick, heavily-staining cell walls immediately adjacent
to the endothelium. The outer integument at this stage
consists of smaller, thin-walled parenchyma cells surrounded
by the cuboidal epidermis. The lateral vascular bundles
have started to form in the outer integument below the
funiculus.
There are numerous small starch granules in the funiculus,
and in the thin- and thick-walled parenchyma of the
integuments, but not in the epidermis or the endothelium
(Fig. 3 B). There are no lipid bodies in the seed coat at this
stage, but a few tiny lipid droplets can be found in the
funiculus, just outside the seed coat proper (not shown).
Six days post anthesis
At 6 dpa, the thick-walled parenchyma of the inner
integument has expanded from an average thickness of
three cells to between four and six cells (Fig. 4 A). Although
this study focused primarily on sections through the hilum
region, we did observe variations in the thickness of the
inner integument in different areas of the seed. In sections
approaching the chalazal end, the thick-walled parenchyma
of the inner integument may become up to 10–15 cell layers
thick. The epidermal cells are gradually losing their cuboidal
shape and are beginning to differentiate into the thickwalled palisade layer. The lateral vascular bundles are
starting to extend, to form what will eventually become the
vascular region of the thin-walled parenchyma of the outer
integument.
Starch is present in the stellate or modified parenchyma
(Corner, 1951) beneath the funiculus. Where the funiculus
meets the seed coat (Fig. 4 B), the palisade and counterpalisade layers are now visible (Corner, 1951). Starch
granules are not detectable in the vascular region, inner
integument, or palisade layers, although a few granules have
appeared in the endothelium. As at 3 dpa, there are no lipid
bodies in the seed coat.
Nine days post anthesis
At 9 dpa, the seed has started to enlarge (Fig. 5). At the
base of the funiculus, the characteristic features of the
Miller et al.—Soybean Seed Coat DeŠelopment
301
however, in the developing palisade (epidermis) throughout
the rest of the seed coat (Fig. 5 D). Although more sparsely
distributed, there are also lipid bodies in the thick-walled
parenchyma of the outer integument and in the outermost
layer of the endosperm, adjacent to the endothelium.
TwelŠe days post anthesis
F. 6. 12 dpa. A, Cross section of outer seed coat near the top, or
hilum region of the seed, stained with PAS and Light Green.
Carbohydrates are fuschia and proteins are green. Note well developed
palisade layer (p) with a few very small starch granules (arrowheads).
The hypodermis (hp) is beginning to differentiate into hourglass cells,
which contain starch granules, as do the thin-walled parenchyma of the
outer integument (pa). Bar l 50 µm. B, Cryo-section of inner seed
coat, stained with Nile Blue. Note the presence of bright yellow lipid
droplets in the outermost layer of the endosperm (em). en, Endothelium ;
ii, crushed walls of inner integument ; k, thick-walled parenchyma of
outer integument. Bar l 50 µm.
At 12 dpa, the palisade is becoming distinct, and the
hourglass cells have started to differentiate from the
hypodermis near the top, or hilum portion, of the seed (Fig.
6 A). The vascular region in the thin-walled parenchyma of
the outer integument is becoming quite prominent, and the
thin-and thick-walled parenchyma are very distinct. The
thick-walled parenchyma of the inner integument continues
to be compressed. The vascular tissue in the outer
integument does not yet approach the bottom of the seed,
and the differentiation of the hourglass cells is also not as
advanced. Inside the seed coat, the endosperm is visible in
close proximity to the endothelium (Fig. 6 B).
Starch is found throughout the thin- and thick-walled
parenchyma of the outer integument, but not in the vascular
region. Starch granules are still visible in the funiculus, but
few are observed in the palisade layers of the hilum region.
Further along the seed coat, i.e. away from the hilum, starch
is visible in the developing hourglass cells, and in the thinwalled parenchyma, and also some very tiny granules in the
epidermis\palisade (Fig. 6 A). In the hilum region, there are
still abundant lipid bodies in the counter-palisade, and
virtually none in the palisade layer, although small amounts
remain in the epidermis\palisade around the periphery of
the seed, and in the thick walled parenchyma. As at 9 dpa,
lipid bodies are present in the outermost layer of the
endosperm (Fig. 6 B).
Fifteen days post anthesis
hilum, including the palisade, counter palisade and tracheid
bar, are becoming distinct (Fig. 5A). The outer integument
has differentiated into an outer layer of thin-walled
parenchyma, containing vascular tissue, and an inner layer
of thick-walled parenchyma. The thick-walled parenchyma
of the inner integument has stretched and become compressed. In the cells opposite the hilum, at the bottom of
the seed (Fig. 5 B), the differentiation of the tissues is not as
advanced ; development occurs in a gradient from the hilum
to the bottom of the seed. Two epidermal layers (epidermis
and hypodermis), that will become the palisade layer and
hourglass cells are visible. The thin- and thick-walled
parenchyma of the outer integument are discernible,
although not as distinct as near the hilum region of the seed.
Compression of the thick-walled parenchyma of the inner
integument is very distinct. Inside the seed coat, the
endosperm is visible adjacent to the endothelium.
Starch is most prevalent in the thin-walled parenchyma of
the outer integument. The size and abundance of lipid
bodies in the funiculus has increased, and lipid bodies are
present in the counter-palisade, but not in the palisade layer
of the hilum region (Fig. 5 C). Lipid bodies are visible,
Between 12 and 15 d after anthesis, the walls of the
palisade layer become thicker, and the hourglass cells
complete their transformation. At 15 dpa (Fig. 7 A), the
thin- and thick-walled parenchyma and vascular region are
still clearly distinguishable, but all that remains of the thickwalled parenchyma of the inner integument is a single darkstaining wall layer just above the endothelium. The thinwalled parenchyma is starting to take on the characteristics
of aerenchyma, with large air spaces between the cells ; the
cells of the endothelium are no longer cuboidal, and have
become thinner, and slightly elongated (Fig. 7 A and B). The
cotyledons, which are initiated at the chalazal end of the
seed, and thus are not visible in hilum sections during the
first few dpa, have expanded, and now almost fill the seed
coat cavity, crowding the endosperm (Fig. 7 C).
Starch is present mostly in the palisade and hourglass
cells, although there are still a few starch granules distributed
throughout the parenchyma of the seed coat. Between 12
and 15 dpa, the cytoplasm of the outermost endosperm cells
becomes denser, and stains more intensely. This is most
clearly shown in fluorescent micrographs stained with
Coriphosphine (Fig. 7 B). This region of the endosperm is
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Miller et al.—Soybean Seed Coat DeŠelopment
F. 8. Cross section through inner seed coat at 18 dpa, stained with
Coriphospine. Note apoptotic nuclei (arrowheads) in the endothelium.
The outermost layer of the endosperm (em) is being compressed, and
stains intensely. A small portion of the cotyledon (c) is visible. en,
Endothelium ; ii, crushed remnants of inner integument ; k, thick-walled
parenchyma of outer integument. Bar l 20 µm.
F. 9. Cross section through inner seed coat at 21 dpa, stained with
Coriphospine. The densely staining cells of the outermost layer of the
endosperm (em) are encroaching upon and crushing the endothelium
(arrows). ii, Crushed remnants of inner integument ; k, thick-walled
parenchyma of outer integument. Bar l 20 µm.
F. 7. 15 dpa. A, Cross section of whole seed coat stained with PAS
and Light Green. Carbohydrates are fuschia, proteins are green.
Palisade layer (p) is well developed, containing numerous starch
granules. Hourglass cells (h) are also fully formed. Thin-walled
parenchyma (pa) is developing large air spaces characteristic of
aerenchyma. vb, Vascular bundle ; k, thick-walled parenchyma ; en,
endothelium ; em, endosperm. Bar l 100 µm. B, Cross section of inner
seed coat, stained with Coriphosphine. Note densely staining cytoplasm
in outermost layer of endosperm (em). en, Endothelium ; ii, crushed
remnants of inner integument ; k, thick-walled parenchyma of outer
integument. Bar l 20 µm. C, Cryo-section of whole soybean seed
stained with Nile Blue. There is very little lipid left in the seed coat
proper (SC), but there is an increasing gradient from the abaxial (ab)
to the adaxial (ad) sides of the cotyledons. Bar l 100 µm.
also enriched in lipid bodies ; there is very little lipid left in
the seed coat itself. In the cotyledons, there is a gradient in
lipid concentration from the adaxial to the abaxial regions
(Fig. 7 C). At this time, protein bodies have started to
appear in the cotyledons as well (not shown).
Eighteen days post anthesis
At 18 dpa, apoptotic nuclei are visible in the endothelium,
adjacent to the endosperm (Fig. 8). The palisade and
hourglass cells are well defined, as are the thin- and thickwalled parenchyma, and vascular region. There are abundant small starch granules in the palisade, hourglass cells
and cotyledons, although few remain in the parenchyma.
There is no lipid remaining in the seed coat. Inside the seed
coat, the endosperm is beginning to compress, with the
cytoplasm of the outermost layer continuing to increase in
density.
Maturation
After 18 dpa, the rate of differentiation slows, and further
changes are more subtle. At 21 dpa, the cotyledons are
expanding out to the seed coat, crushing all but the
outermost layer of the endosperm in the process, which in
Miller et al.—Soybean Seed Coat DeŠelopment
turn crushes the endothelium (Fig. 9). The cytoplasm of
these endosperm cells stains very intensely. At 30 dpa, the
seed coat parenchyma is starting to be compressed,
particularly the thick-walled parenchyma. Only a thin,
deeply staining wall layer remains of the inner integument
and endothelium. Most of the starch has gone from the seed
coat. At 45 dpa (Fig. 1), the seed coat is essentially mature,
although seeds are not typically harvested until 55–60 dpa.
The most prominent features are the hourglass cells and
palisade layer. Only a few layers of slightly flattened thinwalled parenchyma (aerenchyma) remain. The portion of
the outer integument containing the vascular tissue has been
crushed, as have the thick-walled parenchyma and the
endothelium. Adjacent to the remnants of the thick-walled
parenchyma and the endothelium is a layer of thick-walled
cells derived from the endosperm : the aleurone layer.
D I S C U S S I ON
During the first 3 weeks after anthesis, the soybean seed coat
undergoes a sequence of changes, from a single organ of
three tissue layers containing similar cell types, to a multilayered organ with a variety of cell types which persist for
varying lengths of time. The thick-walled parenchyma of the
inner integument, which is the most prominent feature in
the seed coat in the first week after anthesis, has been
compressed almost beyond recognition by the end of the
second week. Before the end of the third week, the outer
integument changes from thin-walled parenchyma a few
cells thick (including the epidermis), to a complex of tissues
comprised of an inner layer of thick-walled parenchyma, an
outer layer of thin-walled parenchyma containing vascular
tissue which has grown down from the lateral vascular
bundles in the hilum region, a hypodermis of hourglass
cells, and palisade layer (epidermis). As the seed approaches
maturity, all that remains of the seed coat proper are a few
layers of partially flattened, thin-walled parenchyma, the
hourglass cells, and the palisade layer.
Perhaps the most disputed area in the literature on
soybean seed coat development is the nature of the innermost layer of the mature seed coat : endothelium (maternal)
or aleurone (endosperm) ? Pamplin (1963) suggested that the
aleurone was most likely derived from the inner integument,
by virtue of having thick cell walls, and the thick walls of the
layer of crushed cells between the aleurone and the embryo.
However, Pamplin’s (1963) studies did not actually extend
to the stage at which the aleurone layer is formed. Later
studies by Thorne (1981) and Baker et al. (1987), using SEM
to examine seed coat development, also supported a
maternal origin for the aleurone layer. Thorne’s (1981)
study, however, also did not encompass the critical stages of
development when the aleurone is actually formed.
The study by Baker et al. (1987) covered the critical time
period, but the SEM was performed largely at relatively low
magnifications, so that critical details were missed. Indeed,
re-examination of Baker’s micrographs supports the endosperm-derived hypothesis. The endothelium is present
(although ignored or incorrectly labelled) throughout the
early stages of development, and the aleurone can be seen to
303
be encroaching on the endosperm as development proceeds.
Other authors have stated that the aleurone is derived from
the endosperm (Winton and Winton, 1932 ; Williams, 1950 ;
Carlson and Lersten, 1987, Yaklich et al., 1992).
The work presented here provides evidence that the
aleurone is actually the outermost layer of the endosperm,
which persists at maturity, supporting the theories of
Winton and Winton (1932), Williams (1950), Carlson and
Lersten (1987) and Yaklich et al. (1992). The density of
staining of the cytoplasm in the outermost layer of the
endosperm, adjacent to the endothelium, increases steadily
from 15 to 21 dpa (Figs 7 B, 8 and 9). At 21 dpa, this densely
staining layer of cells is clearly encroaching upon and
crushing the endothelial layer (Fig. 9). By 18 dpa, the nuclei
in the endothelium show symptoms of apoptosis. This
process of programmed cell death is common in both plants
and animals, in cells and tissues that are lost or replaced
during development (Wang et al., 1996 ; Pennell and Lamb,
1997). The nucleoplasm has fragmented, and apoptotic
bodies can be seen around the periphery of the nucleus (Fig.
8). In the mature seed coat (Fig. 1), the remains of the
endothelial layer are indistinguishable in the crushed layer
of parenchyma above the aleurone ; the crushed remains of
the inner endosperm layers lie between the aleurone and the
embryo. In addition to this physical evidence, convincing
biochemical evidence of the endosperm origin of the
aleurone has been reported (Schmidt, Lindstrom and
Vodkin, 1994). Analysis of the expression of proline-rich
proteins in seed coats and aleurone layers dissected from F
#
seeds showed that the seed coats had the F (maternal)
#
genotype, whereas the aleurone was F (zygotic).
$
In addition to providing a protective covering for the
embryo at maturity, tissues of the soybean seed coat provide
support and nourishment for the developing embryo. In a
variety of legumes, it has been demonstrated that the
composition of solutes (sugars, amino acids, minerals)
secreted by the seed coat into the embryo sac during
development is different from the solute entering the seed
coat (for a review see Murray, 1988), indicating active
metabolism of incoming assimilate by maternal tissues
before delivery to the embryo. A surprising variety of
developmental patterns occur during the growth and
maturation of the seed coat. Cells may become highly
differentiated and develop elaborate structural characteristics, such as the thick-walled hourglass cells. Alternatively,
differentiation may terminate in apoptotic-like processes
where nothing remains of the cell except a crushed and
compressed remnant, as observed for the inner integument.
These contrasting fates undoubtedly arise from contrasting
functional requirements that occur during the course of
development : nutrient transport and metabolism, required
during embryo growth, is superseded by physical and
structural requirements as the seed matures. Although the
precise function(s) of each tissue is not known, examination
of the ultrastructure of certain cells provides clues that
suggest functions in apoplastic and\or symplastic transport
and secretion (Thorne, 1981 ; Yaklich et al., 1986, 1992,
1995). Clearly then, the tissues of the developing seed coat
are logical targets for modification of gene expression in any
plan to modify the properties of the mature seed.
304
Miller et al.—Soybean Seed Coat DeŠelopment
A C K N O W L E D G E M E N TS
Parts of this work were supported by the Ontario Soybean
Grower’s Marketing Board. We thank Susan Winter for
invaluable technical assistance, Lise St. Jean for graphics,
and Lila Vodkin for helpful comments during the preparation of the manuscript. ECORC contribution 991409.
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