Download seed coats

yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Document related concepts

Plant morphology wikipedia , lookup

Arabidopsis thaliana wikipedia , lookup

Glossary of plant morphology wikipedia , lookup

Flowering plant wikipedia , lookup

Gartons Agricultural Plant Breeders wikipedia , lookup

In Vitro Cell. Dev. Biol.—Plant 41:620–644, September– October 2005
q 2005 Society for In Vitro Biology
1054-5476/05 $18.00+0.00
DOI: 10.1079/IVP2005686
Ottawa-Carleton Institute of Biology, Department of Biology, University of Ottawa, P.O. Box 450, Station A, Ottawa, Ontario,
Canada K1N 6N5
BioProducts & BioProcesses, Research Branch, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada K1A OC6
(Received 8 May 2005; accepted 17 May 2005; editor E. C. Pua)
Although seeds have been the subject of extensive studies for many years, their seed coats are just beginning to be
examined from the perspective of molecular genetics and control of development. The seed coat plays a vital role in the life
cycle of plants by controlling the development of the embryo and determining seed dormancy and germination. Within the
seed coat are a number of unique tissues that undergo differentiation to serve specific functions in the seed. A large
number of genes are known to be specifically expressed within the seed coat tissues; however, very few of them are
understood functionally. The seed coat synthesizes a wide range of novel compounds that may serve the plant in diverse
ways, including defense and control of development. Many of the compounds are sources of industrial products and are
components of food and feeds. The use of seed coat biotechnology to enhance seed quality and yield, or to generate novel
components has not been exploited, largely because of lack of knowledge of the genetic systems that govern seed coat
development and composition. In this review, we will examine the recent advances in seed coat biology from the
perspective of structure, composition and molecular genetics. We will consider the diverse avenues that are possible for
seed coat biotechnology in the future. This review will focus principally on the seed coats of the Brassicaceae and Fabaceae
as they allow us to merge the areas of molecular biology, physiology and structure to gain a perspective on the possibilities
for seed coat modifications in the future.
Key words: biotechnology; Brassica; genes; legume; seed coat.
opportunity for enhancing existing seed traits and for adding value
to seeds.
Fundamental to the study of seed coat developmental genetics is
a comprehensive understanding of seed coat morphology. Seed
coats develop from the integuments that surround the ovule prior to
fertilization. Before fertilization, cells of the integuments are
relatively undifferentiated. However, development after fertilization
can include extensive differentiation of the cell layers into
specialized cell types. In addition, some cell layers in the seed coats
may accumulate large quantities of certain substances, such as
mucilage or pigments that can also contribute to overall seed
A number of cell types are found in common in seed coats of both
the Brassicaceae and Fabaceae. Some cell layers will not undergo
any significant differentiation and will remain parenchymatous.
These are often crushed at maturity. Other cells will undergo a
slight thickening of the cell wall and thus become collenchymatous.
Some cell layers will undergo extensive secondary thickening of
some parts of the cell walls and will, thus, become sclerotic. These
layers are often called palisade layers, especially if the cells also
become elongated in the radial plane. Figure 1 demonstrates the
positioning of different cell layer types in Arabidopsis and soybean.
By harboring the embryo, seed coats separate one generation of
plants from the next and ensure the survival of the offspring. Strong
Seeds have played a fundamental role in the development of
civilizations by supplying food, feed, natural products, and
traditional medicines. The application of genetics resulted in
breeding for quality and yield and the adoption of agricultural and
industrial processes for harvesting their valuable components.
Because cultivated seeds remain a vital link to health and
prosperity, acquiring knowledge of plant seed biology has been a
priority for most cultures. Today years of research have generated
new domesticated varieties of plants that have diverged
considerably from their wild ancestors in form and traits, yet we
still understand little about the biological processes that govern
these valuable traits. It is a goal of agricultural research to
accelerate the development of seed quality and yield and to
diversify their traits to satisfy more of our needs. Technologies such
as genomics, proteomics, and metabolomics promise to accelerate
our understanding of seeds and thus open new possibilities for uses.
Emerging technologies for generating transgenic plants provide an
*Author to whom correspondence should be addressed: Email [email protected]
†The authors have contributed equally and are considered first authors.
In this review, we will examine the recent literature on seed coats
to review our understanding, particularly at the level of molecular
biology, and to assess the potential for new seed coat modifications
and uses. We will pay attention to the legume seed, which
represents a well-established system for studying metabolic control
mechanisms, and Arabidopsis, which is the major system for
understanding genetic control mechanisms. Finally, we will
examine a wide range of novel compounds found in the seed
coats of diverse plants and consider the biotechnological
applications for diversifying our use of seed coats.
Role of Seed Coats in Plant Development
FIG . 1. Schematic diagrams illustrating the general organization of the
seed coat of Arabidopsis thaliana and soybean (G. max L. Merrill). a, A.
thaliana. The seed coat at the torpedo stage. mu, Mucilaginous epidermal
cells; p, palisade layer with thickened inner cell walls; pa, parenchymatous
cells; en, endothelium layer. The mucilaginous cells and the palisade layer
comprise the outer integument while the inner integument consists of
parenchymatous cells and the endothelium layer. Adapted from Beeckman
et al. 2000; b, Soybean. A mature seed coat. p, Palisade layer; h, hourglass
cells; pa, partially crushed parenchyma; a, aleurone; em, crushed endosperm
The palisade layer and the hourglass cells comprise the outer integument
while the inner integument consists of parenchymatous cells. Adapted from
Miller et al. (1999).
impermeable seed coats protect the embryos during dormancy and
maintain an environment around the embryo that is conducive for
quiescence (Bewley, 1997). During germination the seed coat must
weaken and break open and may provide components that
contribute to biotic- and abiotic-stress resistance. Recently, a
large body of research has shown that the seed coat is vital for
directing the nutrient supply to the embryo during seed
development (Weber et al., 2005). By governing seed dormancy
and germination the seed coat plays an important role in
determining the optimal environmental conditions for the viability
and growth of the next plant generation. An understanding of these
roles is essential for developing crops suitable for agricultural
production. The seed coat is also a rich source of many valuable
naturally occurring compounds and the use of transgenic
technologies can greatly expand and diversify them. Generally,
the seed coat has not been characterized at the molecular level to
the extent of the embryo and endosperm. It is now recognized that
such knowledge is a prerequisite for the development of plants with
modified seed coat traits.
Although the seed coats of different species vary greatly in
structure and composition, they undergo similar phases of
development in relation to the embryo and endosperm. For
example, in legume seed development the seed coat and endosperm
develop first, followed by the development of the embryo,
maturation of the seed coat, and maturation of the embryo (Weber
et al., 2005). This will be discussed later in detail for Arabidopsis.
The coordination of these events is governed by communication
among the tissues of the seed organs. For example, communication
between the seed coat and endosperm of Arabidopsis has shown to
be particularly important by targeted cell ablation experiments
(Weijers et al., 2003). Early embryo development and differentiation is controlled by the maternal tissues, therefore signals must
be transmitted through the seed coat and endosperm before they can
reach the embryo. For example, specialized cell types called
transfer cells facilitate the transfer of nutrients within the seed
(Thompson et al., 2001). An elaborate model for the maternal
control of embryo development through sugar metabolism has been
developed and well discussed in the literature (Wobus and Weber,
1999; Weber et al., 2005). Such models must also integrate a variety
of other signaling pathways that involve phytohormones (Bewley,
1997), hypoxia (Rolletschek et al., 2002) and carbon dioxide
recycling (Furbank et al., 2004) to mention a few. The
phytohormone and sugar response pathways are known to converge
in seed development and are well studied (Gibson, 2004). Cross talk
among the various pathways must play a major role in the control of
seed development and provide mechanisms for communication
among the various seed organs, which undergo coordinated
development. A number of recent reviews examine these in detail
(Olszewski et al., 2002; Gibson, 2004; Weber et al., 2005). As we
will show later, there is a wide range of seed coat morphologies and
compositions, therefore the details of these processes will likely
differ among species.
Metabolic Controls
Seed coat invertases of legumes play a central role in the maternal
control of seed development. Cell wall invertases facilitate assimilate
unloading by increasing the sucrose gradient in the unloading zones of
the legume seed coat (Weber et al., 1995). The high hexose-to-sucrose
ratios are believed to promote embryo growth by enhancing cell
division (Borisjuk et al., 1998). This may be mediated through sensing
pathways that use D-type cyclins (Weber et al., 2005). The growth of
the embryo within the confines of the legume seed coat is correlated
with the crushing of the inner seed coat cell layers and loss of cell wall
invertase activity and the assimilate supply (Weber et al., 1995).
The expression of yeast invertase in transgenic Vicia narbonensis to
alter the sugar composition in the seed led to alterations in embryo
development and partitioning of carbon into starch as a storage
product (Weber et al., 1998). Data from transgenic tobacco with
artificially prolonged invertase activity revealed no comparable
alterations in development or carbon partitioning to storage lipids,
indicating that the switch in metabolism to storage product
accumulation in the seed is not simply a direct response to the
elevated hexose to sucrose ratio conferred by the invertase activity in
all plants (Tomlinson et al., 2004).
Dormancy and Abscisic Acid
Through seed dormancy the progression of the embryo into a
seedling or plantlet is arrested. This process plays a vital role in the
plant life cycle by allowing progeny to survive adverse
environmental conditions and to coordinate their growth with the
most favorable conditions. In domesticated crops, this is less
necessary and has been eliminated by breeding (Bewley, 1997). The
seed coat is a major determinant of dormancy, particularly in
species with seed coat-imposed dormancy in which the embryo is
physically constrained from developing further. This differs from
embryo dormancy where the embryos of certain species are
dormant. A critical interplay between the seed coat and embryo
occurs that involves the phytohormone abscisic acid (ABA), which
reaches the seed in two phases. In Nicotiana, studies with ABA
mutants revealed that the first phase of ABA is synthesized in the
maternal vegetative tissues and translocated to the seed to initiate
early seed development. The second phase, which is needed for
seed coat maturation, is synthesized in the seed coat itself (Frey
et al., 2004). Similarly, ABA produced in the embryo was needed to
complete embryo dormancy in Arabidopsis (Karssen et al., 1983)
after the arrest of embryo growth (Raz et al., 2001). Mutations in the
ABA synthetic genes can result in major disruptions in seed
development, including dormancy, and, reciprocally, mutations in
seed coat development can result in defects in dormancy (reviewed
by Finkelstein et al., 2002). Control of seed coat development and
dormancy are therefore fundamentally linked.
The signaling networks controlled by ABA in Arabidopsis are very
extensive and it has been estimated that 8 – 10% of the Arabidopsis
genes on a partial chip are ABA-responsive (Finkelstein et al.,
2002). Many of the ABA responses associated with embryo
maturation and stress responses also occur in the seed coat.
Interestingly, an Arabidopsis orthologue of the ABA-responsive gene
rab 28 which is a late embryogenesis abundant protein (LEA), Atrab
28, is expressed selectively in the outer integuments of the seed
coat, embryos and silique epidermis but is not responsive to ABA in
somatic cells (Arenas-Mena et al., 1999). The storage proteins
which accumulate in the embryo during ABA-induced maturation
also accumulate in the seed coats of legumes where they may act as
deterrents to certain predators and provide selection pressure for
the evolution of pests (Silva et al., 2004).
Germination and Gibberellin
Germination, which involves factors that break dormancy, is
initiated with the imbibition of water and resumption of respiration
and metabolic activity. Gibberellin signaling appears to play a role in
this activity and may act antagonistically to ABA (Bewley, 1997).
In soybean, which has a very hard, impermeable seed coat, water
enters through small cracks in the seed coat (Ma et al., 2004a).
Germination is completed once the radicle breaks through the seed
coat. In species with coat-enhanced dormancy, such as Nicotiana,
the constraints imposed by the seed coat and underlying endosperm
must be weakened enzymatically. This occurs sequentially through
the rupture of the seed coat followed by rupture of the endosperm. It
appears that enzymes such as class I b-1,3-glucanase I (bGluI) are
involved in both processes by weakening of the cell walls (LeubnerMetzger, 2002). De novo expression of bGluI in the inner seed coat of
dry seeds appears to be among the first steps in releasing coatenhanced dormancy (Leubner-Metzger, 2005). The signals for the
induction of bGluI appear to involve the decline in ABA and the
onset of GA signaling (Olszewski et al., 2002). Genes coding for other
cell wall-weakening enzymes, such as a-amylase, have been shown
to be induced in seed coats of morning glory coordinately with the
induction of gibberellin 3-oxidases (Nakajima et al., 2004). Control
of GA:ABA balance is likely to have an effect on the embryo in
addition to the seed coat, in that GA may stimulate the resumption of
embryo growth to achieve radical protrusion (Debeaujon and
Koornneef, 2000). In species such as radish and soybean, reactive
oxygen intermediates such as superoxide radicals, hydrogen
peroxide, and hydroxyl radicals are synthesized by the seed coat
and embryos as by-products of the metabolic processes that
accompany germination and presumably provide resistance to
pathogens during seedling emergence (Schopfer et al., 2001).
Oxidases such as peroxidases, oxylate oxidases, or amine oxidases
can be potential sources of hydrogen peroxide. Peroxidases are
commonly found in seed coats, particularly soybean seed coats
(Gijzen, 1993; Welinder and Larsen, 2004) and seed coat-specific
forms of oxylate oxidase have been found in barley (Wu et al., 2000).
Structure and Development of Brassicaceae Seed Coats
The seed coats of the Brassicaceae are becoming very important
for understanding the genetic mechanisms that govern seed coat
development, particularly with the emergence of Arabidopsis as a
model genetic system for plants (Kuang et al., 1996; Beeckman
et al., 2000; Western et al., 2000; Windsor et al., 2000). In the past,
the seed coat has also served as a useful characteristic for taxonomy
(Vaughan and Whitehouse, 1971; Bouman, 1975; Zeng et al., 2004).
Today, it is understood that the family includes a large number of
important crops and that seed coat characteristics are associated
with important agronomic traits, motivating studies on seed coat
structure and development (Bouman, 1975; Van Caeseele et al.,
1981, 1982; Kuang et al., 1996; Beeckman et al., 2000; Western
et al., 2000; Windsor et al., 2000).
From these studies, a common seed coat structure has been
identified within the Brassicaceae, consisting of four distinct layers
(Vaughan and Whitehouse, 1971; Bouman, 1975). The outermost, or
epidermal layer of the outer integument is most frequently one celllayer thick and may or may not contain mucilage (Vaughan and
Whitehouse, 1971; Bouman, 1975). Below the epidermal layer, in
the middle of the outer integument, is a subepidermal layer that may
consist of one, or more cell layers and is typically parenchymatous,
although it may be collenchymatous, or sclerotic (Vaughan and
Whitehouse, 1971; Bouman, 1975). In some species, including
Arabidopsis, this cell layer is entirely absent. The innermost layer of
the outer integument is most often one-cell thick and forms a sclerotic
layer, sometimes known as the palisade layer. This layer is often
characterized by thickened inner tangential and radial walls
(Vaughan and Whitehouse, 1971; Bouman, 1975). These first three
layers are derived from the outer integument of the ovule, while the
fourth layer is derived from the inner integument. The fourth layer,
formed of parenchyma cells compressed at maturity, is referred to as
the pigment layer, as pigments most often accumulate within this
layer (Vaughan and Whitehouse, 1971; Bouman, 1975). At maturity,
the outer layer of the endosperm is closely associated with the inner
integument and forms an aleurone layer (Bouman, 1975).
The epidermal layer of Brassicaceae seed coats may contain
mucilage, a pectic polysaccharide that may contribute to seed
hydration and seed dispersal. In Arabidopsis, mucilage can
contribute to efficient germination and seedling establishment
when the seeds are in an environment of low water potential
(Penfield et al., 2001). Mucilage is present in a dehydrated form in
the mature seeds, but, upon imbibition, it expands, rupturing the
outer cell wall of the epidermal cells enveloping the entire seed.
Mucilage is also present in the seed coats of some Solanaceae,
Linaceae, and Plantaginaceae species. In some agricultural species,
such as yellow mustard, mucilage is beneficial, as it contributes to
the quality of the final product. In other species, the presence of
mucilage is an economical disadvantage, as it impedes the removal
of the seed coat during processing.
Another important feature of Brassicaceae seeds is the presence
of flavonoids found in the inner integument. The role of flavonoids in
plants is diverse and includes protection against UV-B light,
regulation of auxin transport, signaling between plants and
microbes, male fertility, and plant defense through antimicrobial
activity and decreased palatability (Winkel-Shirley, 2001).
Furthermore, flavonoids have recently gained importance for their
role in preventing bloat and controlling internal parasites in
ruminants (Aerts et al., 1999). Some flavonoids are also known to
have antioxidant activity and may, therefore, be beneficial for
human health (Ross and Kasum, 2002). In seeds, flavonoids have
important functions in the induction of seed coat-imposed
dormancy, as well as in seed longevity and quality (Debeaujon
et al., 2000). In many Brassicaceae species, the absence of
flavonoids in yellow-seeded cultivars is correlated with greater oil
yield, higher protein content, and a reduction in undesirable
characteristics such as fiber content (Simbaya, 1995).
The mature seed coat of Arabidopsis is mostly composed of five
cell layers of maternal origin (Beeckman et al., 2000). The outer two
cell layers of the seed coat, which are derived from the outer
integument, form the epidermal and palisade layers. A subepidermal parenchyma layer is absent in the Arabidopsis seed coat. The
inner integument has three cell layers, although the middle layer is
only present in the curving body, such that the inner integument is
composed of two cell layers near the chalaza and micropyle
(Beeckman et al., 2000).
Outer and inner integuments. In Arabidopsis, two integuments,
both epidermal in origin, initiate close to the chalaza and grow to
surround the ovule (Robinson-Beers et al., 1992; Schneitz et al.,
1995). The outer integument has two cell layers with prominent
vacuoles (Schneitz et al., 1995). Cells of the outer layer of the outer
integument are wider on their convex side, which, in addition to the
greater proliferation of cells on this side, contributes to the
curvature of the integument. Furthermore, the convex side of the
ovule experiences a more rapid growth of the cells of the outer
integument, which further increases the curvature (Robinson-Beers
et al., 1992; Schneitz et al., 1995). This asymmetrical growth of the
outer integuments initiates the abaxial – adaxial axis of the
developing ovule (Balasubramanian and Schneitz, 2002).
Initially, the inner integument is also composed of two cell layers
(Schneitz et al., 1995). The outer layer of the inner integument
undergoes vacuolization and ultimately appears most similar to the
two cell layers of the outer integument (Schneitz et al., 1995). The
inner layer of the inner integument, however, develops into a
distinctive endothelium, which is closely connected to the embryo
sac. The endothelium has cuboid cells with little vacuolization and
a compact appearance (Schneitz et al., 1995).
Just prior to fertilization, a third cell layer of the inner
integument is initiated and becomes the middle layer of the inner
integument. This new layer, however, only surrounds part of the
embryo sac (Schneitz et al., 1995). Ultimately, these cells are also
vacuolated, to an even greater extent than the outer integument and
the outer layer of the inner integument (Schneitz et al., 1995).
The epidermal layer. Following fertilization, cells of the outer
integument initially have a large central vacuole (Beeckman et al.,
2000; Western et al., 2000; Windsor et al., 2000). Both layers also
begin to accumulate starch grains early in development, around the
globular stage of embryo development (Beeckman et al., 2000;
Western et al., 2000). They accumulate near the outer cell wall in the
outer layer and near the inner cell wall of the inner layer (Windsor
et al., 2000). The outer cell layer, or epidermis initially increases in
size immediately following fertilization, as does the central vacuole.
Around the torpedo stage, the epidermal layer begins to produce
mucilage, which is deposited between the primary radial cell wall and
the protoplasm. The mucilage is pectinaceous and mostly composed
of unsubstituted rhamnogalacturonan I (RGI), with rhamnose and
uronic acid being the main sugars (Goto, 1985; Penfield et al., 2001).
Other monosaccharides are present in lower ratios. As mucilage
deposition continues, the protoplasm is compressed into a column
located in the center of the cell, a structure known as the columella
(Western et al., 2000; Windsor et al., 2000). The primary cell wall
remains attached to the plasma membrane along the inner tangential
wall, as well as the center of the outer tangential wall and the inner
sections of the radial walls. As the protoplasm continues to compress,
the vacuole shrinks and disappears. The starch grains also begin to
disappear, coincident with the deposition of a secondary cell wall
around the columella (Western et al., 2000; Windsor et al., 2000).
This temporal sequence likely indicates that the starch granules are
the precursors to the polysaccharides required for the reinforcement
of cell walls. At maturity, the columellae appear to entirely consist of
cell wall material and spread along the inner tangential wall and up
parts of the radial walls, providing reinforcement in these areas
(Western et al., 2000). The cytoplasm is typically no longer
observable in this layer (Kuang et al., 1996; Western et al., 2000).
At maturity, the mucilage has dried into a thin, compressed layer
under the primary cell wall, causing the cell wall to drape over the
columella (Western et al., 2000; Windsor et al., 2000). During
imbibition, the tangential primary cell walls likely break first as the
mucilage swells, while the radial walls and the top of the columellae
remain attached to the outer wall (Western et al., 2000; Windsor
et al., 2000).
The palisade layer. In the inner layer of the outer integument,
known as the palisade layer, the starch granules first enlarge, while
the central vacuole divides into two or three smaller vacuoles. As
the starch granules begin to shrink during the torpedo stage, the
inner tangential cell walls are thickened or reinforced (Beeckman
et al., 2000; Western et al., 2000; Windsor et al., 2000). Again, the
starch granules likely contribute to cell wall reinforcement. At
maturity, the palisade layer is largely collapsed, although it may
persist in some areas (Beeckman et al., 2000).
The pigment layer. At fertilization, the innermost layer of the
inner integument, known as the endothelium, is composed of
isodiametric cells with dense cytoplasm and few starch grains
(Kuang et al., 1996). Immediately following fertilization, this layer
then becomes vacuolated (Beeckman et al., 2000). During the onecell embryo stage, the endothelial cells form large central vacuoles,
which are subsequently filled with light yellow pigments during the
two-cell embryo stage. The majority of these pigments are
proanthocyanidins (PAs) (Devic et al., 1999). The pigments initially
fill the vacuole, but ultimately accumulate in the cytoplasm as well.
Pigment accumulation is maximal at the early torpedo stage of
embryo development. In the late torpedo stage, the pigment begins
to disappear from the center of the cells, remaining only at the
periphery (Beeckman et al., 2000). At maturity, the endothelial
layer is broken down, such that only dead cells remain, although
these may be crushed and indistinguishable from the layers above
(Beeckman et al., 2000).
The other two layers of the inner integument are highly
vacuolated, especially on the abaxial side of the seed (Beeckman
et al., 2000). Beginning in the bent-cotyledon stage, these layers
begin to shrink and disintegrate as the embryo grows (Kuang et al.,
1996; Beeckman et al., 2000). At maturity, these layers are
completely crushed and, with the remains of the inner layer, form a
brown pigment layer (Beeckman et al., 2000).
Flavonoids accumulate in all the three layers of the inner
integument (Debeaujon et al., 2000). At maturity, the seed coat is
desiccated, allowing the oxidation of the flavonoids found in these
layers. The oxidation process causes the flavonoids to turn brown,
which is the source of the color of the mature seed coat.
Other Brassicaceae
Many Brassica species have a seed coat structure that is similar
to that of Arabidopsis. Seed coat development also proceeds through
many of the same steps.
Brassica campestris. The outer integument of the seed coat of
Brassica campestris L. cv. Candle has been studied in great detail
(Van Caeseele et al., 1981, 1982). The palisade layer develops a
secondary thickening of the inner tangential cell wall throughout
development, much like Arabidopsis (Van Caeseele et al., 1982).
Unlike Arabidopsis, some parts of the radial cell walls are also
thickened, becoming progressively thinner as they approach the
outer tangential cell walls. As thickening occurs, the inner
tangential cell walls of the palisade layer become closely associated
with the outer tangential wall of the outer layer of the inner
integument (Van Caeseele et al., 1982). The palisade cells elongate
along the radial plane, in regions that have retained the primary cell
wall, and these areas are associated with numerous vacuoles (Van
Caeseele et al., 1982). At maturity, sections of the radial walls
without secondary thickening collapse into folds. The thickened
tangential walls appear fused to the adjacent region of the inner
integument (Van Caeseele et al., 1982).
B. campestris, like Arabidopsis, has a mucilaginous epidermal
layer; however, unlike Arabidopsis, the epidermal cells do not form
the distinct columella structure and, at maturity, this layer is
compressed, much like the parenchyma cells of the inner
integument (Van Caeseele et al., 1981). Initially following
pollination, there is little change in the epidermal cell structure,
with the exception of the amyloplasts, which further develop starch
grains. However, by 15 d after pollination, degraded organelles,
membranes, and possibly endoplasmic reticulum and tonoplasts are
sometimes visible in the cytoplasm. As the compression of the
epidermal layers continues, mucilage becomes deposited and
appears stratified, likely as a result of layered deposition (Van
Caeseele et al., 1981). The cytoplasm becomes strongly
disorganized by 25 d after pollination and the radial cell walls of
the epidermis begin to collapse. Ultimately, cytoplasm is no longer
visible and mucilage deposition is completed, forming a meniscuslike structure in the middle of each elongated cell. Along the inner
tangential wall, the mucilage appears to be thickest but the entire
lumen of the cell is filled (Van Caeseele et al., 1981). At maturity,
the outer tangential cell walls of the epidermal layer are easily
Capsella bursa-pastoris. The mature seed coat of C. bursapastoris is almost identical to that of Arabidopsis. It has a
mucilaginous epidermal layer that forms central columns (Bouman,
1975). The subepidermal layer is absent and the outer integument
remains two-cell layered throughout development. Adjacent to the
epidermal layer, therefore, is the palisade layer, which is sclerotized
(Bouman, 1975). A minor difference from Arabidopsis is that both
the inner tangential cell walls and all of the radial walls of the
palisade layer are secondarily thickened, and these cells are
elongated along the tangential plane (Vaughan and Whitehouse,
1971). All three layers of the inner integument are crushed at
maturity and partially resorbed, although the outermost two layers
are crushed before the pigmented endothelial layer (Bouman, 1975).
Sinapis alba and Brassica nigra. The seed coat of S. alba and
B. nigra are very similar with the exception of the inner integument,
which does not accumulate pigments in B. nigra (Bouman, 1975). In
these two species, the outer integument originates subdermally into
three-cell layers and even four-cell layers in some areas (Bouman,
1975). The epidermal layer is mucilaginous, although it does not
form central columns, as seen in Arabidopsis and C. bursa-pastoris
(Bouman, 1975). A subepidermal layer is present in the outer
integument and is formed of parenchyma cells that may appear
collenchymatous at times. Like C. bursa-pastoris, the palisade layer
displays secondary thickening of both the inner tangential and the
radial cell walls, although the cells remain isodiametric (Vaughan
and Whitehouse, 1971; Bouman, 1975). The inner integument has
two cell layers in some areas and multiple cell layers in others and
is completely crushed at maturity, such that cells are no longer
distinguishable (Bouman, 1975).
Lunaria annua. The seed coat of Lunaria annua exhibits a rare
vascularization within the innermost layer of the inner integument
(Bouman, 1975). The innermost layer, in this species, demonstrates
characteristics of an integumentary tapetum, which may play a
special role in nutrition in the seed (Bouman, 1975). The somewhat
mucilaginous epidermal layer of the L. annua seed coat is composed
of large cells that likely collapse at maturity. Cells of the palisade
layer are cubic and the walls are thickened on both the inner and
the outer tangential walls (Vaughan and Whitehouse, 1971;
Bouman, 1975). Both the subepidermal layer and the outer layers of
the inner integument are crushed and partially resorbed at maturity
(Bouman, 1975). The innermost layer, however, forming the
integumentary tapetum, develops into sclereids, with cell wall
thickening along the inner tangential wall and parts of the radial
walls (Bouman, 1975). On the dorsal side of the seed, the crushing
of the parenchyma layers leads to the formation of a seed wing
(Bouman, 1975).
Molecular Genetics of Brassicaceae Seed Coats
Several genetic systems specific to the seed coat in Arabidopsis
were first identified by mutations followed by the identification of
the genes. As a model, Arabidopsis has become very important for
understanding the genetic processes that govern development of the
seed coat and that are responsible for seed coat qualities, such as
mucilage production and pigment accumulation. These genes are
very important for future studies that aim to alter or modify seeds
through biotechnological strategies. It is assumed that the most
basic genetic regulatory systems governing Arabidopsis seed coat
development will also apply to the other Brassicaceae species;
however, each will have to be functionally assessed individually.
A list of some of the seed coat genes studied in Arabidopsis is given
in Table 1.
Genes Involved in Ovule Integument Development
Structure and patterning of the mature seed coat is often
dependent upon the correct initiation, growth, and differentiation of
the integuments. In Arabidopsis, the proximal – distal and abaxial –
adaxial poles found in the mature seed coat are formed during
integument development. Furthermore, after fertilization there is no
change in the number of cell layers in the seed coat, such that the
development of the seed coat is largely a process of differentiation
as opposed to proliferation. Therefore, the regulation of development of the integuments prior to fertilization can have profound
effects on final seed coat structure, composition, and function.
The control of ovule development has been reviewed in a number
of papers (Grossniklaus and Schneitz, 1998; Schneitz, 1999;
Skinner et al., 2004). The following section will focus on genes
specifically involved in the development of the integument, with an
emphasis on recent work highlighting the importance of these genes
for future seed and seed coat development.
Integument identity. BELL1 (BEL1) is an Arabidopsis homeodomain protein that contributes to ovule integument identity (Reiser
et al., 1995). bel1 mutant plants produce only a single integumentlike structure (Robinson-Beers et al., 1992; Modrusan et al., 1994).
Ovule integument development
Reiser et al., 1995
Villanueva et al., 1999
TT18/ TDS4
Homeodomain protein
Member of the YABBY family of
putative transcription factors
Zinc finger domain protein
AP2 domain protein
Novel nuclear protein
Multidomain ribonuclease
Zinc finger domain protein
MYB domain protein
Dihydroflavonol 4-reductase
Chalcone synthase
Chalcone isomerase
Flavonone 3-hydroxylase
Flavonone 30 -hydroxylase
bHLH domain protein
Multidrug secondary transporter-like
MADS box protein
Leucoanthocyanidin dioxygenase
Putative glutathione S-transferase
WD40 repeat protein
WRKY domain protein
Anthocyanidin reductase
Plasma membrane Hþ-ATPase
bHLH domain protein
Homeodomain-leucine zipper protein
MYB domain protein
MYB domain protein
Putative NDP-L -rhamnose synthase
Homeotic regulatory protein
Putative asparaginyl endopeptidase
Flavonoid biosynthesis
Mucilage production
APETALA2 programmed cell death
Sakai et al., 1995
Elliott et al., 1996; Klucher et al., 1996
Schiefthaler et al., 1999
Golden et al., 2002
Sagasser et al., 2002
Nesi et al., 2001
Shirley et al., 1992
Feinbaum and Ausubel, 1988
Shirley et al., 1992
Wisman et al., 1998
Schoembohm et al., 2000
Nesi et al., 2000
Debeaujon et al., 2001
Nesi et al., 2002
Abrahams et al., 2003;
Shikazono et al., 2003
Kitamura et al., 2004
Walker et al., 1999
Johnson et al., 2002
Devic et al., 1999; Xie et al., 2003
Baxter et al., 2005
Zhang et al., 2003
Di Cristina et al., 1996
Penfield et al., 2001
Matsui et al., 2005
Usadel et al., 2004; Western et al., 2004
Jofuku et al., 1994
Nakaune et al., 2005
BEL1 may function by suppressing expression of AGAMOUS (AG), a
homeotic gene that promotes floral meristem, carpel and stamen
identity (Ray et al., 1994; Reiser et al., 1995). In the initial stages of
ovule development, BEL1 is restricted to the region from which the
integuments initiate. Although most bel1 mutants are female-sterile,
viable seed is produced occasionally and these seeds have altered
seed coat morphology, indicating the dependence of seed coat
structure on proper integument development (Modrusan et al.,
1994). bel1 mutants also have altered embryo sac development,
although it is not clear if this is a result of altered integument
development, or a pleiotropic phenotype of the bel1 mutant
(Robinson-Beers et al., 1992; Modrusan et al., 1994; Western and
Haughn, 1999).
Integument patterning. A number of genes expressed in the
integuments have been found to play important roles in ovule
patterning. These genes include BEL1, INNER NO OUTER (INO),
BEL1 and NZZ redundantly specify the chalazal region from which
the integuments initiate, contributing to the proximal – distal axis of
the ovule primordium (Balasubramanian and Schneitz, 2000). ANT
encodes an AP2 domain protein and its expression is limited to the
distal chalaza and funiculus just prior to integument initiation and
then to the integument primordial and distal funiculus once
integument cell divisions begin (Elliott et al., 1996; Klucher et al.,
1996; Balasubramanian and Schneitz, 2000). This spatial pattern of
expression indicates that it may play an important role in forming
the proximal – distal axis.
INO encodes a member of the YABBY family of transcription
factors, and mutations in this gene result in ovules with an inner
integument but no outer integument (Baker et al., 1997; Villanueva
et al., 1999). In wild-type plants, INO is expressed in the ovule
primordium in the cells that will give rise to the outer integument,
but only on the abaxial side and is, therefore, important for the
formation of the abaxial – adaxial axis (Villanueva et al., 1999). Also
important for the abaxial – adaxial axis is SUP, which encodes a zinc
finger transcription factor (Sakai et al., 1995). sup mutants develop
an outer integument that grows equally on both the abaxial and
adaxial side and thus may play a role in the maintenance of the
abaxial – adaxial axis as opposed to its initiation (Gaiser et al., 1995;
Sakai et al., 1995).
NZZ, in addition to specifying the proximal– distal axis, plays a
role in the regulation of INO expression, restricting it spatially to
the abaxial epidermis (Balasubramanian and Schneitz, 2000, 2002).
NZZ, which encodes a novel nuclear protein, may represent a
molecular link between the proximal –distal axis and the abaxial –
adaxial axis during ovule development (Schiefthaler et al., 1999;
Balasubramanian and Schneitz, 2002). A recent study found that
NZZ could directly bind INO, indicating an additional level of
regulation that may also contribute to the formation of the abaxial –
adaxial polarity (Sieber et al., 2004).
Morphogenesis and differentiation. INO also appears to function
in the growth of the outer integument, as weak ino mutants can
initiate an outer integument but the integument exhibits reduced
growth compared to wild type (Villanueva et al., 1999). Another
gene, SHORT INTEGUMENTS2 (SIN2) also plays a role in
integument growth by promoting cell division (Broadhvest et al.,
2000). SIN1, important for integument growth, is also known as
DICER-LIKE1 (DCL1). Mutations in SIN1 lead to the development
of integuments that are shorter than wild-type integuments, largely
as a result of reduced cell elongation as opposed to cell division
(Robinson-Beers et al., 1992). Recently, SIN1 was cloned and found
to encode a protein related to the Drosophila melanogaster gene
Dicer, which functions in RNA silencing and plays an important
role in animal development on a temporal scale (Golden et al.,
2002). Dicer encodes a multidomain ribonuclease that specifically
digests double-stranded RNA. Interestingly, sin1 mutants demonstrate defects in embryo development that are maternally inherited,
indicating that expression of SIN1 in the maternal sporophyte is
controlling some aspects of embryo development (Ray et al., 1996).
However, it is not clear if it is the expression of SIN1 in the
integuments or in the funiculus that ultimately affects embryonic
A mutant exhibiting heart-shaped seeds, as opposed to the wildtype oval shape was named aberrant testa shape (ats) (LéonKloosterziel et al., 1994). Microscopic analysis indicated that at the
apical end of the seed, ats mutants have only three cell layers, as
opposed to the five normally visible in wild-type seeds and, within
the three layers, there is no clear distinction between the outer and
the inner integument. In addition, ATS may also function in postfertilization development as the epidermal layer of the seed coat of
ats mutants is abnormal, composed of fewer larger cells compared to
wild type. These epidermal cells also produce very little mucilage
and the columellae are absent (Léon-Kloosterziel et al., 1994).
The ats mutation is maternally inherited and, therefore,
demonstrates the importance of the seed coat in determining seed
shape (Léon-Kloosterziel et al., 1994). Mutant ats plants also
exhibit reduced dormancy, supporting a role for the seed coat in
controlling this aspect of development (Léon-Kloosterziel et al.,
Genes Involved in Flavonoid Biosynthesis
The Arabidopsis seed coat has served as a model for the study of
flavonoid biosynthesis. This largely arose because Arabidopsis
mutants defective in flavonoid biosynthesis could be easily
identified by changes in seed color. Mature Arabidopsis seeds are
brown as a result of the accumulation and subsequent oxidation of
PAs, also known as condensed tannins, within the endothelial layer
of the seed coat. To date, 21 such mutants, known as transparent
testa (tt), have been identified. Two of these are known as
transparent testa glabra (ttg) because they also demonstrate a
hairless phenotype. An additional six mutants named tannindeficient seed (tds) have also been identified (Abrahams et al., 2002).
Many of these mutations affect the dormancy of the seeds,
indicating the important role of pigments in this aspect of
development (Debeaujon et al., 2000).
Flavonoid biosynthesis. Genetic analysis of the tt mutants has
identified a number of enzymes in the flavonoid biosynthesis
pathways. TT4 and TT5 encode chalcone synthase (CHS) and
chalcone isomerase (CHI), two enzymes at the beginning of the
biosynthesis pathways (Feinbaum and Ausubel, 1988; Shirley et al.,
1992). TT3 encodes dihydroflavonol 4-reductase (DFR), TT6
encodes flavonone 3-hydroxylase (F3H), and TT7 encodes flavonoid
30 -hydroxylase (F30 H) (Shirley et al., 1992; Wisman et al., 1998;
Schoenbohm et al., 2000). TT18 encodes leucoanthocyanidin
dioxygenase (LDOX) (Shikazono et al., 2003). tds4, the only tds
mutant characterized to date, was also found to be mutated at this
locus (Abrahams et al., 2003). Mutations in all of these genes also
affect anthocyanin production in vegetative tissues as the enzymes
are common to both the anthocyanin and PA pathways.
Arabidopsis plants with seeds exhibiting purple coloration were
found to have a mutation in the gene BANYULS (BAN) which
encodes anthocyanidin reductase (AR), an enzyme located at a
major branch point between anthocyanin and PA production (Devic
et al., 1999; Debeaujon et al., 2003; Xie et al., 2003). Besides BAN,
no other biosynthetic genes specific to the PA pathway have been
In Brassica carinata, yellow-seeded cultivars have reduced
pigments in the seed coat and the seeds have increased oil
concentrations and fiber, all of which are desirable agronomic traits
in this species (Simbaya et al., 1995). Comparing brown-seeded and
yellow-seeded cultivars of B. carinata revealed that the yellowseeded lines have reduced or absent expression of DFR, indicating
that this locus is important in determining pigmentation levels
(Marles et al., 2003a). The identification of this relationship has
important implications for the generation of yellow-seeded, low fiber
cultivars of B. napus, which has proven difficult in typical breeding
approaches (Marles et al., 2003a).
Regulation of flavonoid biosynthesis. Proteins that regulate the
flavonoid biosynthesis pathway are encoded by another set of TT
genes. These regulatory proteins include TTG1, a WD40-repeat
protein; TT1, a protein belonging to the WIP subfamily of zinc finger
proteins; TT2, an R2R3 MYB domain protein; TT8, a basic helix –
loop – helix (bHLH) domain protein; TTG2, a WRKY transcription
factor; and TT16, an ARABIDOPSIS BSISTER MADS domain
protein (Walker et al., 1999; Nesi et al., 2000, 2001, 2002; Johnson
et al., 2002; Sagasser et al., 2002). Another regulator subsequently
identified is ENHANCER OF GLABRA3 (EGL3), a bHLH domain
protein (Zhang et al., 2003).
These regulatory proteins control a number of the biosynthetic
genes discussed above. Largely through gene expression studies, it
has been determined that TT2, TT8, and TTG1 regulate DFR; TT2,
and TTG1 also regulate LDOX; and TT2, TT8, TTG1, and TT16
regulate BAN (Shirley et al., 1995; Nesi et al., 2000, 2001, 2002;
Debeaujon et al., 2003). In addition, some of these genes may play a
function in seed coat endothelial cell differentiation. For example,
tt16 and tt1 mutant endothelial cells both display aberrant
morphology, which suggests that they may regulate differentiation of
these cells as well as regulating PA production (Nesi et al., 2002;
Sagasser et al., 2002). ttg2 mutants have reduced seed size, due to a
reduced endosperm size and this phenotype is sporophytic maternal
(Garcia et al., 2005). The reduction in size is suggested to be the
result of reduced cell elongation in the integuments, possibly
resulting from changes in cell wall composition because of the PA
deficiency in these mutants (Garcia et al., 2005).
TTG1 appears to play an important role in the regulation of
epidermal cell morphogenesis in a number of tissues. Some recent
studies have demonstrated the interplay between TTG1, bHLH
domain proteins, and MYB domain proteins. In the control of seed
coat pigmentation, TTG1 interacts with the bHLH protein TT8 and
the R2R3 MYB protein TT2 (Zhang et al., 2003). There is evidence
that TTG1, TT8, and TT2 can form a ternary complex and that their
combined activity is required for maximum BAN expression (Baudry
et al., 2004). The bHLH proteins do not appear to be specific to one
function. TT8 also regulates anthocyanin production in vegetative
tissues, as does EGL3, a bHLH protein that functions with TTG1 to
regulate mucilage production (Zhang et al., 2003). However, there
is evidence that it is mainly through these bHLH proteins that TTG1
acts (Baudry et al., 2004). The MYB proteins, however, are often
associated with only one pathway and may account for the
specificity of each of the various pathways (Zhang et al., 2003).
TTG1 also regulates anthocyanin production, as well as trichome
and root development, and, for each of these functions, it appears to
interact with different combinations of bHLH proteins and MYB
proteins (Zhang et al., 2003).
Vacuolar transport. During Arabidopsis seed coat development,
pigments are stored in vacuoles and two other tt mutants apparently
have defects in machinery for the vacuolar sequestration of the PAs.
TT12 was found to encode a member of the multidrug and toxic
compound extrusion (MATE) family (Debeaujon et al., 2001). TT19
was found to encode a member of the glutathione S-transferase
(GST) gene family (Shikazono et al., 2003; Kitamura et al., 2004). In
addition, mutations in the gene AHA10, encoding a plasma
membrane Hþ-ATPase, were also found to result in a reduction in
seed coat PAs (Baxter et al., 2005). When all three of these genes
are mutated, multiple small vacuoles, as opposed to a single large
vacuole, accumulate, indicating that these mutants are also
defective in vacuole biogenesis (Kitamura et al., 2004; Baxter
et al., 2005).
Genes that are specific to the PA branch of flavonoid
biosynthesis, or that regulate this branch, are typically expressed
exclusively within the seed coat, particularly the endothelium.
These include the transcription factors TT1 and TT2 (Nesi et al.,
2001; Sagasser et al., 2002), the vacuolar sequestration proteins
TT12 and AHA10 (Debeaujon et al., 2001; Baxter et al., 2005), and
the biosynthetic enzyme BAN (Devic et al., 1999; Debeaujon et al.,
2003). This is likely due to the fact that PAs are only produced in
the seed coat in Arabidopsis plants. Accordingly, homologues of
many of these genes found in other species are not exclusively
expressed in seed coats (Winkel-Shirley, 2001; Marles et al.,
2003b). For example, the genetics of flavonoid biosynthesis have
also been studied in petunia and snapdragon flowers and maize
Genes Involved in Mucilage Production
As mentioned above, the epidermal cells of the Arabidopsis seed
coat produce mucilage that is excreted upon imbibition. Several
genes involved in this process have been identified.
TTG1, TTG2, TT8, EGL3 and GL2. Interestingly, a number of
mutants with defects in flavonoid biosynthesis also demonstrate
defects in the mucilage-secreting epidermal cells. These include
TTG1, TTG2, and TT8. TTG1 affects mucilage production in the
Arabidopsis seed coat, possibly through interactions with EGL3 and
a MYB domain protein (Koornneef, 1981; Penfield et al., 2001;
Zhang et al., 2003).
TTG1, EGL3 and the MYB domain protein may control mucilage
production through GL2 (Western et al., 2001, 2004). Mutations in
GL2, a homeodomain-leucine zipper protein, produce defects in
mucilage production that are very similar to those of ttg1 mutants
(Koornneef 1981; Di Cristina et al., 1996). Furthermore, GL2
expression in the seed coat is reduced in ttg1 mutants, supporting
the role of TTG1 in regulating GL2 (Western et al., 2004).
The role of TT8 in mucilage production was only identified
recently, through the analyses of double mutants. Mucilage
production is normal in tt8 mutants, partially reduced in egl3
mutants, but completely absent in tt8 egl3 double mutants, which
also demonstrate collapsed columellae (Zhang et al., 2003). This
indicates that TT8 and EGL3 likely have redundant roles in
mucilage production.
ttg2 mutants also lack mucilage and columellae (Johnson et al.,
2002). In the seed coats of ttg1 mutants, TTG2 expression is
reduced, suggesting that TTG2 acts downstream of TTG1 in
mucilage production (Western et al., 2004). TTG2 expression is not
affected by mutations in GL2, however, indicating that TTG2 and
GL2 may regulate mucilage production through independent
pathways (Johnson et al., 2002; Western et al., 2004). This is
supported by the finding that expression of MUM4, another gene
involved in mucilage production that is discussed below, is reduced
in ttg1 and gl2 mutants but not in ttg2 mutants (Western et al.,
MYB61. The myb61 mutant lacks mucilage extrusion and the
columellae have a reduced stature (Penfield et al., 2001). The seeds
have reduced levels of polysaccharides as well as rhamnose and
galacturonic acid, indicating a reduction in pectin levels (Penfield
et al., 2001). During development, mucilage deposition is abnormal,
with dense mucilage deposited only against the outer tangential cell
wall and the rest of the space filled with more diffuse mucilage
(Penfield et al., 2001). It has been suggested that MYB61 may
interact with TTG1 and EGL3 in the regulation of mucilage
production (Zhang et al., 2003). However, the myb61 mutant
phenotype is unique from that of the ttg1 mutant and myb61 ttg1
double mutants demonstrate an additive effect, indicating that they
may function in independent pathways (Pendfield et al., 2001).
Furthermore, as with ttg2 mutants, expression of MUM4 is not
affected in myb61 mutants, as it is in ttg1 and gl2 mutants, again
suggesting independent pathways (Western et al., 2004).
AtMYB23. Another MYB protein, AtMYB23, also regulates
mucilage production in epidermal seed coat cells. A chimeric
AtMYB23 repressor inhibited the deposition of mucilage in
transgenic Arabidopsis plants, in addition to a number of other
defects (Matsui et al., 2005). It has been suggested that AtMYB23
may be a better candidate than MYB61 for the MYB domain protein
that interacts with TTG1 and EGL3 to regulate mucilage production
(Western et al., 2004). This was supported in the study by Matsui
et al. (2005) by the finding that the AtMYB23 repressor was able to
suppress GL2 expression in Arabidopsis siliques but did not affect
expression of TTG1.
MUM1– 5. Five other mutants were identified because of the
absence of mucilage after imbibition and were named mum1 – mum5
(Western et al., 2001). mum1 and mum2 are deficient in mucilage
extrusion. The composition and amount of mucilage is comparable
to wild type, but upon imbibition, the mucilage is not excreted.
There is, however, an increase in the levels of methylation of the
mucilage and possibly the cell walls, as well. This increased
methylation, or some other as yet unidentified alteration, likely
alters the cell walls, or the mucilage, preventing release of mucilage
upon imbibition (Western et al., 2001).
mum4, as well as ttg1 and gl2, have reduced peaks for
galacturonic acid, rhamnose, fucose, and galactose, the major
components of pectin, as well as a reduction in the overall amount
of mucilage produced (Western et al., 2001). The secondary cell
wall in these three mutants is deposited, but in a peaked dome over
the vacuole, as opposed to the volcano-shaped deposition visible in
wild-type cells. These mutants appear to have defects in
cytoplasmic rearrangement and possibly construction, as well as
defects in mucilage production (Western et al., 2001).
MUM4, also known as RHM2, has been cloned and found to
encode a putative NDP-L -rhamnose synthase, required for the
synthesis of RGI (Usadel et al., 2004; Western et al., 2004). MUM4
belongs to a family of putative nucleotide sugar interconversion
factors, which includes two other members, RHM1 and RHM3.
MUM4 gene expression was found to be regulated by AP2, TTG1,
and GL2, which is also supported by the fact that mutations in all of
these genes result in very similar seed coat epidermal cell mutant
phenotypes (Western et al., 2004).
mum3 and mum5 have mucilage with altered staining properties
(Western et al., 2001). Reduction in the levels of rhamnose and
fucose, or other defects, may affect the basic structure of the
mucilage in these mutants, and, therefore, its branching and crosslinking properties (Western et al., 2001).
Mutations in the APETALA2 (AP2) gene give rise to many
different phenotypes, resulting from defects in many different
aspects of flower and ovule development, including mucilage
production. Epidermal cells of ap2 mutants show altered
morphology, including lack of a columella, larger size, and irregular
shape (Jofuku et al., 1994; Western et al., 2001). A study of the
development of the seed coat in ap2 cells indicates that the outer
integument begins development normally, but halts before mucilage
production and cytoplasmic rearrangements begin. At maturity, the
pigmented layer is the only discernable cell layer in the seed coats.
The epidermal and palisade layers appear absent, and only crushed
remnants of the cells surround the pigmented layer (Western et al.,
The AP2 gene was first cloned by Jofuku et al. (1994), and found
to encode an unknown protein with a number of interesting
domains. These include a serine-rich acidic domain, a putative
nuclear localization signal, and two copies of a 68 amino acid direct
repeat. The last domain was named the AP2 domain and can be
found in transcription factors in many different plant species
(Jofuku et al., 1994). The AP2 domains may be able to form
amphipathic a-helical structures that could potentially mediate
protein – protein interactions (Jofuku et al., 1994).
AP2 is a gene of particular interest because recent studies have
indicated that ap2 mutant seeds demonstrate increased seed mass
accompanied by increases in both total seed protein and total seed
oil (Jofuku et al., 2005; Ohto et al., 2005). This is unusual because
typically total seed protein and total seed oil vary inversely. ap2
embryos have altered cell number and cell size, which contributes
to the overall increase in seed mass (Ohto et al., 2005). AP2 may
affect seed mass by controlling source-sink relations in the seed
(Jofuku et al., 2005; Ohto et al., 2005). Ohto et al. (2005) examined
hexose and sucrose accumulation in ap2 mutants and found that ap2
seeds accumulated higher levels of hexose that decreased more
slowly compared to wild-type seeds (Ohto et al., 2005). It has been
suggested that the changes in hexose to sucrose ratios may result in
extended cell division, leading to the increase in embryo size (Ohto
et al., 2005). AP2 controls seed mass through the maternal
sporophyte and endosperm (Jofuku et al., 2005; Ohto et al., 2005).
However, there is some evidence that AP2 activity in the maternal
sporophyte, potentially including the seed coat, may be more
important than endosperm AP2 activity in controlling seed mass
(Ohto et al., 2005).
Genes Involved in Programmed Cell Death
As described above, many of the seed coat layers of Arabidopsis,
and other Brassicaceae species, are compressed at maturity, as a
result of the expansion of the embryo. Cells of the outer two layers of
the inner integument are compressed during seed development in
Arabidopsis, such that the seed coat diameter decreases by at least
half by maturity (Nakaune et al., 2005). DAPI staining indicates
that cells in these layers undergo programmed cell death while
being compressed (Nakaune et al., 2005). This process has not been
extensively studied in seed coats, but some initial studies have
highlighted the importance of cysteine proteinases.
dVPE. A vacuolar processing enzyme, dVPE, from the cysteine
proteinase family, was found to be expressed exclusively in the
outer two layers of the inner integument of the Arabidopsis seed coat
(Nakaune et al., 2005). Its expression increases rapidly and
transiently during early embryogenesis, coordinate with the process
of programmed cell death. dVPE is likely synthesized as an inactive
protein and was subsequently found to be inhibited by caspase-1
inhibitors, indicating that it has caspase-1 like activity (Nakaune
et al., 2005). These results indicate that dVPE may be involved in
the process of programmed cell death in the inner integument
(Nakaune et al., 2005).
BnCysP1. Cells in the inner integument of B. napus seed coats
also undergo programmed cell death (Wan et al., 2002). The gene
BnCysP1 encodes a protein with similarity to cysteine proteinases
and its expression is temporally correlated with programmed cell
death in the inner integument (Wan et al., 2002). Its similarity to
papaya papain indicates it may be synthesized as a preproprotein,
much like dVPE (Wan et al., 2002). These results again support a
role for a cysteine proteinase in the process of programmed cell
death in the seed coat.
Structure of Legume Seed Coats
The seed coats of legumes such as soybean, common bean and
pea are relatively large and complex. As discussed above, they are
well suited to the study of the physiological and biochemical
processes that govern embryo nutrition and composition (Rochat
and Boutin, 1991; Zeng et al., 2004; Wang and Grusak, 2005); for
example, the control of the aqueous and gaseous environment
around the embryo (Gijzen et al., 1999b; Souza and Marcos-Filho,
2001), or the protection of the embryo against pests and diseases
(Ndakidemi and Dakora, 2003). The morphological features of the
legume seed coat are relatively insensitive to environmental
conditions and therefore are sufficiently distinctive to be used for
taxonomy (Souza and Marcos-Filho, 2001). Yet, information on the
differentiation and development of specific cells and tissues of seed
coats is generally lacking. Soybean seed coat development is one of
the few that have been examined in detail (Miller et al., 1999).
The features of the mature seed coat have been described by
Corner (1951). Outermost is the epidermal layer, which consists of a
single layer of palisade cells (macrosclereids). They are elongated
perpendicular to the surface of the seed. Inside the palisade layer is
an hourglass cell layer, which is composed of thick-walled
osteosclereids. The innermost portion of the seed coat proper is a
multicellular layer of partially flattened parenchyma. The aleurone
layer is immediately inside the inner parenchyma. Each of the
above layers is maternally derived from the outer, or inner
integuments (Fig. 1). The next innermost layer is the endosperm,
derived by double fertilization. As shown in Fig. 1, it is tightly
compressed against the seed coat by the expansion of the embryo
cotyledons (Miller et al., 1999).
Recently, the seed coat structure of the legume Medicago
truncatula has been characterized (Wang and Grusak, 2005). It is
similar to that of soybean. It features an epidermal layer of
macrosclereids, a subepidermal layer of osteosclereids, and 2 – 5
rows of internal parenchyma cells. The parenchymal layer is
thinnest at the end of the seed coat opposite the hilum. As discussed
later, M. truncatula is an important model legume that may
facilitate the functional identification of legume genes involved in
seed coat development and composition.
Cuticle and Epidermal (Palisade Cell) Layer
The outermost layer of the legume seed coat is the waxy cuticle,
which represents the first barrier to imbibition. It is variable in
thickness. Actually, two layers of waxy deposits, one very stable and
the other environmentally labile are suggested for soybean seeds
(Ragus, 1987; Souza and Marcos-Filho, 2001). In several Glycine
species the membranous inner endocarp epidermis of the pod wall
detaches and adheres to the seed coat surface, becoming part of the
mature seed coat (Gijzen et al., 1999b).
The epidermis arises from the outer cell layer of the outer
integument (Zeng et al., 2004). It is composed of a layer of
thick-walled cells. Only one palisade layer is found, except under
the hilum, where two can occur. The external layer is called the
counter-palisade and originates from the funiculus (Souza and
Marcos-Filho, 2001). In Trifolium pratense L., the cytoplasm of the
macrosclereids contains small and medium-sized vacuoles and
several organelles such as mitochondria, rough endoplasmic
reticulum, and ribosomes. The cell vacuoles are completely or
incompletely filled with tannins, indicating that the macrosclereid
cells may play a role in the hardening of seed coats. Studies showed
that cell walls of the palisade layer contribute to the mechanical
strength of the seed coat (Algan and Büyükkartal, 2000).
Hourglass Cell Layer
The hypodermis is formed from a single layer of cells called
hourglass cells, pillar cells, osteosclereids, or lagenosclereids,
depending on their pattern of cell-wall thickness and shape. The
hourglass cells of the seed coat arise from the outer-cell layer of the
inner integument (Zeng et al., 2004). They are usually larger than
adjacent cell layers and are separated by wide intercellular spaces,
except under the hilum cleft where they are absent (Souza and
Marcos-Filho, 2001). The osteosclereid layer is composed of large
vacuolated cells and is densely cytoplasmic. The presence of
numerous starch grains in the hourglass cells during embryogenesis
indicates that the seed coat could synthesize nutrients for the
developing embryo (Algan and Büyükkartal, 2000; Wang and
Grusak, 2005). Like palisade cells, hourglass cell walls also play
a role in the mechanical strength of the seed coat.
The hourglass cells in the soybean seed coat are interesting in
that they appear to serve as a reservoir for proteins. A single
isozyme of seed coat peroxidase accumulates in large amounts (5%
total soluble protein) in the hourglass cell vacuoles (Gillikin and
Graham, 1991; Gijzen et al., 1993). Mutants (epep) deficient in this
peroxidase produced normal-looking hourglass cells and did not
under-perform compared with wild-type (EPEP) soybean varieties.
Parenchyma Layers
Adjacent to the hourglass cells is the interior parenchyma,
formed by 6 – 8 layers of thin-walled, tangentially elongated
parenchyma cells. They are uniformly distributed throughout the
seed coat, except in the area of the hilum, where a smaller number
of layers can be distinguished. In mature seed coats, the interior
parenchyma is often crushed or partially crushed (Miller et al.,
1999) as the embryo expands.
An important function of the seed coat is to deliver nutrients to
the embryo. In the parenchyma layer, three sublayers can be
discerned: chlorenchyma, ground parenchyma, and branched
parenchyma in pea (Pisum sativum L.). Light- and cryo-scanning
electron microscopy (cryo-SEM) from the late pre-storage phase to
the end of seed filling showed that solutes imported by the phloem
moved into the chlorenchyma and ground parenchyma, but not into
the branched parenchyma (van Dongen et al., 2003). It is believed
that chlorenchyma and ground parenchyma may be involved in the
post-phloem symplasmic transport of nutrients in the seed coat.
In the developing red clover seed coat, the parenchyma cells contain
numerous organelles, such as amyloplasts, rough ER, ribosomes,
plastids, dictyosomes, mitochondria, lipids, and protein bodies (Algan
and Büyükkartal, 2000), suggesting that this tissue is in a metabolically
active state. The ER system in the parenchyma cells suggests that these
cells may produce nutrients destined for the developing embryo. It was
shown that many intracellular spaces in the seed coat parenchyma are
filled with an aqueous solution, suggesting that it facilitates the
diffusion of nutrients from the site of unloading towards the cotyledons
(van Dongen et al., 2003). The parenchyma cells degenerate later and
become a major source of nutrients for the embryo.
It is uncertain whether the seed coat vascular system lies within
the parenchyma layer. It should be noted that seed coat vascular
systems, which are responsible for transporting the nutrients from the
maternal organs to the embryo, vary structurally in the legumes. Some
species possess extensive vascular systems that anastomose to form
reticulated networks throughout the entire seed coat; for example,
soybean. Other species have relatively simple vascular systems with
only a single chalazal vascular bundle and two lateral branches
extending into the seed coats; for example, M. truncatula. The
nutrients are almost exclusively imported through the phloem and
include the main organic nutrients, sucrose, and amino acids, as well
as potassium and micronutrients (Patrick and Offler, 2001). Sucrose
leaves the seed coat unaltered, but it is hydrolyzed by extracellular
invertases during the pre-storage phase. Amino acids imported via
the phloem undergo elaborate metabolic conversion in the seed coat
before release into the apoplast (van Dongen et al., 2003).
Aleurone Cell Layer and Compressed Endosperm Layer
As the seed coat matures, the endosperm cell layers adjacent to
the embryo degenerate and eventually appear as compressed wall
materials at seed maturity but the outermost endosperm layer
remains intact and differentiates into what has been known as the
aleurone layer, which is the only portion of the endosperm that is
clearly visible (Miller et al., 1999). The aleurone layer of soybean
and a few other legumes is known for its role in the enzymatic
mobilization of seed reserves, such as carbohydrates, during
germination (Ma et al., 2004b); however, its role during seed
development is not known. Yaklich et al. (1992) observed that
aleurone cells in a developing soybean seed have features of
secretory cells and speculate that they are able to convert
photosynthate into substances for export to the milieu (apoplast)
bathing the embryo during seed fill.
A mature soybean seed contains endosperm remnants as a
vestigially crushed layer inside the aleurone and by convention it is
considered to be part of the seed coat (Miller et al., 1999). The
endosperm is formed initially as a coenocytium around the young
embryo and starts to compartmentalize into cells when the embryo is
at its late globular stage (Chamberlin et al., 1994). Its function is to
serve as a nutrient source. The antipit of seed coat located in the
abaxial center of each cotyledon is composed of additional
endosperm cells situated between the aleurone layer and the
compressed endosperm tissue. It was postulated that the hilum of
the legume seed is a weak spot and, thus, can be a potential site for
bacterial invasion. However, Ma et al. (2004b) suggested that the pit
region might be resistant to pathogen attack due to the thick cuticle
along with thick periclinal walls of its epidermal cells.
Alternatively, this thickened area of seed coat endosperm might
have a physical role in helping to pin the embryo in position within
the seed coat (M. Gijzen, personal communication).
Molecular Characterization of the Legume Seed Coats
Legumes comprise one of the most important agricultural taxa
worldwide, providing a major source of protein and oil. Soybean
accounts for over 50% of the world oilseed production each year
and alfalfa (Medicago sativa) ranks high in acreage planted and
dollar value among forage crops (Cook, 1999; Harrison, 2000).
However, both have limitations for molecular genetic studies, such
as large genome sizes, abundant repetitive DNA, complex ploidy,
and difficulties with regeneration of transgenic plants for functional
studies of genes (Cook, 1999; Maguire et al., 2002). Arabidopsis, as
a model plant, has accelerated the development of plant genetics in
general but it is inadequate for addressing many of the legumespecific properties of legume seeds (VandenBosch and Stacey,
2003). M. truncatula and Lotus japonicus have been recommended
as more appropriate models (Harrison, 2000; Bell et al., 2001;
Young et al., 2003). Attributes of M. truncatula includes its small,
diploid genome (5 £ 108 bp); self-fertility; prolific seed production;
and rapid generation time (Cook, 1999). The legume information
system (LIS) ( has been set up
to integrate comparative genetic and molecular data from multiple
legume species, which include Medicago, Lotus, and soybean, and
Arabidopsis (Gonzales et al., 2005).
In the NCBI database, as of March 2005, researchers have
deposited 355 863 ESTs for Glycine max, 216 645 ESTs for M.
truncatula and 111 471 ESTs for Lotus corniculatus (http://www. It was shown that
2525 legume-specific ESTs contigs were found after comparing
unigene sets from Medicago, Lotus, and soybean with and rice and
Arabidopsis genomic sequences (Graham et al., 2004). Soybean seed
coat ESTs have also been generated and analyzed (Shoemaker et al.,
2002). In an immature seed coat cDNA library (Gm-c1019), 644
contigs were assembled, and the unigene percentage was 71%. In a
mature seed coat cDNA library (Gm-c1023), 188 contigs were
assembled and the unigene percentage was 82%.
The occurrence of a large number of seed-coat-specific genes was
confirmed by microarray analyses performed with different soybean
organs, such as seed coat, embryo, flower, and pod (Maguire et al.,
2002; Thibaud-Nissen et al., 2003). According to the NSF soybean
functional genomics website (http://soybeangenomics.cropsci.uiuc.
edu/files/NSFWeb Overview.pdf) 2163 unigenes from soybean seed
coat were identified. Alkharouf and Matthews (2004) have set up the
the soybean genomics and microarry database (SGMD), which
contains genomic, EST and microarray data with embedded
analytical tools, allowing correlation of soybean ESTs with their
gene expression profiles (
Legume Seed Coat Composition
Seed coats possess a wide assortment of novel compounds. Upon
imbibition, a complex mixture of chemicals is released from the
seed coat, which consists of flavonoids, proteins, peptides, amino
acids, alkaloids, terpenoids, steroids, etc. (Ndakidemi and Dakora,
2003). Of these, the isoflavonoids are best known for their role as
anti-microbial phytoalexins and phytoanticipins (Dakora and
Phillip, 1996). It is known that proanthocyanins and the
isoflavonoid, glycitin, contribute to resistance to legume weevils
by inhibiting their reproduction (Oigiangbe and Onigbinde, 1996).
Thus, many seed coat components play important roles in defense.
Moreover, the components of seed coat tissues affect the overall
quality and value of legume food or feed products, and are the
sources of novel compounds for industry and medicine. In this
section, we discuss legume seed coat composition and the
importance of some compounds to agriculture, food quality and
human health. A list of some of the genes involved in these
processes is given in Table 2.
Functions and uses. Polyphenolic compounds, including
phenolic acids and derivatives, tannins, and flavonoids, represent
the largest group of natural products in the plant kingdom. They
confer color to fruit and seed. They play a significant role in plant
disease resistance. They have diverse roles in plant development
and interactions with the environment (Salunkhe et al., 1982;
Harborne, 1988; Dixon and Paiva, 1995; Paiva, 2000). For example,
seed and seed coat phenolic compounds participate in nodulation
by acting as chemoattractants; promoting rhizobial growth; and
inducing transcription of nodulation genes in symbiotic bacteria
(Ndakidemi and Dakora, 2003).
The developing seed is a sink for many products synthesized in
other plant tissues. However, Dhaubhadel et al. (2003) demonstrated that IFS1 and IFS2, genes encoding 2-hydroxyisoflavonoid
synthase, are expressed not only in a variety of plant vegetative
organs, but also in developing embryos and seed coats. Moreover,
reciprocal crosses of cultivars, with high and low isoflavonoid
content revealed that the biosynthesis of isoflavonoids in maternal
tissues of soybean seeds may contribute substantially to total seed
isoflavonoid content at maturity (Dhaubhadel et al., 2003).
In recent years, polyphenols in many edible plant products have
received increasing attention due to their influence on the
nutritional and aesthetic quality of foods and pharmacological
implications. As they possess both antioxidant and antimutagenic
activity, they are implicated in the prevention of many diseases,
including atherosclerosis and cancer (Stavric, 1994; Karakaya and
Kavas, 1999; Hou, 2003; Martı́nez et al., 2003). The majority of
phenolic compounds found in the seeds of legumes are located in
the seed coat. Phenolics of the cotyledon and the seed coat of lentils
and peas have been used in studies of dietary intake of these
compounds (Bekkara et al., 1998; Wang et al., 1998; Dueñas et al.,
2002, 2004).
In soybean and common bean, the concentration of phenolic
compounds, such as flavonoids and anthocyanins, correlates with
seed coat color (Hungria and Phillips, 1993; Yaklich and BarlaSzabo, 1993; Nakamura et al., 2003; Benitez et al., 2004).
Legumes with dark seed coats, such as soybean, broad beans,
faba beans, lentils, and peas, possess high antioxidant activity
mainly due to the proanthocyanidins (Cardador et al., 2002;
Dueñas et al., 2002, 2004; Martı́nez et al., 2003). They are
natural sources of antioxidants that could replace the synthetic
antioxidants in foods.
Tannins, such as proanthocyanidins, might be less favored
because they form complexes that may inactivate enzymes, or
precipitate proteins, thereby, reducing food protein quality (Tan
et al., 1983; Cabrera and Martin, 1986). Furthermore, a high
content of tannins in sorghum was shown to be associated with a
marked degree of resistance to preharvest seed germination (Harris
and Burns, 1970). Tannins are also responsible for retarding
seedling growth by decreasing the rate of starch and protein
degradation in germinating high-tannin seeds, possibly by
inactivating hydrolytic enzymes (Salunkhe et al., 1982).
The accumulation of phenolics can decrease the agronomic value
of soybean due to increased cracking of the seed coat (Yaklich and
Barla-Szabo, 1993; Nakamura et al., 2003; Benitez et al., 2004).
Two types of cracking have been reported: Type I with irregular
cracks, and Type II with net-like cracks. Both result from separation
of epidermal and hypodermal tissues exposing the underlying
parenchyma tissue (Wolf and Baker, 1972; Yaklich and BarlaSzabo, 1993). Genetic control of cracking has been well
characterized and closely linked to the pigmented seed coat
which results from the accumulation of anthocyanin in epidermal
cells (Yaklich and Barla-Szabo, 1993; Nakamura et al., 2003;
Benitez et al., 2004).
Genetics of seed coat color. Several independent genetic loci,
including I, T, R and O, control the color and distribution of
pigments (Bernard and Weiss, 1973; Palmer and Kilen, 1987). The
best characterized I locus (for inhibitor) inhibits the production and
accumulation of anthocyanins and proanthocyanidins in the
epidermal layer of the seed coat. It consists of four alleles with
the following phenotypes: I results in complete absence of seed coat
pigment; i i limits pigment to the narrow hilum area where seed and
pod are attached; i k restricts pigment to a saddle-shaped region
over two-thirds of seed coat, and i results in a completely pigmented
seed coat (Bernard and Weiss, 1973). Most cultivated soybean
varieties are homozygous for a dominant form of the I gene resulting
in a yellow seed coat. However, spontaneous mutations from yellow
seed to dark-colored seed with i/i genotype arise frequently within
highly inbred soybean varieties (Wilcox, 1988).
Gene, Accession
Gene product
CHS1 BG509422
Glycine max
Chalcone synthase
CHS2 X65636
G. max
Chalcone synthase
Akada et al., 1991;
Shimizu et al., 1999
Akada et al., 1993a
CHS3 X16186
CHS4 X52097
CHS5 L07647
G. max
G. max
G. max
Chalcone synthase
Chalcone synthase
Chalcone synthase
CHS6 L03352
G. max
Chalcone synthase
CHS7 BE800791
G. max
Chalcone synthase
CHS8 AY237728
G. max
Chalcone synthase
SF3 0 H1 BF069512
G. max
G. max
G. max
CHIA1 AF202731
G. max
30 -hydroxylase
Proline-rich cell
wall protein 1 precursor
Proline-rich cell
wall protein 1 precursor
Chitinase class I
HPS AF100159
G. max
Hydrophobic seed
protein precursor
VfVINV1 Z35162
Vicia faba
Cell wall invertase I
SUS X98598
P. sativum
Sucrose synthesis
Flavonoid biosynthesis, response to
UV light and elicitors
Flavonoid biosynthesis, promoter contains
sugar response elements
Flavonoid biosynthesis
Flavonoid biosynthesis
Flavonoid biosynthesis, response to
UV-B irradiation
Flavonoid biosynthesis, response to
UV-B irradiation
Flavonoid biosynthesis, responsible
for seed coat pigmentation; promoter contains
regulatory element
Flavonoid biosynthesis, responsible
for seed coat pigmentation
Hydroxylation of 30 position in B-ring
of flavonoids, control of pubescence color
Developmentally regulated, with
organ-specific and stage-specific expression.
Possible role in plant development
Developmentally regulated, with
organ-specific and stage-specific expression.
Possible role in plant development
Expressed late in seed development, involved
in plant defense against
Protection against pathogens, or predators,
attachment of the endocarp to
the seed surface
Cleavage of the incoming sucrose within the
apoplast at a stage when mitosis
is active in the embryo
Sucrose synthesis
Seed coat
intrinsic protein
P. sativum
intrinsic protein
P. sativum
Tonoplast intrinsic
P. sativum
intrinsic protein
PsGNS2 AJ251646
P. sativum
SCB1 AY075133
G. max
SCS1 A276710
G. max
Seed coat
BURP domain protein
Putative subtilisin
G. max
Seed coat peroxidase
Aquaglyceroprotein which plays a role
in water absorption during seed
Aquaporin, expressed in the cotyledons
of developing and germinating seed, involved
in the release of water from
seed coat symplast
Aquaporin, expressed in the cotyledons
of developing and germinating seed, involved
in the release of water from
seed coat symplast
Only detected in seed coat. Encodes
aquaglyceroporin, involved in
water absorption and formation of
water and glycerol channels
Developmental regulation possibly
via callose hydrolysis, release of
oligosaccharides and/or degradation
of thin-walled parenchyma and
endosperm layers during development
Expressed in the developing soybean
seed coat, plays a role in the differentiation of
seed coat parenchyma cells
Expressed in thick-walled parenchyma,
is involved in a signal transduction, and
differentiation of soybean seed coat cells
Possibly defense via oxidative processes
Wingender et al., 1989
Akada et al., 1990
Akada et al., 1995;
Shimizu et al., 1999
Akada et al., 1993b;
Shimizu et al., 1999
Akada et al., 1993c;
Tuteja et al., 2004
Akada et al., unpublished;
Tuteja et al., 2004
Shoemaker et al. 1999a;
Toda et al., 2002
Shoemaker et al., 1999b;
Keller, 1993
Shoemaker et al., 1999c;
Keller, 1993
Gijzen et al., 2001
Gijzen et al., 1999b; 2003
Weber et al., 1995
Dejardin et al., 1997
Schuurmans et al., 2003
Schuurmans et al., 2003
Schuurmans et al., 2003
Schuurmans et al., 2003
Buchner et al., 2002
Bachelor et al., 2002
Bachelor et al., 2000
Gijzen, 1997
TABLE 2 Continued
MADS box
Gene, Accession
PRX2, AF039027
G. max
Leginsulin gene,
lup-leg1, U74383
P. sativum
Gene product
Function unknown
Gijzen et al., 1999a
Possible regulation of development of
seed coat
Signaling pathways for the transport
of nutrients to the embryo
Signaling pathways for the transport
of nutrients to the embryo
Buchner and Boutin, 1998
G. max
Cationic peroxidase
MADS box
transcription factor
At the molecular level the I locus has been extensively
analyzed and shown to be a naturally occurring duplication of
chalcone synthase (CHS) genes (Wang et al., 1994; Todd and
Vodkin, 1996; Senda et al., 2002a, b). CHS is the first
committed enzyme of the multibranched pathway of flavonoid/
isoflavonoid biosynthesis and it also plays a significant role in
the synthesis of secondary metabolites functioning as UV
protectants, phytoalexins, insect deterrents, and symbiosis
initiators in various plant tissues. Three of the seven members
of the multigene family (CHS1, CHS3, and CHS4) are located in
tandem within a 10-kb duplication region (Akada and Dube,
1995). Paradoxically, deletions of the CHS promoter sequences
allow higher levels of CHS mRNA accumulation and restore
pigmentation to the seed coat (Todd and Vodkin, 1996). The
restoration upon deletion points to the involvement of the
homology-dependent gene silencing processes (Todd and Vodkin,
1996; Senda et al., 2004; Tuteja et al., 2004).
Tuteja et al. (2004) demonstrated that the presence of the yellow
dominant (I) allele significantly decreases CHS mRNA levels in
seed coat but not in pods, leaves, stem, roots, and cotyledons.
Moreover, their investigations on the relative expression profile of
CHS gene family members show that the three CHS genes
comprising the I locus are transcribed at comparable although low
levels in both the pigmented and the non-pigmented isolines. The
increase of total CHS mRNA levels in the seed coats of I to i
mutations is primarily because of an increase in CHS7/CHS8
transcript levels (Tuteja et al., 2004). Thus, the dominant alleles
inhibit pigmentation in a trans-dominant manner. It is possible that
relative orientation of the two genes generates inverted repeat,
between CHS3 and CHS4, resulting in dsRNA (Tuteja et al., 2004).
If cleaved into small siRNAs (Hamilton and Baulcombe, 1999),
sequence-specific post-transcriptional degradation of CHS7/CHS8
could be generated in a tissue-specific manner to inhibit
pigmentation of the seed coats (Tuteja et al., 2004). A detailed
understanding of this system may reveal mechanisms of tissuespecific gene silencing which could be applied to the targeted
silencing of other seed coat genes.
Another locus, T, consists of two alleles, the dominant T and the
recessive t, which produce brown and gray pubescence,
respectively (Palmer and Kilen, 1987). Further, T generally
darkens hilum and/or seed coat color in combination with other
genotypes, and thus, induces seed coat cracking (Palmer and Kilen,
1987). The T locus encodes flavonoid 30 -hydroxylase (F30 H), which
is necessary for the formation of quercetin from kaempferol and is
responsible for the hydroxylation of the 30 position of flavonoids,
Venâncio et al., 2003;
Oliveira et al., 2004
Ilgoutz et al., 1997
leading to the production of cyanidin-based pigments (Forkman,
1991). The SF3 0 H1 cDNA from soybean represents the only fulllength sequence of F30 H from leguminous plants (Toda et al.,
2002). The sequence analysis of this gene from a pair of isogenic
lines for T (TT brown and tt gray) revealed that they are different
only by a single C deletion in the coding region of SF3 0 H1 (Toda
et al., 2002). The truncated protein lacks the conserved region of
F30 H and the heme-binding domain and probably lacks F30 H
activity. This may result in the absence of quercetin and its
derivatives in tissues and consequently produces the grey
pubescence color (Toda et al., 2002).
Seed coat color is a commercially important trait for other
legumes such as common bean, lentil, vetch, or pea. A common
vetch with low toxins could provide a valuable source of proteins for
animals. However, the linkage of color and other important
agronomic traits in such species is not yet evident. Five genes in
common bean, at least two genes in lentil, and one gene in common
vetch were shown to be involved in the control of seed coat color
(Brady et al., 1998; Emami and Sharma, 2000; Chowdhury et al.,
2004). However, these data were obtained from genetic crosses and
little is known about regulation of pigmentation in these species at
the molecular level.
Involvement of proline-rich proteins. Seed coat cracking in
pigmented soybean lines is also correlated with the accumulation of
proline-rich proteins (PRP1 and PRP2) in the cell walls. PRPs
represent a class of plant cell wall proteins characterized by high
proline content. They are composed of small tandem repeats, such
as PPPVYK, or PPVEK, where the second proline is often
hydroxyproline (Marcus et al., 1991; Keller, 1993; Showalter,
1993). PRPs exist as a soluble form but can also be insolubilized in
the wall over time (Kleis-San Francisco and Tierney, 1990; Bradley
et al., 1992). The two genes are expressed in different stages of
developing seed coats, hypocotyls and roots of soybeans. Expression
of the SbPRP1 gene is high in young seed coats and later during
seed desiccation (Lindstrom and Vodkin, 1991). The abundance of
PRP1 protein is affected by the I locus in soybean which controls
distribution of pigment in the seed coat. Interestingly, pigmented
varieties possessing lower levels of soluble PRP1 also display a netlike pattern of seed coat cracking (Lindstrom and Vodkin, 1991,
Nicholas et al., 1993). Both SbPRP1 and SbPRP2 cytoplasmic
mRNA were found in the net-defective seed coat, suggesting that
the absence of soluble PRP polypeptides in the defective net lines
is due to post-translational regulation, or due to a more rapid and/or
premature insolubilization of PRP polypeptides within the cell wall
matrix (Percy et al., 1999).
Protein and Carbohydrate Toxins
Globulins. Legume seed resistance to pests and pathogens may
also involve factors other than the phenolics. For example, in the
seed coat of common bean (Phaseolus vulgaris L.) neither thickness
nor the levels of phenolic compounds such as tannins and tannic
acids alone were important for resistance (Silva et al., 2004).
Vicilin-like 7S storage globulins, such as canavalin, concanavalin
A, canatoxin and phaseolin, reported in Jack bean (Canavalia
ensiformis), Lima bean (Phaseolus lunatus) and common bean (P.
vulgaris) have been implicated (Oliveira et al., 1999a; Moraes et al.,
2000; Silva et al., 2004). Canatoxin was shown to be toxic to some
insects (Carlini et al., 1997) and plant pathogenic fungi (Oliveira
et al., 1999a). Canavalin inhibits spore germination of several fungi.
Furthermore, both phaseolin and canavalin have detrimental effects
on larval development in bruchids (Oliveira et al., 1999a, b; Moraes
et al., 2000; Silva et al., 2004).
Albumins. The insecticidal properties of the pea albumin 1b
peptide(s) has opened new possibilities for seed protection against
cereal weevils. Although the mechanism of action of this toxin is
still unknown, binding to insect protein extracts occurs (Gressent
et al., 2003). This albumin is the first entomotoxic cystine-knot
peptide identified. It might belong to a multi-gene family, as at least
five isoforms of the peptide exist within a single pea genotype.
Moreover, it seems to be widespread among legumes (Higgins et al.,
1986; Gressent et al., 2003). The cystine-knot structural motif is
present in peptides and proteins from a variety of species and
appears to be a highly efficient motif for structure stabilization
(Craik et al., 2001).
Chitinase. Gijzen et al. (2001) isolated a class I chitinase from
soybean seed coat. Although chitin is absent in plants it is a major
component of fungal cell walls; therefore, chitinase may play a role
in plant defense against pathogens (Schlumbaum et al., 1986). The
seed coat chitinase is expressed late in seed development, with
particularly high expression levels in the seed coat. Moreover,
expression is associated with senescence, ripening and response to
pathogen infection (Gijzen et al., 2001).
Polysaccharides. Applebaum et al. (1970) demonstrated that a
polysaccharide fraction isolated from P. vulgaris seeds, present at a
level of c. 1% dry weight, increases larval mortality and reduces
rate of larval development. Gatehouse et al. (1987) observed that
carbohydrates from P. vulgaris seeds reduced Acanthoscelides
obtectus adult emergence and this activity was due, at least in part,
to the presence of a heteropolysaccharide which has an unusually
high content of arabinose and fructose. Recent work of Oliveira et al.
(2001) demonstrated the presence of the polysaccharide galactorhamnan in the innermost cell layer of the seed coat and also in
cotyledons of Jack bean (C. ensiformis). The concentration of this
polysaccharide in the seed coat (c. 2%) is sufficient by itself to
protect the seeds from attack by Callosobruchus maculatus (Oliveira
et al., 2001).
Soybean is known to have at least 15 allergenic proteins (Ogawa
et al., 1991). The majority of these allergens are oil body proteins
(Ogawa et al., 1993). Surface proteins associated with the seed coat
luster phenotype are also responsible for seed dust allergenicity
(Gonzalez et al., 1995; Gijzen et al., 1999b). Recently, the
hydrophobic protein (HPS) which is synthesized in the pod
endocarp and subsequently deposited to the seed surface was found
to be a major soybean dust allergen (Swanson et al., 1991; Gonzalez
et al., 1995; Gijzen et al., 1999b). The hydrophobicity and
topography of the surface of soy varieties expressing high levels of
HPS could affect pathogen attachment and penetration, influence
the water-absorptive properties of the seed (Gijzen et al., 1999b)
and/or mediate the attachment of the endocarp to the seed surface
(Gijzen et al., 2003).
Recently, Herman et al. (2003) demonstrated that transgene
silencing could be used to remove a major soybean allergen, the Gly
m Bd 30 K protein found in the seed globulin protein fraction. This
approach may prove to be useful for the reduction, or elimination of
allergens deposited upon the seed coat.
Insulin-like Proteins
Collip (1923) discovered the presence of insulin-like hypoglycemic activity in plant materials such as green tops of onions,
lettuce and bean leaves, barley and beet roots, and others. A few
decades later proteins from plant tissues were found which were
potentially beneficial to diabetic patients and had properties similar
to those of insulin (Khanna et al., 1981; Collier et al., 1987). The
insulin-like peptide and its receptor were isolated from a number of
legume seeds, such as soybean, cowpea, Jack bean, winged bean,
French bean, and lupin (Komatsu and Hirano, 1991; Ilgoutz et al.,
1997; Hanada et al., 2003; Venâncio et al., 2003; Yamazaki et al.,
2003; Hanada and Hirano, 2004). The finding that insulin-like
protein and insulin receptor-like protein with tyrosine protein
kinase activity were localized to the innermost region of the seed
coats may indicate that they are involved in signaling pathways for
the transport of nutrients to the embryo (Venâncio et al., 2003;
Oliveira et al., 2004). Interestingly, insulin has been shown to
stimulate the activity of enzymes involved in the conversion of
stored fat to carbohydrates in the fat-storing seed of sunflower,
watermelon, and cucumber, as well as the legume Jack bean
(Goodman and Davis, 1993; Oliveira et al., 2004). The presence of
insulin-like antigens in the leaves of several plants, from bryophytes
to angiosperms, as well as in a red alga, fungi, and cyanobacterium,
suggests that insulin-dependent sugar transport may have been
conserved during evolution (Silva et al., 2002; Xavier-Filho et al.,
Proteins Involved in Cellular Differentiation
Invertases. As reviewed extensively (Weber et al., 2005), seed
coats play a very important role in the metabolic control of seed
development. Not surprisingly, many seed coat proteins are
associated with sucrose metabolism (Kuo et al., 1997), including
invertase which regulates the hexose:sucrose ratios (Weber et al.,
1995; Borisjuk et al., 1998), or sucrose synthase, which is
responsible for 37% of neosynthesized sucrose in pea seed coat
cells. There are two kinds of invertase found in the maternal tissue
of young legume seeds. Soluble invertase, in the vacuole, is
important for the control of the hexose:sucrose ratio. Insoluble
invertase, embedded in cell walls, is active in growing zones and
extending tissues (Weber et al., 1996). Sequential expression of first
soluble and then insoluble invertases has been found during legume
seed development (Weber et al., 1995). In Vicia faba, the gene
VfVINV1 is expressed in the unloading area of the seed coat and
functions in the cleavage of incoming sucrose within the apoplast at
a stage when mitosis is active in the embryo. High levels of hexoses
in the cotyledons and the apoplastic endospermal space are
correlated with activity of cell wall-bound invertase in the seed coat
during the prestorage phase. In situ hybridization showed that the
gene, VfCWINV1, is expressed exclusively in the chalazal vein and
the inner rows of the thin-walled parenchyma of the seed coat.
These cells represent the end of the sieve element system.
VfWINV1 plays an important role in providing developing embryos
with hexoses and contributing to the establishment of sink strength
in young seeds.
Aquaporins. Unloading of nutrients from the seed coat is a vital
function and must be associated with a wide range of other
processes such as release of water from the seed coat during the
transfer of the solutes. These processes may involve the family of
major intrinsic proteins (MIP) which include the aquaporins. Four
full-length aquaporin-related cDNAs were cloned and sequenced
from a cDNA library of developing pea seed coats (P. sativum L.).
The cDNA of PsPIP1-1 appears to be a turgor-responsive gene.
PsNIP-1 is only detected in the seed coat, while PsPIP1-1, PsPIP21, and PsTIP 1-1 are expressed in cotyledons of developing and
germinating seeds, and seedling roots and shoots. In mature dry
seeds, strong hybridization signals were detected with the probe for
PsPIP1-1, but no transcripts of PsPIP2-1, PsTIP1-1, and PsNIP-1
were found. Functional analysis in Xenopus oocytes confirmed that
PsPIP2-1 and PsTIP1-1 are aquaporin whereas PsNIP-1 is an
aquaglyceroporin. It is suggested that PsPIP1-1 could play a role in
water absorption during the seed imbibition. PsPIP2-1 together with
PsPIP1-1 could be involved in the release of water from the seed
coat symplast that is finally related to release of nutrients for the
embryo (Schuurmans et al., 2003).
Glucanases. Plant b-1,3-glucanases represent a highly diverse
family of hydrolytic enzymes, which are generally induced in
response to pathogen attack or environmental stress (Kauffmann
et al., 1987; Simmons et al., 1992). Nevertheless, tissue-specific
and developmentally regulated but non-pathogen-induced
expression of b-1,3-glucanase genes have also been reported
(Memelink et al., 1990; Hird et al., 1993). The expression of
PsGNS2, encoding b-1,3-glucanase, was demonstrated to be
spatially and temporally regulated in P. sativum seed coats
(Buchner et al., 2002). The high abundance of the transcripts
observed when the embryo reached the late heart stage, remained
until the mid seed-filling stage and was restricted to a strip of the
inner parenchyma tissue of the seed coat (Buchner et al., 2002). It
was suggested that PsGNS2 could function in maintaining routes for
lateral transport and cell – cell communication through plasmodesmata possibly via callose hydrolysis (Buchner et al., 2002).
Alternatively, it might be associated with the release of
oligosaccharides which are important for embryogenesis (Berger,
1999; Buchner et al., 2002). Finally, both seed coat thin-walled
parenchyma and endosperm layers are tissues subjected to partial
degradation during development (Weber et al., 1995; Berger, 1999;
Miller et al., 1999) and these apoptotic processes may also involve
glucanases (Buchner et al., 2002).
BURP-domain proteins. Expressed sequence tags (ESTs)
exhibiting homology to a BURP domain-containing gene family
were identified in G. max (L.) Merr (Granger et al., 2002). These
ESTs were assembled into 16 contigs of variable sizes and lengths.
The soybean family members exhibit 35 – 98% similarity in a
, 100-amino acid C-terminal region. Soybean BURP-domain
proteins have diverse expression patterns. Batchelor et al. (2002)
investigated SCB1, a BURP-domain protein gene, which is
expressed in developing soybean seed coats. It is known that
changes in the cell wall structure are a distinguishing features of
cell differentiation in the seed coats (Miller et al., 1999). The BURP
domain may represent a general motif for localization of proteins
within the cell wall matrix. SCB1 mRNA accumulates first within
the developing thick-walled parenchyma cells of the inner
integument and later in the thick- and thin-walled parenchyma
cells of the outer integument. This occurs prior to the period of seed
coat maturation and seed filling and before either of the layers start
to degrade. In addition, the SCB1 protein appears to be located
within cell walls, and thus, may play a role in the differentiation of
the seed coat parenchyma cells.
Seed coat subtilisin. Another seed coat specific gene is SCS1
(seed coat subtilisin 1) that belongs to a small gene family of
genes with sequence similarity to subtilisin, a serine protease
(Bachelor et al., 2000). Northern blot analysis and in situ
hybridization studies revealed that accumulation of SCS1 mRNA
is restricted to thick-walled parenchyma cells of the soybean
seed coat and reaches maximal levels at 12 d post-anthesis,
preceding the final stages of seed coat differentiation. The thickwalled parenchyma, which is derived from the inner integument,
is very prominent during the first week but is rapidly degraded
by the second week. These cells are important in the apoplastic
translocation of nutrients en route to the embryo from the
vascular tissues. As serine proteases are often involved in signal
transduction, SCS1 could play a role in cell differentiation in
seed coats.
Seed coat peroxidases. Soybean seed coat peroxidase (SBP) is
another important enzyme found in seed coats. This enzyme belongs
to class III plant peroxidases that function in cell wall biosynthesis,
defense, and other oxidative processes (Welinder, 1992). Class III
plant peroxidases can oxidize a wide variety of organic and
inorganic substrates using hydrogen peroxide (Henriksen et al.,
Peroxidase activity in the seed coat of soybean is controlled by
the Ep locus. Such activity from seed coats of EpEp cultivars is 100fold higher than that from epep cultivars. In seed coat extracts,
peroxidase is the most abundant soluble protein in EpEp cultivars,
whereas this enzyme was present only in trace amounts in epep
cultivars (Gijzen et al., 1993). No obvious difference in the structure
of the seed coat was associated with the Ep locus (Gijzen et al.,
1993). The mutation in the peroxidase-deficient line was a deletion
in the promoter region of the Ep peroxidase genes (Gijzen, 1997).
Histochemical localization of peroxidase activity revealed that the
enzyme accumulates predominately in the hourglass cells of the
subepidermis. A single isozyme of the seed coat peroxidase
constitutes about 5% of total soluble protein in the seed coat of
EpEp cultivars (Gillikin and Graham, 1991; Gijzen et al., 1993). No
putative plant peroxidases are orthologous to soybean peroxidase.
Soybean peroxidase shows more than 70% amino acid sequence
identity to peroxidases from other legumes involved in various
defense responses (Welinder and Larsen, 2004). For instance, the
soybean sequence is very closely related to four peroxidase cDNAs
isolated from alfalfa, with 65 – 67% identity at the amino acid level
(Gijzen, 1997).
It was once thought that soybean seed coat peroxidase might be
important in lignification, or suberization, hardness, and permeability (Gillikin and Graham, 1991; Gijzen et al., 1993). Now, the
similar phenotypes of EpEp and epep cultivars, and the existence of
vacuolar targeting signals on the protein argue against the
involvement of the peroxidase in the synthesis or modification of
extracellular polymers. It may have a function in defense, when
released from hourglass cells during seeds imbibition (M. Gijzen,
personal communication).
Due to its high stability and activity, SBP provides an alternative
to horseradish peroxidase in many industrial applications. SBP is
present at high concentration in the soybean seed coat depending
on the cultivar (Gijzen, 1997; Gijzen et al., 1993). Due to its high
thermo- and pH-stability it can be easily purified from soybean
hulls (Henriksen et al., 2001; Nissum et al., 2001; Welinder and
Larsen, 2004).
Gijzen et al. (1999a) isolated a second peroxidase gene, Prx2 that
is also highly expressed in developing seed coat tissues. Sequence
analysis of Prx2 cDNA indicates that this transcript encodes a
cationic peroxidase. By comparing the abundance and localization
of the EP and Prx2 transcripts, it was shown that the expression of
EP begins in a small number of cells flanking the vascular bundle
in the seed coat, spreads to encircle the seed, and then migrates to
the hourglass cells as they develop. Expression of Prx2 occurs
throughout development in all cell layers of the seed coat, and is
also evident in the pericarp and embryo. The Prx2 enzyme is either
insoluble in a catalytically inactive form, or is subjected to
degradation during seed maturation.
MADS box proteins. MADS box genes represent a large family of
highly conserved transcription factors. In plants, most of these factors
seem to be involved in the control of floral development. However, the
pea gene paeMFT1-encoding MADS box transcription factor was
shown to be specifically expressed in the seed coats during seed
development with weak floral expression (Buchner and Boutin, 1998).
Maternally controlled defects in seed development after downregulation of MADS box genes in petunia (Colombo et al., 1997)
suggest that MADS box proteins might play important roles in the
seed coat development (Buchner and Boutin, 1998).
Seed Coat Biotechnology
The modification of seed quality and use of seeds for the
expression and storage of foreign proteins has been widely
investigated in plant systems, including crop species. Seeds have
several major advantages, including the availability of regulatory
signals derived from studies of seed storage proteins and their
genes, and the agricultural systems designed to harvest, store, and
process them. The types of foreign proteins that have been
expressed include proteins to improve the amino acid content,
enzymes, antigens, and biopharmaceuticals among others. In theory,
any protein could be expressed in the seed, as could non-protein
materials such as vitamin A precursors in rice (Xudong et al., 2000)
if the metabolic pathways in the developing seed were modified.
Many of the advantages relating to seeds (e.g., harvesting,
storage, processing) would also accrue to seed coats. Although seed
coats in soybean may constitute ,8% of the seed by weight, this
amount still represents a very substantial source of material for
processing when considering that the production of soybean seed in
2004 was estimated to be , 190 £ 106 Mt (
default.asp). Although most proteins expressed in seeds could also
be expressed in seed coats, including pharmaceuticals, the greatest
potential for the technology may be in the use of biotechnology to
solve problems in industrial, environmental (‘white biotechnology’),
or agricultural processes (‘green biotechnology’). Genetically
modified seed coats may provide a biodegradable, environmentally
friendly source of enzymes and biochemicals either in a purified
or partially purified form. Many uses for seed coat technology
have been described and these can be scrutinized on government,
industry and growers’ web sites (, http://www.,, http://, and many more) but here we
will focus on examples of non-medical applications, such as
industrial processing, pollution control, and feed modification. Fig. 2
provides an overview linking together many of the traditional seed
traits that can be improved through biotechnology, and new
opportunities that exist for generating novel bioproducts and
Germination & dormancy
Nutrition & allergenicity
Processing qualities
Seed color
Seed size & shape
Seed coat
Novel products
FIG . 2. Modification of soybean seed coats by transgenic routes including value-added properties.
Industrial and Environmental Applications
Peroxidases and laccases are oxidoreductases that have potential
applications in industrial processes and environmental technologies. Peroxidases are heme-containing enzymes that use hydrogen
peroxide as the electron donor, while copper-containing laccases
employ molecular oxygen as the electron donor. These enzymes are
widespread in nature and catalyze one-electron oxidations of
suitable substrates. Many potential uses for these enzymes have
been identified, ranging from bleaching denim, to pulp and paper
bleaching, waste water treatment, soil remediation, food conditioning, and diagnostic kits (, http://www.vtt.
fi/bel/indexe.htm, And there is
evidence that SBP can substitute for formaldehyde in the formation
of phenolic resins used for the production of plywood and particle
board (Enzymol International, USA, US patent 5,491,085).
SBP is better suited for industrial applications than most
peroxidases, having a relatively high thermal stability (active at
708C) and being active over a broad pH range. Many laccases have
been isolated from fungi and other organisms, their properties
investigated and shown to be potentially useful. The major factor
that limits the adoption of these technologies is the cost of the
enzymes. The seed coat expression system for the production of
heterologous enzymes of potential industrial relevance offers great
promise to overcome this limitation by lowering the cost. Further
advantages include the targeting of enzymes to the hourglass cells
that inherently store proteins, thus avoiding potential problems with
proteolytic degradation. Soybean seed coats are already harvested
as part of the processing of soybeans, so there is no additional cost
associated with harvesting. The potential for high yield, low cost
downstream processing is good, since the target enzyme will likely
be the predominant protein in the extracts when the host plant does
not express the endogenous Ep peroxidase. Since the amount of
hulls produced worldwide approaches , 15 £ 106 Mt yr21, a cheap,
stable source of raw materials can be provided through
The feasibility of such applications is illustrated by the work of
Bassi and Gijzen (Flock et al., 1999; Geng et al., 2004) who studied
the degradation of phenols and chlorophenols. Their work
demonstrated that crude hulls were superior to purified SBP for
the degradation of pollutants in batch reactors and removed 96% of
the phenol within 20 min and 98.5% of the 2-chlorophenol within
15 min. These studies point to the potential cost savings by the use
of unpurified, or partially purified enzymes for industrial/
environmental purposes.
Feed Modification
Soybean in the form of meal, oil, or pellets is a major source of
protein and nutrient in animal feed for livestock, poultry, and pets.
This market is growing and could expand to include fish, exotic
animal farming, and many other applications. Seed coats can be
added back to feed as a source of fiber and nutrients. Thus modified
seed coats could be the means to improve the nutritional quality of
the feed by introducing enzymes, or compounds that aid digestion,
specific micronutrients, or proteins with antibiotic activity. For
example, PNPI ( has proposed
that seed coats could be a source of phytase, an enzyme that
improves phosphorus utilization in feed for poultry, swine and other
monogastric animals by breaking down the phytin that binds
phosphorus in plant material. The improved efficiency of phosphate
utilization has an additional benefit, i.e., reduced phosphorus
pollution when the manure is used as a fertilizer (Golovan et al.,
2001). This example illustrates a general strategy by which a seed
coat supplement containing digestive enzymes could be used to
modify feed stocks and improve nutrition.
Animal feeds are sometimes used as a means to introduce
antibiotics and other growth-promoting compounds. Recent
concerns about antibiotic resistance have spurred a search for
alternatives. Antimicrobial peptides (AMPs) expressed in seed
coats could provide a viable substitute. AMPs are small (12 – 45
amino acids) cationic peptides that at low concentrations (0.5 –
4.0 mg ml21) have antimicrobial activity against bacteria, fungi, and
viruses (Hancock and Lehrer, 1998). Their antibacterial mode of
action is to assemble within the bacterial or fungal membrane to
create pores, leading to leakage of the cell contents and death.
Naturally occurring AMPs are encoded by genes and are produced
as a precursor protein that is processed to give the final active
polypeptide. They are non-immunogenic when ingested but may
stimulate an immune response if injected. Several groups have
demonstrated that plants can express AMP transgenes at low levels
and that the AMPs retain their biological activity (Osusky et al.,
2004; P.G. Arnison, SynGene Biotek Inc., personal communication). Since seed coats have reduced digestibility when compared
to meal, the active AMP inside the hourglass cell may be protected
from proteolytic activity while the seed coat slowly breaks down,
releasing AMP in a manner similar to a slow-release pill. The recent
in vitro experiments (Geng et al., 2004) described above suggest
that this may be a viable strategy for feed supplements.
Vectors for Transformation
Soybean can be effectively transformed with a variety of vector
types, including the CAMBIA series ( For
expression of the transgene in specific cell types, such as the HGCs
of seed coats, these vectors must be modified by the addition of an
HGC-specific promoter. If accumulation of the protein in a
particular location (vacuole, endoplasmic reticulum, cell wall) is
desired, then additional protein targeting signals must be added to
the transgene to create a fusion protein with these signals.
Ep peroxidase is expressed in HGCs and the enzyme is
translocated to the protein storage vacuole (PSV) via the
endomembrane system with concomitant removal of the appropriate
signal peptides (Welinder and Larsen, 2004). Thus sequences for an
HGC-specific promoter, an amino-terminal ER signal peptide and a
carboxy-terminal VSP signal may be supplied from the Ep gene.
While the addition of these signals requires more steps in transgene
construction, the extra effort of targeting the Escherichia coli Lt-B
toxin to the PSV resulted in a higher expression level in maize
(Hood, 2004). The choice of target will depend on many factors
including the properties of the protein and its ease of purification.
Preliminary experiments suggest that the Ep peroxidase promoter
region can direct the HGC-specific expression of GUS in transgenic
soybean (Gijzen and Simmonds, unpublished) but the ability of the
signal peptides to correctly target a foreign protein to the vacuole
has not yet been verified experimentally. Many potential cis-acting
elements within the Ep promoter can be identified by bioinformatics
tools, but a functional analysis of the promoter has not yet been
performed. From the point of view of optimizing the expression of
transgenes, the identification of sequences that determine tissuespecific expression would be a necessary prelude to the construction
of hybrid promoters that would retain elements for tissue-specificity
while introducing elements from stronger promoters, such as tCUP
(Malik et al., 2002). Future experiments employing CHIPs to
analyze the soybean transcriptome (Stacey et al., 2004) will most
certainly identify more seed coat-specific genes and their regulatory
sequences, including promoters and enhancers. The availability of
such a toolbox would allow much more flexibility in the design and
expression of transgenes.
Targeting of heterologous proteins may also lead to problems, and
again SBP can be used as an example. In the mature SBP, 18% of
the mass is heterogeneous glycan (Welinder and Larsen, 2004)
attached though N-linkages at asparagines residues within the
consensus sequence Asn-X-Ser/Thr. They are added in the ER and
modified in the Golgi during protein processing (Helenius and Aebi,
2001). While expression of the transgene in the cytoplasm does not
lead to this type of modification, targeting to the ER, vacuole and
cell wall most certainly will, and could change the properties of the
protein, including activity and antigenicity. It would be necessary to
carefully analyze each protein product to investigate whether
modifications will influence the desired properties.
Additional Considerations for Exploitation
To this point the discussion has centered on the use of HGCs in
soybean as a vehicle for the expression of a single gene. The
expression of multiple genes (‘gene stacking’) would extend the
utility of this system and allow the accumulation of combinations of
enzymes for industrial or environmental purposes, or the
construction of metabolic pathways for the synthesis of novel
compounds. For some purposes, high levels of SBP normally stored
in the HGCs may prove to be problematic and epep lines in which
the SBP is not expressed (Gijzen, 1997) could be substituted.
Worldwide, soybeans are the major legume crop and thus could
potentially generate the greatest amount of seed coat for processing.
Indeed large-scale production of seed, oil, and feed implies that a
processing system is in place. Other legumes are significant sources
of seed and forage in many countries around the world and potential
for the use of other minor legumes as alternatives to soybean should
be considered for certain geographic regions.
Hourglass cells (or osteosclereids, pillar cells, lagenosclereids)
have been described in the seed coats from a large number of
legumes such as lupin (Clements et al., 2004), pea (Harris, 1984),
milkvetch (Miklas et al., 1987), common bean (Yeung, 1990), white
popinac (Serrato-Valenti et al., 1995), yam bean (Ene-Obong and
Okoye, 1993), pulse (Gupta et al., 1985), and many others (Irving,
1984; Manning and van Staden, 1987; Trivedi and Gupta, 1987;
Pandey and Jha, 1988; Izaguirre et al., 1994; Sharma and Sharma,
1994; Paria et al., 1997; Sornsathapornkul and Owens, 1999). Their
wide distribution suggests that legumes have similar seed coat
anatomy and that the regulatory signals for transcription and protein
targeting that function in soybean may also function correctly in
closely related species. If such speculation is in fact true in practice,
then the use of alternatives to soybean becomes not a technical
problem but a socio-economic one. The choice of system depends
upon the ability of the local economy to supply seed coats, the
presence of the appropriate processing and purification systems and
the demand for the products. Where seed legumes are grown, their
future use as a bioreactor must successfully compete with the present
use for seed coats. For example, although the price of seed coats
may be low, if they are added back to feed after crushing to
remove the oil, then their effective price is the price of meal ($160–
170 US/ton in 2004 ,http://usda.manlib/cornell/edu/data-sets/
crops/89002/.). As new uses for legumes are developed then
thought should be given to the potential of the seed coat system and to
the merging of technologies, e.g., lupin seeds could be processed in
such a way that allows the easy isolation of seed coats for
further exploitation (
04/ sands.htm).
Not only has the seed coat developed highly sophisticated
processes for the protection of embryos and germinating seedlings
from biotic and abiotic stresses, but it also plays a pivotal role in the
control of development. The ability to manipulate any of these
processes with cloned genes has the potential to dramatically alter
the yield and composition of seeds for traditional uses and for the
production of novel products for new uses (Fig. 2). In this review we
have tried to summarize our understanding of seed coat form,
composition, and genetics. It is clear that our knowledge of the
genes that regulate seed coat development or the pathways that
produce their valuable compounds is very poor, and inadequate to
realize the full potential of biotechnology at this time. However,
many of the biological tools needed for biotechnology are well
developed. Arabidopsis is an important model species that may
accelerate our acquisition of knowledge but it may not be sufficient
to provide the information that is specific to many of our crops.
There is a need for more basic research on the seed coat, a novel
and fascinating developmental system in its own right that has been
ignored for too long.
The authors are grateful to Drs Mark Gijzen, Shea Miller, and Tamara
Western for reviewing the manuscript prior to submission. The authors are
grateful to C.L. Johnson for drawing the figures. J. M. is the recipient of an
NSERC postgraduate scholarship. S. H. is the recipient of an Ontario
Graduate Scholarship. The research was supported in part by an NSERC
Strategic Grant awarded to D. A. J. and by Agriculture and Agri-Food Canada.
Eastern Cereal and Oilseed Research Centre publication number 05520.
Abrahams, S.; Lee, E.; Walker, A. R.; Tanner, G. J.; Larkin, P. J.; Ashton,
A. R. The Arabidopsis TDS4 gene encodes leucoanthocyanidin
dioxygenase (LDOX) and is essential for proanthocyanidin synthesis
and vacuole development. Plant J. 35:624–636; 2003.
Abrahams, S.; Tanner, G. J.; Larkin, P. J.; Ashton, A. R. Identification and
biochemical characterization of mutants in the proanthocyanidin
pathway in Arabidopsis. Plant Physiol. 130:561–576; 2002.
Aerts, R. J.; Barry, T. N.; McNabb, W. C. Polyphenols and agriculture:
beneficial effects of proanthocyanidins in forages. Agric. Ecosyst.
Environ. 75:1– 12; 1999.
Akada, S.; Dube, S. K. Organization of soybean chalcone synthase gene
clusters and characterization of a new member of the family. Plant
Mol. Biol. 29:189– 199; 1995.
Akada, S.; Kung, S.-D.; Dube, S. K. The nucleotide sequence of gene 3 of the
soybean chalcone synthase multigene family. Nucleic Acids Res.
18:5899; 1990.
Akada, S.; Kung, S.-D.; Dube, S. K. The nucleotide sequence of gene 1 of the
soybean chalcone synthase multigene family. Plant Mol. Biol.
16:751–752; 1991.
Akada, S.; Kung, S.-D.; Dube, S. K. Nucleotide sequence and putative
regulatory elements of gene 2 of the soybean (Glycine max) chalcone
synthase multigene family. Plant Physiol. 102:317–319; 1993a.
Akada, S.; Kung, S.-D.; Dube, S. K. Nucleotide sequence of a soybean
chalcone synthase gene with a possible role in ultraviolet-B
sensitivity, Gmchs6. Plant Physiol. 102:699–701; 1993b.
Akada, S.; Kung, S.-D.; Dube, S. K. Nucleotide sequence and putative
regulatory elements of a nodule-development-specific member of the
soybean (Glycine max) chalcone synthase multigene family, Gmchs7.
Plant Physiol. 102:321–323; 1993c.
Algan, G.; Büyükkartal, H. N. B. Ultrastructure of seed coat development in
the natural tetraploid Trifolium pratense L. J. Agron. Crop Sci.
184:205– 213; 2000.
Alkharouf, N. W.; Matthews, B. F. SGMD: the soybean genomics and
microarray database. Nucleic Acids Res. 32:D398–D400; 2004.
Applebaum, S. W.; Tadmor, U.; Podoler, H. The effect of starch and of a
heteropolysaccharide fraction from Phaseolus vulgaris on development and fecundity of Callosobruchus chinensis (Coleoptera –
Bruchidae). Entomol. Exp. Applicata 13:61–70; 1970.
Arenas-Mena, C.; Raynal, M.; Borrell, A.; Varoquaux, F.; Cutanda, M. C.;
Stacy, R. A. P.; Pagès, M.; Delseny, M.; Culiáñez-Macià, F. A.
Expression and cellular localization of Atrab28 during Arabidopsis
embryogenesis. Plant Mol. Biol. 40:355–363; 1999.
Baker, S. C.; Robinson-Beers, K.; Villanueva, J. M.; Gaiser, J. C.; Gasser,
C. S. Interactions among genes regulating ovule development in
Arabidopsis thaliana. Genetics 145:1109–1124; 1997.
Balasubramanian, S.; Schneitz, K. NOZZLE regulates proximal–distal
pattern formation, cell proliferation and early sporogenesis during
ovule development in Arabidopsis thaliana. Development
127:4227– 4238; 2000.
Balasubramanian, S.; Schneitz, K. NOZZLE links proximal– distal and
adaxial–abaxial pattern formation during ovule development in
Arabidopsis thaliana. Development 129:4291–4300; 2002.
Batchelor, A. K.; Boutilier, K.; Miller, S. S.; Hattori, J.; Bowman, L. A.; Hu,
M.; Lantin, S.; Johnson, D. A.; Miki, B. L. A. SCB1, a BURP-domain
protein gene from developing soybean seed coats. Planta
215:523– 532; 2002.
Batchelor, A. K.; Boutilier, K.; Miller, S. S.; Labbé, H.; Bowman, L. A.; Hu,
M.; Johnson, D. A.; Gijzen, M.; Miki, B. L. A. The seed coat-specific
expression of a subtilisin-like gene, SCS1, from soybean. Planta
211:484– 492; 2000.
Baudry, A.; Heim, M. A.; Dubreucq, B.; Caboche, M.; Weisshaar, B.;
Lepiniec, L. TT2, TT8, and TTG1 synergistically specify the
expression of BANYULS and proanthocyanidin biosynthesis in
Arabidopsis thaliana. Plant J. 39:366–380; 2004.
Baxter, I. R.; Young, J. C.; Armstrong, G.; Foster, N.; Bogenschutz, N.;
Cordova, T.; Peer, W. A.; Hazen, S. P.; Murphy, A. S.; Harper, J. F. A
plasma membrane Hþ-ATPase is required for the formation of
proanthocyanidins in the seed coat endothelium of Arabidopsis
thaliana. Proc. Natl Acad. Sci. USA 102:2649–2654; 2005.
Beeckman, T.; De Rycke, R.; Viane, R.; Inzé, D. Histological study of seed
coat development in Arabidopsis thaliana. J. Plant Res.
113:139– 148; 2000.
Bekkara, F.; Jay, M.; Viricel, M. R.; Rome, S. Distribution of phenolic
compounds within seed and seedlings of two Vicia faba cvs differing
in their seed tannin content, and study of their seed and root
phenolic exudations. Plant Soil 203:27–36; 1998.
Bell, C. J.; Dixon, R. A.; Farmer, A. D.; Flores, R.; Inman, J.; Gonzales, R.
A.; Harrison, M. J.; Paiva, N. L.; Scott, A. D.; Weller, J. W.; May, G.
D. The Medicago genome initiative: a model legume database.
Nucleic Acids Res. 29:114–117; 2001.
Benitez, E. R.; Funatsuki, H.; Kaneko, Y.; Matsuzawa, Y.; Bang, S. W.;
Takahashi, R. Soybean maturity gene effects on seed coat
pigmentation and cracking in response to low temperatures. Crop
Sci. 44:2038–2042; 2004.
Berger, F. Endosperm development. Curr. Opin. Plant. Biol. 2:28–32; 1999.
Bernard, R. L.; Weiss, M. G. Qualitative genetics. In: Caldwell, B. E., ed.
Soybeans: Improvement, production and uses, 1st ed. Madison, WI:
American Society of Agronomy; 1973:117– 149.
Bewley, J. D. Seed germinatin and dormancy. Plant Cell 9:1055–1066;
Borisjuk, L.; Walenta, S.; Weber, H.; Mueller-Klieser, W.; Wobus, U. High
resolution histographical mapping of glucose concentrations in
developing cotyledons of Vicia faba in relation to mitotic activity and
storage processes: glucose as a possible developmental trigger. Plant
J. 15:583–591; 1998.
Bouman, F. Integument initiation and testa development in some Cruciferae.
Bot. J. Linn. Soc. 70:213– 229; 1975.
Bradley, D. J.; Kjellbom, P.; Lamb, C. J. Elicitor- and wound-induced
oxidative cross-linking of a proline-rich plant cell wall protein: a
novel, rapid defense response. Cell 70:21–30; 1992.
Brady, L.; Bassett, M. J.; McClean, P. E. Molecular markers associated with
T and Z, two genes controlling partly colored seed coat patterns in
common bean. Crop Sci. 38:1073–1075; 1998.
Broadhvest, J.; Baker, S. C.; Gasser, C. S. SHORT INTEGUMENTS 2
promotes growth during Arabidopsis reproductive development.
Genetics 155:899–907; 2000.
Buchner, P.; Boutin, J.-P. A MADS box transcription factor of the
AP1/AGL9 subfamily is also expressed in the seed coat of pea
(Pisum sativum) during development. Plant Mol. Biol.
38:1253–1255; 1998.
Buchner, P.; Rochat, C.; Wuillème, S.; Boutin, J.-P. Characterization of a
tissue-specific and developmentally regulated b-1,3-glucanase gene
in pea (Pisum sativum). Plant Mol. Biol. 49:171–186; 2002.
Cabrera, A.; Martin, A. Variation in tannin content in Vicia faba L. J. Agric.
Sci. 106:377– 382; 1986.
Cardador, M. A.; Castaño, T. E.; Loarca, P. G. Antimutagenic activity of
natural phenolic compounds present in common bean (Phaseolus
vulgaris) against aflatoxin B1. Food Addit. Contam. 19:62– 69; 2002.
Carlini, C. R.; Oliveira, A. E. A.; Azambuja, P.; Xavier-Filho, J.;
Wells, M. A. Biological effects of canatoxin in different insect
models: Evidence for a proteolytic activation of the toxin by insect
cathepsinlike enzymes. J. Econ. Entomol. 90:340–348; 1997.
Chamberlin, M. A.; Horner, H. T.; Palmer, R. G. Early endosperm, embryo,
and ovule development in Glycine max (L.) Merr. Int. J. Plant. Sci.
155:421–436; 1994.
Chowdhury, D. M. S.; Rathjen, J. M.; Tate, M. E.; McDonald, G. Genetics of
colour traits in common vetch (Vicia sativa L.). Euphytica
136:249–255; 2004.
Clements, J. C.; Zvyagin, A. V.; Silva, K. K. M. B.; Wanner, T.; Sampson, D.
D.; Cowling, W. A. Optical coherence tomography as a novel tool for
non-destructive measurement of the hull thickness of lupin seeds.
Plant Breed. 123:266–270; 2004.
Collier, E.; Watkinson, A.; Cleland, C. F.; Roth, J. Partial purification and
characterization of an insulin-like material from spinach and Lemna
gibba G3. J. Biol. Chem. 262:6238–6247; 1987.
Collip, J. B.; Glucokinin. A new hormone present in plant tissue. Preliminary
paper. J. Biol. Chem. 56:513– 543; 1923.
Colombo, L.; Franke, J.; Van der Krol, A. R.; Wittich, P. E.; Dons, H. J. M.;
Angenent, G. C. Downregulation of ovule-specific MADS box genes
from Petunia results in maternally controlled defects in seed
development. Plant Cell 9:703– 715; 1997.
Cook, D. R. Medicago truncatula – a model in the making! Curr. Opin. Plant
Biol. 2:301–304; 1999.
Corner, E. J. H. The leguminous seed. Phytomorphology 1:117–150;
Craik, D. J.; Daly, N. L.; Waine, C. The cystine knot motif in toxins and
implications for drug design. Toxicon 39:43–60; 2001.
Dakora, F. D.; Phillip, D. A. Diverse functions of isoflavonoids in legumes
transcend anti-microbial definitions of phytoalexins. Physiol. Mol.
Plant Pathol. 49:1–20; 1996.
Debeaujon, I.; Koornneef, M. Gibberellin requirement for Arabidopsis seed
germination is determined both by testa characteristics and
embryonic abscisic acid. Plant Physiol. 122:415– 424; 2000.
Debeaujon, I.; Léon-Kloosterziel, K. M.; Koornneef, M. Influence of the testa
on seed dormancy, germination, and longevity in Arabidopsis. Plant
Physiol. 122:403–413; 2000.
Debeaujon, I.; Nesi, N.; Perez, P.; Devic, M.; Grandjean, O.; Caboche, M.;
Lepiniec, L. Proanthocyanidin-accumulating cells in Arabidopsis
testa: regulation of differentiation and role in seed development.
Plant Cell 15:2514–2531; 2003.
Debeaujon, I.; Peeters, A. J. M.; Léon-Kloosterziel, K. M.; Koornneef, M.
The TRANSPARENT TESTA12 gene of Arabidopsis encodes a
multidrug secondary transporter-like protein required for flavonoid
sequestration in vacuoles of seed coat endothelium. Plant Cell
13:853–871; 2001.
Dejardin, A.; Rochat, C.; Maugenest, S.; Boutin, J. P. Purification,
characterization and physiological role of sucrose synthase in the pea
seed coat (Pisum sativum L.). Planta 201:128–137; 1997.
Devic, M.; Guilleminot, J.; Debeaujon, I.; Bechtold, N.; Bensaude, E.;
Koornneef, M.; Pelletier, G.; Delseny, M. The BANYLUS gene
encodes a DFR-like protein and is a marker of early seed coat
development. Plant J. 19:387–398; 1999.
Dhaubhadel, S.; McGarvey, B. D.; Williams, R.; Gijzen, M. Isoflavonoid
biosynthesis and accumulation in developing soybean seeds. Plant
Mol. Biol. 53:733–743; 2003.
Di Cristina, M.; Sessa, G.; Dolan, L.; Linstead, P.; Baima, S.; Ruberti, I.;
Morelli, G. The Arabidopsis Athb-10 (GLABRA2) is an HD-Zip
protein required for regulation of root hair development. Plant J.
10:393–402; 1996.
Dixon, R. A.; Paiva, N. L. Stress-induced phenylpropanoid metabolism.
Plant Cell 7:1085– 1097; 1995.
Dueñas, M.; Estrella, I.; Hernández, T. Occurence of phenolic compounds in
the seed coat and the cotyledon of peas (Pisum sativum L.). Eur.
Food Res. Technol. 219:116–123; 2004.
Dueñas, M.; Hernández, T.; Estrella, I. Phenolic composition of the
cotyledon and the seed coat of lentils (Lens culinaris L.). Eur. Food
Res. Technol. 215:478– 483; 2002.
Elliott, R. C.; Betzner, A. S.; Huttner, E.; Oakes, M. P.; Tucker, W. Q. J.;
Gerentes, D.; Perez, P.; Smyth, D. R. AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule
development and floral organ growth. Plant Cell 8:155–168; 1996.
Emami, M. K.; Sharma, B. Inheritance of black testa colour in lentil (Lens
culinaris Medik.). Euphytica 115:43–47; 2000.
Ene-Obong, E. E.; Okoye, F. I. Effect of seed coat on water permeability in
the African yam bean, Sphenostylis stenocarpa. Nigerian J. Bot.
6:43–51; 1993.
Feinbaum, R. L.; Ausubel, F. M. Transcriptional regulation of the
Arabidopsis thaliana chalcone synthase gene. Mol. Cell Biol.
8:1985–1992; 1988.
Finkelstein, R. R.; Gampala, S. S. L.; Rock, C. D. Abscisic acid signaling in
seeds and seedlings. Plant Cell 14:S15–S45; 2002.
Flock, C.; Bassi, A.; Gijzen, M. Removal of aqueous phenol and
2-chlorophenol with purified soybean peroxidase and raw soybean
hulls. J. Chem. Technol. Biotechnol. 74:303–309; 1999.
Forkman, G. Flavonoids as flower pigments: the formation of the natural
spectrum and its extension by genetic engineering. Plant Breed.
106:1–26; 1991.
Frey, A.; Godin, B.; Bonnet, M.; Sotta, B.; Marion-Poll, A. Maternal
synthesis of abscisic acid controls seed development and yield in
Nicotiana plumbaginifolia. Planta 218:958–964; 2004.
Furbank, R. T.; White, R.; Palta, J. A.; Turner, N. C. Internal recycling of
respiratory CO2 in pods of chickpea (Cicer arietinum L.): the role of
pod walls, seed coat, and embryo. J. Exp. Bot. 55:1687–1696; 2004.
Gaiser, J. C.; Robinson-Beers, K.; Gasser, C. S. The Arabidopsis
SUPERMAN gene mediates asymmetric growth of the outer
integuments of ovules. Plant Cell 7:333– 345; 1995.
Garcia, D.; Fitz Gerald, J. N.; Berger, F. Maternal control of integument
cell elongation and zygotic control of endosperm growth are
coordinated to determine seed size in Arabidopsis. Plant Cell
17:52–60; 2005.
Gatehouse, A. M. R.; Dobie, P.; Hodges, R. J.; Meik, J.; Pusztai, A.; Boulter,
D. Role of carbohydrates in insect resistance in Phaseolus vulgaris.
J. Insect Physiol. 33:843–850; 1987.
Geng, Z.; Bassi, A. S.; Gijzen, M. Enzymatic treatment of soils contaminated
with phenols and chlorophenols using soybean seedhulls. Water, Air,
Soil Pollut. 154:151–166; 2004.
Gibson, S. I. Sugar and phytohormone response pathways: navigating a
signaling network. J. Exp. Bot. 55:253–264; 2004.
Gijzen, M. A deletion mutation at the ep locus causes low seed coat
peroxidase activity in soybean. Plant J. 12:991– 998; 1997.
Gijzen, M.; Kuflu, K.; Qutob, D.; Chernys, J. T. A class I chitinase from
soybean seed coat. J. Exp. Bot. 52:2283–2289; 2001.
Gijzen, M.; Miller, S. S.; Bowman, L.-A.; Batchelor, A. K.; Boutilier, K.;
Miki, B. L. A. Localization of peroxidase mRNAs in soybean seeds
by in situ hybridization. Plant Mol. Biol. 41:57–63; 1999a.
Gijzen, M.; Miller, S. S.; Kuflu, K.; Buzzell, R. I.; Miki, B. L. A. Hydrophobic
protein synthesized in the pod endocarp adheres to the seed surface.
Plant Physiol. 120:951–959; 1999b.
Gijzen, M.; van Huystee, R.; Buzzell, R. I. Soybean seed coat peroxidase: a
comparison of high and low activity genotypes. Plant Physiol.
103:1061–1066; 1993.
Gijzen, M.; Weng, C.; Kuflu, K.; Woodrow, L.; Yu, K.; Poysa, V. Soybean
seed lustre phenotype and surface protein cosegregate and map to
linkage group E. Genome 46:659– 664; 2003.
Gillikin, J. W.; Graham, J. S. Purification and developmental analysis of the
major anionic peroxidase from the seed coat of Glycine max. Plant
Physiol. 96:214– 220; 1991.
Golden, T. A.; Schauer, S. E.; Lang, J. D.; Pien, S.; Mushegian, A. R.;
Grossniklaus, U.; Meinke, D. W.; Ray, A. SHORT INTEGUMENTS1/SUSPENSOR1/CARPEL FACTORY, a dicer homolog, is a
maternal effect gene required for embryo development in
Arabidopsis. Plant Physiol. 130:808– 822; 2002.
Golovan, S. P.; Meidinger, R. G.; Ajakaiye, A.; Cottrill, M.; Wiederkehr, M.
Z.; Barney, D. J.; Plante, C.; Pollard, J. W.; Fan, M. Z.; Hayes, M.;
Laursen, J.; Hjorth, J. P.; Hacker, R. R.; Phillips, J. P.; Forsberg, C.
W. Pigs expressing salivary phytase produce low-phosphorus
manure. Nat. Biotechnol. 19:741–745; 2001.
Gonzales, M. D.; Archuleta, E.; Farmer, A.; Gajendran, K.; Grant, D.;
Shoemaker, R.; Beavis, W. D.; Waugh, M. E. The legume
information system (LIS): an integrated information resource for
comparative legume biology. Nucleic Acids Res. 33:D660– D665;
Gonzalez, R.; Valera, J.; Carreira, J.; Polo, F. Soybean hydrophobic protein
and soybean hull allergy. Lancet 346:48– 49; 1995.
Goodman, D. B. P.; Davis, W. L. Insulin accelerates the post germinative
development of several fat-storing seeds. Biochem. Biophys. Res.
Commun. 190:440–446; 1993.
Goto, N. A mucilage polysaccharide secreted from testa of Arabidopsis
thaliana. Arabid. Inf. Serv. 22:143–145; 1985.
Graham, M. A.; Silverstein, K. A. T.; Cannon, S. B.; VandenBosch, K. A.
Computational identification and characterization of novel genes
from legumes. Plant Physiol. 135:1179–1197; 2004.
Granger, C.; Coryell, V.; Khanna, A.; Keim, P.; Vodkin, L.; Shoemaker, R. C.
Identification, structure, and differential expression of members of a
BURP domain containing protein family in soybean. Genome
45:693–701; 2002.
Gressent, F.; Rahioui, I.; Rahbé, Y. Characterization of a high-affinity
binding site for the pea albumin 1b entomotoxin in the weevil
Sitophilus. Eur. J. Biochem. 270:2429–2435; 2003.
Grossniklaus, U.; Schneitz, K. The molecular and genetic bases of ovule and
megagametophyte development. Semin. Cell Dev. Biol. 9:227–238;
Gupta, V.; Lamba, L. C.; Goel, J. P. Comparative study on the seed of two
major pulses vis-à-vis their common adulterant. Plant Sci.
95:283–289; 1985.
Hamilton, A. J.; Baulcombe, D. C. A species of small antisense RNA in
posttranscriptional gene silencing in plants. Science 286:950–952;
Hanada, K.; Hirano, H. Interaction of a 43-kDa receptor-like protein with a
4-kDa hormone-like peptide in soybean. Biochemistry
43:12105–12112; 2004.
Hanada, K.; Nishiuchi, Y.; Hirano, H. Amino acid residues on the surface of
soybean 4-kDa peptide involved in the interaction with its binding
protein. Eur. J. Biochem. 270:2583–2592; 2003.
Hancock, R. E.; Lehrer, R. Cationic peptides: a new source of antibiotics.
Trends Biotechnol. 16:82–88; 1998.
Harborne, J. B. The flavonoids: Advances in research since 1980. New York:
Chapman and Hall; 1988.
Harris, H. B.; Burns, R. E. Influence of tannin content on preharvest seed
germination in sorghum. Agron. J. 62:835–836; 1970.
Harris, W. M. On the development of osteosclereids in seed coats of Pisum
sativum L. New Phytol. 98:135–141; 1984.
Harrison, M. J. Molecular genetics of model legumes. Trends Plant Sci.
5:414–415; 2000.
Helenius, A.; Aebi, M. Intracellular functions of N-linked glycans. Science
291:2364– 2369; 2001.
Henriksen, A.; Mirza, O.; Indiani, C.; Teilum, K.; Smulevich, G.; Welinder,
K. G.; Gajhede, M. Structure of soybean seed coat peroxidase: a
plant peroxidase with unusual stability and haem-apoprotein
interactions. Protein Sci. 10:108–115; 2001.
Herman, E. M.; Helm, R. M.; Jung, R.; Kinney, A. J. Genetic modification
removes an immunodominant allergen from soybean. Plant Physiol.
132:36–43; 2003.
Higgins, T. J. V.; Chandler, P. M.; Randall, P. J.; Spencer, D.; Beach, L. R.;
Blagrove, R. J.; Kort, A. A.; Inglis, A. S. Gene structure, protein
structure and regulation of the synthesis of a sulfur-rich protein in
pea seeds. J. Biol. Chem. 261:11124–11130; 1986.
Hird, D. L.; Worrall, D.; Hodge, R.; Smartt, S.; Paul, W.; Scott, R. The
anther-specific protein encoded by the Brassica napus and
Arabidopsis thaliana A6 gene displays similarity to b-1,3glucanases. Plant J. 4:1023–1033; 1993.
Hood, E. E. Where, oh where has my protein gone? Trends Biotechnol.
22:53–55; 2004.
Hou, D.-X. Potential mechanisms of cancer chemoprevention by
anthocyanins. Curr. Mol. Med. 3:149–159; 2003.
Hungria, M.; Phillips, D. A. Effects of a seed color mutation on rhizobial
nod-gene-inducing flavonoids and nodulation in common bean. Mol.
Plant–Microbe Interact. 6:418–422; 1993.
Ilgoutz, S. C.; Knittel, N.; Lin, J. M.; Sterle, S.; Gayler, K. R. Transcription of
genes for conglutin g and a leginsulin-like protein in narrow-leafed
lupin. Plant Mol. Biol. 34:613–627; 1997.
Irving, D. W. Seed structure and histochemisty of Prosopis velutina
(Leguminosae). Bot. Gaz. 145:340–345; 1984.
Izaguirre, P.; Mérola, S.; Beyhaut, R. Seed ontogeny in Adesmia
securigerifolia (Fabaceae-Adesmieae). Nord. J. Bot. 14:547–556;
Jofuku, K. D.; den Boer, B. G. W.; Van Montagu, M.; Okamuro, J. K. Control
of Arabidopsis flower and seed development by the homeotic gene
APETALA2. Plant Cell 6:1211– 1225; 1994.
Jofuku, K. D.; Omidyar, P. K.; Gee, Z.; Okamuro, J. K. Control of seed mass
and seed yield by the floral homeotic gene APETALA2. Proc. Natl
Acad. Sci. USA 102:3117–3122; 2005.
Johnson, C. S.; Kolevski, B.; Smyth, D. R. TRANSPARENT TESTA
GLABRA2, a trichome and seed coat development gene of
Arabidopsis, encodes a WRKY transcription factor. Plant Cell
14:1359– 1375; 2002.
Karakaya, S.; Kavas, A. Antimutagenic activities of some foods. J. Sci. Food
Agric. 79:237–242; 1999.
Karssen, C. M.; Brinkhorst-van der Swan, D. L. C.; Breekland, A. E.;
Koornneef, M. Induction of dormancy during seed development by
endogenous abscisic acid: studies on abscisic acid deficient
genotypes of Arabidopsis thaliana (L.) Heynh. Planta
157:158– 165; 1983.
Kauffmann, S.; Legrand, M.; Geoffroy, P.; Fritig, B. Biological function of
‘pathogenesis-related’ proteins: four PR proteins from tobacco have
1,3-b-glucanase activity. EMBO J. 6:3209–3212; 1987.
Keller, B. Structural cell wall proteins. Plant Physiol. 101:1127–1130;
Khanna, P.; Jain, S. C.; Panagariya, A.; Dixit, V. P. Hypoglycemic
activity of polypeptide-p from a plant source. J. Nat. Prod.
44:648–655; 1981.
Kitamura, S.; Shikazono, N.; Tanaka, A. TRANSPARENT TESTA 19 is
involved in the accumulation of both anthocyanins and proanthocyanidins in Arabidopsis. Plant J. 37:104– 114; 2004.
Kleis-San Francisco, S. M.; Tierney, M. L. Isolation and characterization of a
proline-rich cell wall protein from soybean seedlings. Plant Physiol.
94:1897– 1902; 1990.
Klucher, K. M.; Chow, H.; Reiser, L.; Fischer, R. L. The AINTEGUMENTA
gene of Arabidopsis required for ovule and female gametophyte
development is related to the floral homeotic gene APETALA2. Plant
Cell 8:137– 153; 1996.
Komatsu, S.; Hirano, H. Plant basic 7S globulin-like proteins have insulin
and insulin-like growth factor binding activity. FEBS Lett.
294:210– 212; 1991.
Koornneef, M. The complex syndrome of ttg mutants. Arabid. Inf. Serv.
18:45–51; 1981.
Kuang, A.; Xiao, Y.; Musgrave, M. E. Cytochemical localization of reserves
during seed development in Arabidopsis thaliana under spaceflight
conditions. Ann. Bot. 78:343–351; 1996.
Kuo, T. M.; Lowel, C. A.; Smith, P. T. Changes in soluble carbohydrates and
enzymatic activities in maturing soybean seed tissues. Plant Sci.
125:1– 11; 1997.
Léon-Kloosterziel, K. M.; Keijzer, C. J.; Koornneef, M. A seed shape mutant
of Arabidopsis that is affected in integument development. Plant Cell
6:385– 392; 1994.
Leubner-Metzger, G. Seed after-ripening and over-expression of class I b1,3-glucanase confer maternal effects on tobacco testa rupture and
dormancy release. Planta 215:959–968; 2002.
Leubner-Metzger, G. b-1,3-glucanase gene expression in low-hydrated seeds
as a mechanism for dormancy release during tobacco after-ripening.
Plant J. 41:133–145; 2005.
Lindstrom, J. T.; Vodkin, L. O. A soybean cell wall protein is affected by
seed color genotype. Plant Cell 3:561–571; 1991.
Ma, F.; Cholewa, E.; Mohamed, T.; Petersen, C. A.; Gijzen, M. Cracks in the
palisade cuticle of soybean seed coats correlate with their
permeability to water. Ann. Bot. 94:213–228; 2004a.
Ma, F.; Peterson, C. A.; Gijzen, M. Reassessment of the pits and antipits in
soybean seeds. Can. J. Bot. 82:654–662; 2004b.
Maguire, T. L.; Grimmond, S.; Forrest, A.; Iturbe-Ormaetxe, I.; Meksem, K.;
Gresshoff, P. Tissue-specific gene expression in soybean (Glycine
max) detected by cDNA microarry analysis. J. Plant Physiol.
159:1361–1374; 2002.
Malik, K.; Wu, K.; Li, X.-Q.; Martin-Heller, T.; Hu, M.; Foster, E.; Tian, L.;
Wang, C.; Ward, K.; Jordan, M.; Brown, D.; Gleddie, S.; Simmonds,
D.; Zheng, S.; Simmonds, J.; Miki, B. A constitutive gene expression
system derived from the tCUP cryptic promoter sequence. Theor.
Appl. Genet. 105:505–514; 2002.
Manning, J. C.; van Staden, J. The functional differentiation of the testa in
seed of Indigofera parvivlara (Leguminosae: Papilionoideae). Bot.
Gaz. 148:23–34; 1987.
Marcus, A.; Greenberg, J.; Averyhart-Fullard, V. Repetitive proline-rich
proteins in the extracellular matrix of the plant cell. Physiol. Plant.
81:273–279; 1991.
Marles, M. A. S.; Gruber, M. Y.; Scoles, G. J.; Muir, A. D. Pigmentation in
the developing seed coat and seedling leaves of Brassica carinata is
controlled at the dihydroflavonol reductase locus. Phytochemistry
62:663–672; 2003a.
Marles, M. A. S.; Ray, H.; Gruber, M. Y. New perspectives on
proanthocyanidin biochemistry and molecular regulation. Phytochemistry 64:367–383; 2003b.
Martı́nez, C. J.; Loarca-Piña, G.; Ortı́z, G. D. Antimutagenic activity of
phenolic compounds, oligosaccharides and quinolizidinic alkaloids
from Lupinus campestris seeds. Food Addit. Contam. 20:940–948;
Matsui, K.; Hiratsu, K.; Koyama, T.; Tanaka, H.; Ohme-Takagi, M. A
chimeric AtMYB23 repressor induces hairy roots, elongation of
leaves and stems, and inhibition of the deposition of mucilage on
seed coats in Arabidopsis. Plant Cell Physiol. 46:147–155; 2005.
Memelink, J.; Linthorst, H. J.; Schilperoort, R. A.; Hoge, J. H. Tobacco
genes encoding acidic and basic isoforms of pathogenesis-related
proteins display different expression patterns. Plant Mol. Biol.
14:119–126; 1990.
Miklas, P. N.; Townsend, C. E.; Ladd, S. L. Seed coat anatomy and the
scarification of cicer milkvetch seed. Crop Sci. 27:766–772; 1987.
Miller, S. S.; Bowman, L. A.; Gijzen, M.; Miki, B. L. A. Early development of
the seed coat of soybean (Glycine max). Ann. Bot. 84:297–304;
Modrusan, Z.; Reiser, L.; Feldmann, K. A.; Fischer, R. L.; Haughn, G. W.
Homeotic transformation of ovules into carpel-like structures in
Arabidopsis. Plant Cell 6:333–349; 1994.
Moraes, R. A.; Sales, M. P.; Pinto, M. S. P.; Silva, L. B.; Oliveira, A. E. A.;
Machado, O. L. T.; Fernandes, K. V. S.; Xavier-Filho, J. Lima bean
(Phaseolus lunatus) seed coat phaseolin is detrimental to the cowpea
weevil (Callosobruchus maculates). Braz. J. Med. Biol. Res.
33:191–198; 2000.
Nakajima, M.; Nakayama, A.; Xu, Z.-J.; Yamaguchi, I. Gibberellin induces
a-amylase gene in seed coat of Ipomoea nil immature seeds. Biosci.
Biotechnol. Biochem. 68:631–637; 2004.
Nakamura, T.; Yang, D.; Kalaiselvi, S.; Uematsu, Y.; Takahashi, R. Genetic
analysis of net-like cracking in soybean coats. Euphytica
133:179–184; 2003.
Nakaune, S.; Yamada, K.; Kondo, M.; Kato, T.; Tabata, S.; Nishimura, M.;
Hara-Nishimura, I. A vacuolar processing enzyme, dVPE, is involved
in seed coat formation at the early stage of seed development. Plant
Cell 17:876–887; 2005.
Ndakidemi, P. A.; Dakora, F. D. Legume seed flavonoids and nitrogenous
metabolites as signals and protectants in early seedling development.
Funct. Plant Biol. 30:729–745; 2003.
Nesi, N.; Debeaujon, I.; Jond, C.; Pelletier, G.; Caboche, M.; Lepiniec, L.
The TT8 gene encodes a basix Helix-Loop-Helix domain protein
required for expression of DFR and BAN genes in Arabidopsis
siliques. Plant Cell 12:1863–1878; 2000.
Nesi, N.; Debeaujon, I.; Jond, C.; Stewart, A. J.; Jenkins, G. I.; Caboche, M.;
Lepiniec, L. The TRANSPARENT TESTA16 locus encodes the
ARABIDOPSIS BSISTER MADS domain protein and is required for
proper development and pigmentation of the seed coat. Plant Cell
14:2463–2479; 2002.
Nesi, N.; Jond, C.; Debeaujon, I.; Caboche, M.; Lepiniec, L. The Arabidopsis
TT2 gene encodes an R2R3 MYB domain protein that acts as a key
determinant for proanthocyanidin accumulation in developing seed.
Plant Cell 13:2099–2114; 2001.
Nicholas, C. D.; Lindstrom, J. T.; Vodkin, L. O. Variation of proline rich cell
wall proteins in soybean lines with anthocyanin mutations. Plant
Mol. Biol. 21:145–156; 1993.
Nissum, M.; Schiodt, C.; Welinder, K. G. Reaction of soybean peroxidase
and hydrogen peroxide pH 2.4-12.0, and veratryl alcohol at pH 2.4.
Biochim. Biophys. Acta 1545:339– 348; 2001.
Ohto, M.; Fischer, R. L.; Goldberg, R. B.; Nakamura, K.; Harada, J. J.
Control of seed mass by APETALA2. Proc. Natl Acad. Sci. USA
102:3123–3128; 2005.
Ogawa, T.; Bando, N.; Tsuji, H.; Okajima, H.; Nishikawa, K.; Sasaoka, K.
Investigation of the IgE-binding proteins in soybean by immunobloting with the sera of the soybean-sensitive patients with atopic
dermatitis. J. Nutr. Sci. Vitaminol. 37:555– 565; 1991.
Ogawa, T.; Tsuji, H.; Bando, N.; Kitamura, K.; Zhu, Y. L.; Hirano, H.;
Nishikawa, K. Identification of the soybean allergenic protein, Gly m
Bd 30K, with the soybean seed 34-kDa oil-body-associated protein.
Biosci. Biotechnol. Biochem. 57:1030– 1033; 1993.
Oigiangbe, N. O.; Onigbinde, A. O. The association between some physicochemical characteristics and susceptibility of cowpea (Vigna
unguiculata (L.) Walp) to Callasobruchus maculates (F). J. Stored
Prod. Res. 32:7–11; 1996.
Oliveira, A. E. A.; Gomes, V. M.; Sales, M. P.; Fernandes, K. V. S.; Carlini,
C. R.; Xavier-Filho, J. The toxicity of Jack bean [Canavalia
ensiformis (L.) DC.] canatoxin to plant pathogenic fungi. Rev. Brasil.
Biol. 59:59–62; 1999a.
Oliveira, A. E. A.; Ribeiro, E. S.; da Cunha, M.; Gomes, V. M.; Fernandes,
K. V. S.; Xavier-Filho, J. Insulin accelerates seedling growth of
Canavalia ensiformis (Jack bean). Plant Growth Regul. 43:57–62;
Oliveira, A. E. A.; Sales, M. P.; Machado, O. L. T.; Fernandes, K. V. S.; XavierFilho, J. The toxicity of Jack bean (Canavalia ensiformis) cotyledon and
seed coat proteins to the cowpea weevil (Callosobruchus maculatus).
Entomol. Exp. Appl. 92:249–255; 1999b.
Oliveira, A. E. A.; Sassaki, G. L.; Iacomini, M.; da Cunha, M.; Gomes, V. M.;
Fernandes, B. K. V. S.; Xavier-Filho, J. Isolation and characterization
of a galactorhamnan polysaccharide from the seed coat of Canavalia
ensiformis that is toxic to the cowpea weevil (Callosobruchus
maculatus). Entomol. Exp. Appl. 101:225–231; 2001.
Olszewski, N.; Sun, T.-P.; Gubler, F. Gibberellin signaling: biosynthesis,
catabolism, and response pathways. Plant Cell 14:S61–S80; 2002.
Osusky, M.; Osuska, L.; Hancock, R. E.; Kay, W. W.; Misra, S. Transgenic
potatoes expressing a novel cationic peptide are resistant to late
blight and pink rot. Transgenic Res. 13:181–190; 2004.
Paiva, N. L. An introduction to the biosynthesis of chemicals used in
plant–microbe communication. J. Plant Growth Regul. 19:131–143;
Palmer, R. G.; Kilen, T. C. Qualitative genetics. In: Wilcox, J. R., ed.
Soybeans: Improvement, production and uses, 2nd ed, Madison, WI:
American Society of Agronomy; 1987:135–209.
Pandey, A. K.; Jha, S. S. Development and structure of seeds in
some Genisteae (Papilionoideae-Leguminosae). Flora 181:415–424;
Paria, N.; Deb, D. K.; Chattopadhyay, S. P. Seed-coat anatomy of some
Indian leguminous taxa. J. Plant Anat. Morphol. 7:46–55; 1997.
Patrick, J. W.; Offler, C. E. Compartmentation of transport and transfer event
in developing seeds. J. Exp. Bot. 52:551–564; 2001.
Penfield, S.; Meissner, R. C.; Shoue, D. A.; Carpita, N. C.; Bevan, M. W.
MYB61 is required for mucilage deposition and extrusion in the
Arabidopsis seed coat. Plant Cell 13:2777–2791; 2001.
Percy, J. D.; Philip, R.; Vodkin, L. O. A defective seed coat pattern (Net) is
correlated with the post-transcriptional abundance of soluble
proline-rich cell wall proteins. Plant Mol. Biol. 40:603–613; 1999.
Ragus, L. N. Role of water absorbing capacity in soybean germination and
seedling vigour. Seed Sci. Technol. 15:285–296; 1987.
Ray, S.; Golden, T.; Ray, A. Maternal effects of the short integument
mutation on embryo development in Arabidopsis. Dev. Biol.
180:365–369; 1996.
Ray, A.; Robinson-Beers, K.; Ray, S.; Baker, S. C.; Lang, J. D.; Preuss, D.;
Milligan, S. B.; Gasser, C. S. Arabidopsis floral homeotic gene BELL
(BEL1) controls ovule development through negative regulation of
AGAMOUS gene (AG). Proc. Natl Acad. Sci. USA 91:5761–5765;
Raz, V.; Bergervoet, J. H. W.; Koornneef, M. Sequential steps for
developmental arrest in Arabidopsis seeds. Development
128:243–252; 2001.
Reiser, L.; Modrusan, Z.; Margossian, L.; Samach, A.; Ohad, N.; Haughn, G.
W.; Fischer, R. L. The BELL1 gene encodes a homeodomain protein
involved in pattern formation in the Arabidopsis ovule primordium.
Cell 83:735–742; 1995.
Robinson-Beers, K.; Pruitt, R. E.; Gasser, C. S. Ovule development in wildtype Arabidopsis and two female-sterile mutants. Plant Cell
4:1237–1249; 1992.
Rochat, C.; Boutin, J. P. Metabolism of phloem-borne amino acids in
maternal tissues of fruit of nodulated or nitrate-fed pea plants (Pisum
sativum L.). J. Exp. Bot. 42:207–214; 1991.
Rolletschek, H.; Borisjuk, L.; Koschorreck, M.; Wobus, U.; Weber, H.
Legume embryos develop in a hypoxic environment. J. Exp. Bot.
53:1099–1107; 2002.
Ross, J. A.; Kasum, C. M. Dietary flavonoids: bioavailability, metabolic
effects, and safety. Annu. Rev. Nutr. 22:19–34; 2002.
Sagasser, M.; Lu, G.-H.; Hahlbrock, K.; Weisshaar, B. A. thaliana
TRANSPARENT TESTA1 is involved in seed coat development and
defines the WIP subfamily of plant zinc finger proteins. Genes Dev.
16:138–149; 2002.
Sakai, H.; Medrano, L. J.; Meyerowitz, E. M. Role of SUPERMAN in
maintaining Arabidopsis floral whorl boundaries. Nature
378:199–203; 1995.
Salunkhe, D. K.; Jadhav, S. J.; Kadam, S. S.; Chavan, J. K. Chemical,
biochemical, and biological significance of polyphenols in cereals
and legumes. Crit. Rev. Food Sci. Nutr. 17:277– 305; 1982.
Schiefthaler, U.; Balasubramanian, S.; Sieber, P.; Chevalier, D.; Wisman, E.;
Schneitz, K. Molecular analysis of NOZZLE, a gene involved in
pattern formation and early sporogenesis during sex organ
development in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA
96:11664–11669; 1999.
Schlumbaum, A.; Mauch, F.; Vögeli, U.; Boller, T. Plant chitinases are
potent inhibitors of fungal growth. Nature 324:365–367; 1986.
Schneitz, K. The molecular and genetic control of ovule development. Curr.
Opin. Plant Biol. 2:13–17; 1999.
Schneitz, K.; Hülskamp, M.; Pruitt, R. E. Wild-type ovule development in
Arabidopsis thaliana: a light microscope study of cleared wholemount tissue. Plant J. 7:731–749; 1995.
Schoenbohm, C.; Martens, S.; Eder, C.; Forkmann, G.; Weisshaar, B.
Identification of the Arabidopsis thaliana flavonoid 30 -hydroxylase
gene and functional expression of the encoded P450 enzyme. Biol.
Chem. 381:749– 753; 2000.
Schopfer, P.; Plachy, C.; Frahry, G. Release of reactive oxygen intermediates
(superoxide radicals, hydrogen peroxide, and hydroxyl radicals)
and peroxidase in germinating radish seeds controlled
by light, gibberellin, and abscisic acid. Plant Physiol.
125:1591–1602; 2001.
Schuurmans, A. M. J.; van Dongen, J. T.; Rutjens, B. P. W.; Boonman, A.;
Pieterse, C. M. J.; Borstlap, A. C. Members of the aquaporin family in
the developing pea seed coat include representatives of the PIP, TIP,
and NIP subfamilies. Plant Mol. Biol. 53:655–667; 2003.
Senda, M.; Jumonji, A.; Yumoto, S.; Ishikawa, R.; Harada, T.; Niizeki, M.;
Akada, S. Analysis of the duplicated CHS1 gene related to the
suppression of the seed coat pigmentation in yellow soybeans. Theor.
Appl. Genet. 104:1086–1091; 2002a.
Senda, M.; Kasai, A.; Yumoto, S.; Akada, S.; Ishikawa, R.; Harada, T.;
Niizeki, M. Sequence divergence at chalcone synthase gene in
pigmented seed coat soybean mutants of the Inhibitor locus. Genes
Genet. Syst. 77:341–350; 2002b.
Senda, M.; Masuta, C.; Ohnishi, S.; Goto, K.; Kasai, A.; Sano, T.; Hong, J.-S.;
MacFarlane, S. Patterning of virus-infected Glycine max seed coat is
associated with suppression of endogenous silencing of chalcone
synthase genes. Plant Cell 16:807–818; 2004.
Serrato-Valenti, G.; DeVries, M.; Cornara, L. The hilar region in Leucaena
leucocephala Lam. (De Wit) seed: structure, histochemistry and the
role of the lens in germination. Ann. Bot. 75:569–574; 1995.
Sharma, N. K.; Sharma, K. C. Development and structure of seedcoat in
Tephrosia Pers. (Leguminosae). Feddes Repert. 105:287–292; 1994.
Shikazono, N.; Yokota, Y.; Kitamura, S.; Suzuki, C.; Watanabe, H.; Tano, S.;
Tanaka, A. Mutation rate and novel tt mutants of Arabidopsis
thaliana induced by carbon ions. Genetics 163:1449–1455; 2003.
Shimizu, T.; Akada, S.; Senda, M.; Ishikawa, R.; Harada, T.; Niizeki, M.;
Dube, S. K. Enhanced expression and differential inducibility of
soybean chalcone synthase genes by supplemental UV-B in darkgrown seedlings. Plant Mol. Biol. 39:785–795; 1999.
Shirley, B. W.; Hanley, S.; Goodman, H. M. Effects of ionizing radiation on a
plant genome: analysis of two Arabidopsis transparent testa
mutations. Plant Cell 4:333–347; 1992.
Shirley, B. W.; Kubasek, W. L.; Storz, G.; Bruggemann, E.; Koornneef, M.;
Ausubel, F. M.; Goodman, H. M. Analysis of Arabidopsis mutants
deficient in flavonoid biosynthesis. Plant J. 8:659–671; 1995.
Shoemaker, R.; Keim, P.; Vodkin, L.; Erpelding, J.; Coryell, V.; Khann, A.;
Bolla, B.; Marra, M.; Hillier, L.; Kucaba, T.; Martin, J.; Beck, C.;
Wylie, T.; Underwood, K.; Steptoe, M.; Theising, B.; Allen, M.;
Bowers ,Y.; Person, B.; Swaller, T.; Gibbons, M.; Pape, D.; Harvey,
N.; Schurk,R.; Ritter, E.; Kohn, S.; Shin, T.; Jackson, Y.; Cardenas,
M.; McCann,R.; Waterston, R.; Wilson, R. Public Soybean EST
Project. (Available at website:
viewer.fcgi?db ¼ nucleotide&val ¼ 10846676); 1999a.
Shoemaker, R.; Keim, P.; Vodkin, L.; Erpelding, J.; Coryell, V.; Khann, A.;
Bolla, B.; Marra, M.; Hillier, L.; Kucaba, T.; Martin, J.; Beck, C.;
Wylie, T.; Underwood, K.; Steptoe, M.; Theising, B.; Allen, M.;
Bowers ,Y.; Person, B.; Swaller, T.; Gibbons, M.; Pape, D.; Harvey,
N.; Schurk, R.; Ritter, E.; Kohn, S.; Shin, T.; Jackson, Y.; Cardenas,
M.; McCann, R.; Waterston, R.; Wilson, R. Public Soybean EST
Project. (Available at website:
viewer.fcgi?db ¼ nucleotide&val ¼ 7589170); 1999b.
Shoemaker, R.; Keim, P.; Vodkin, L.; Erpelding, J.; Coryell, V.; Khann, A.;
Bolla, B.; Marra, M.; Hillier, L.; Kucaba, T.; Martin, J.; Beck, C.;
Wylie, T.; Underwood, K.; Steptoe, M.; Theising, B.; Allen, M.;
Bowers ,Y.; Person, B.; Swaller, T.; Gibbons, M.; Pape, D.; Harvey,
N.; Schurk, R.; Ritter, E.; Kohn, S.; Shin, T.; Jackson, Y.; Cardenas,
M.; McCann, R.; Waterston, R. and Wilson, R. Public Soybean EST
Project. (Available at website:
viewer.fcgi?db ¼ nucleotide&val ¼ 14259925); 1999c.
Shoemaker, R.; Keim, P.; Vodkin, L.; Retzel, E.; Clifton, S. W.; Waterston,
R.; Smoller, D.; Coryell, V.; Khana, A.; Erpelding, J.; Gai, X.;
Brendel, V.; Raph-Schmidt, C.; Shoop, E. G.; Vielweber, C. J.;
Schmatz, M.; Pape, D.; Bowers, Y.; Theising, B.; Martin, J.; Dante,
M.; Wylie, T.; Granger, C. A compilation of soybean ESTs:
generation and analysis. Genome 45:329–338; 2002.
Showalter, A. M. Structure and function of plant cell wall proteins. Plant Cell
5:9–23; 1993.
Sieber, P.; Petrascheck, M.; Barberis, A.; Schneitz, K. Organ polarity in
Arabidopsis. NOZZLE physically interacts with members of the
YABBY family. Plant Physiol. 135:2172– 2185; 2004.
Silva, L. B.; Sales, M. P.; Oliveira, A. E. A.; Machado, O. L. T.; Fernandes, K. V.
S.; Xavier-Filho, J. The seed coat of Phaseolus vulgaris interferes with
the development of the cowpea weevil [Callosobruchus maculatus (F.)
(Coleoptera: Bruchidae)]. Ann. Braz. Acad. Sci 76:57–65; 2004.
Silva, L. B.; Santos, S. S. S.; Azevedo, C. R.; Cruz, M. A. L.; Venâncio, T. M.;
Cavalcante, C. P.; Uchôa, A. F.; Astolfi Filho, S.; Oliveira, A. E. A.;
Fernandes, K. V. S.; Xavier-Filho, J. The leaves of green plants as
well as a cyanobacterium, a red alga, and fungi contain insulin-like
antigens. Braz. J. Med. Biol. Res. 35:297–303; 2002.
Simbaya, J.; Slominski, B. A.; Rakow, G.; Campbell, L. D.; Downey, R. K.;
Bell, J. M. Quality characteristics of yellow-seeded Brassica seed
meals: Protein, carbohydrates, and dietary fiber components. J. Agric.
Food Chem. 43:2062–2066; 1995.
Simmons, C. R.; Litts, J. C.; Huang, N.; Rodriguez, R. L. Structure of a rice
b-glucanase gene regulated by ethylene, cytokinin, wounding,
salicylic acid and fungal elicitors. Plant Mol. Biol. 18:33– 45; 1992.
Skinner, D. J.; Hill, T. A.; Gasser, C. S. Regulation of ovule development.
Plant Cell 16:32–45; 2004.
Sornsathapornkul, P.; Owens, J. N. Zygotic embryo development in a tropical
Acacia hybrid (Acacia mangium Willd £ A. auriculiformis A. Cunn.
ex Benth.). Int. J. Plant Sci. 160:445– 458; 1999.
Souza, F. H. D. D.; Marcos-Filho, J. The seed coat as a modulator of seed–
environment relationships in Fabaceae. Rev. Bras. Bot. 24:365–375;
Stacey, G.; Vodkin, L.; Parrott, W. A.; Shoemaker, R. C. National science
foundation-sponsored workshop report. Draft plan for soybean
genomics. Plant Physiol. 135:59–70; 2004.
Stavric, B. Antimutagens and anticarcinogens in foods. Food Chem. Toxicol.
32:79– 90; 1994.
Swanson, M. C.; Li, J. T.; Wentz-Murtha, P. E.; Trudeau, W. L.; FernandezCaldas, E.; Greife, A.; Rodrigo, M. A. J.; Morell, F.; Reed, C. E.
Source of the aeroallergen of soybean dust: a low molecular mass
glycopeptide from the soybean tela. J. Allergy Clin. Immunol.
87:783–788; 1991.
Tan, N.-H.; Rahim, Z. H. A.; Khor, H.-T.; Wong, K.-C. Winged bean
(Psophocarpus tetragonolobus) tannin level, phytate content and
hemagglutinating activity. J. Agric. Food Chem. 31:916–917; 1983.
Thibaud-Nissen, F.; Shealy, R. T.; Khanna, A.; Vodkin, L. O. Clustering of
microarray data reveals transcript patterns associated with somatic
embryogenesis in soybean. Plant Physiol. 132:118–136; 2003.
Thompson, R. D.; Hueros, G.; Becker, H.-A.; Maitz, M. Development and
functions of seed transfer cells. Plant Sci. 160:775– 783; 2001.
Toda, K.; Yang, D.; Yamanaka, N.; Watanaba, S.; Harada, K.; Takahashi, R.
A single-base deletion in soybean flavonoid 30 -hydroxylase gene is
associated with gray pubescence color. Plant Mol. Biol. 50:187–196;
Todd, J. J.; Vodkin, L. O. Duplications that suppress and deletions that
restore expression from a chalcone synthase multigene family. Plant
Cell 8:687–699; 1996.
Tomlinson, K. L.; McHugh, S.; Labbe, H.; Grainger, J. L.; James, L. E.;
Pomeroy, K. M.; Mullin, J. W.; Miller, S. S.; Dennis, D. T.; Miki, B.
L. A. Evidence that the hexose-to-sucrose ratio does not control the
switch to storage product accumulation in oilseeds: analysis of
tobacco seed development and effects of overexpressing apoplastic
invertase. J. Exp. Bot. 55:2291–2303; 2004.
Trivedi, B. S.; Gupta, M. T. Seed coat structure in some species of Atylosia
phaseoleae cajaninae. Scan. Microscopy 1:1465– 1474; 1987.
Tuteja, J. H.; Clough, S. J.; Chan, W.-C.; Vodkin, L. O. Tissue-specific gene
silencing mediated by a naturally occurring chalcone synthase gene
cluster in Glycine max. Plant Cell 16:819–835; 2004.
Usadel, B.; Kuschinsky, A. M.; Rosso, M. G.; Eckermann, N.; Pauly, M.
RHM2 is involved in mucilage pectin synthesis and is required for
the development of the seed coat in Arabidopsis. Plant Physiol.
134:286–295; 2004.
Van Caeseele, L.; Mills, J. T.; Sumner, M.; Gillespie, R. Cytology of
mucilage production in the seed coat of candle canola (Brassica
campestris). Can. J. Bot. 59:292–300; 1981.
Van Caeseele, L.; Mills, J. T.; Sumner, M.; Gillespie, R. Cytological study of
palisade development in the seed coat of candle canola. Can. J. Bot.
60:2469–2475; 1982.
VandenBosch, K. A.; Stacey, G. Summaries of legume genomics projects
from around the globe. Community resources for crops and models.
Plant Physiol. 131:840–865; 2003.
van Dongen, J. T.; Ammerlaan, A. M. H.; Wouterlood, M.; van Aelst, A. C.;
Borstlap, A. C. Structure of the developing pea seed coat and the postphloem transport pathway of nutrients. Ann. Bot. 91:729– 737; 2003.
Vaughan, J. G.; Whitehouse, J. M. Seed structure and the taxonomy of the
Cruciferae. Bot. J. Linn. Soc. 64:383–409; 1971.
Venâncio, T. M.; Oliveira, A. E. A.; Silva, L. B.; Machado, O. L. T.;
Fernandes, K. V. S.; Xavier-Filho, J. A protein with amino acid
sequence homology to bovine insulin is present in the legume Vigna
unguiculata (cowpea). Braz. J. Med. Biol. Res. 36:1167–1173; 2003.
Villanueva, J. M.; Broadhvest, J.; Hauser, B. A.; Meister, R. J.; Schneitz, K.;
Gasser, C. S. INNER NO OUTER regulates abaxial–adaxial
patterning in Arabidopsis ovules. Genes Dev. 13:3160–3169; 1999.
Walker, A. R.; Davison, P. A.; Bolognesi-Winfield, A. C.; James, C. M.;
Srinivasan, N.; Blundell, T. L.; Esch, J. J.; Marks, M. D.; Gray, J. C.
The TRANSPARENT TESTA GLABRA1 locus, which regulates
trichome differentiation and anthocyanin biosynthesis in Arabidopsis,
encodes a WD40 repeat protein. Plant Cell 11:1337– 1349; 1999.
Wan, L.; Xia, Q.; Qiu, X.; Selvaraj, G. Early stages of seed development in
Brassica napus: a seed coat-specific cysteine proteinase associated
with programmed cell death of the inner integument. Plant J.
30:1–10; 2002.
Wang, C.-S.; Todd, J. J.; Vodkin, L. O. Chalcone synthase mRNA and
activity are reduced in yellow soybean seed coats with dominant I
alleles. Plant Physiol. 105:739–748; 1994.
Wang, H. L.; Grusak, M. A. Structure and development of Medicago
truncatula pod wall and seed coat. Ann. Bot. 95:737–747; 2005.
Wang, X.; Warkentin, T. D.; Briggs, C. J.; Oomah, B. D.; Campbell, C. G.;
Woods, S. Total phenolics and condensed tannins in field pea (Pisum
sativum L.) and grass pea (Lathyrus sativus L.). Euphytica
101:97–102; 1998.
Weber, H.; Borisjuk, L.; Heim, U.; Buchner, P.; Wobus, U. Seed coat
associated invertases of fava bean control both unloading and storage
functions: cloning of cDNAs and cell type-specific expression. Plant
Cell 7:1835–1846; 1995.
Weber, H.; Borisjuk, L.; Wobus, U. Controlling seed development and seed
size in Vicia faba: a role for seed coat-associated invertases and
carbohydrate state. Plant J. 10:823–834; 1996.
Weber, H.; Borisjuk, L.; Wobus, U. Molecular physiology of legume seed
development. Annu. Rev. Plant Biol. 56:253– 279; 2005.
Weber, H.; Heim, U.; Golombek, S.; Borisjuk, L.; Manteuffel, R.; Wobus, U.
Expression of a yeast-derived invertase in developing cotyledons of
Vicia narbonensis alters the carbohydrate state and affects storage
functions. Plant J. 16:163– 172; 1998.
Weijers, D.; van Hamburg, J.-P.; van Rijn, E.; Hooykaas, P. J. J.; Offringa,
R. Diphtheria toxin-mediated cell ablation reveals interregional
communication during Arabidopsis seed development. Plant Physiol.
133:1882–1892; 2003.
Welinder, K. G. Superfamily of plant, fungal and bacterial peroxidases. Curr.
Opin. Struct. Biol. 2:388– 393; 1992.
Welinder, K. G.; Larsen, Y. B. Covalent structure of soybean seed coat
peroxidase. Biochim. Biophys. Acta 1698:121–126; 2004.
Western, T. L.; Burn, J.; Tan, W. L.; Skinner, D. J.; Martin-McCaffrey, L.;
Moffat, B. A.; Haughn, G. W. Isolation and characterization of
mutants defective in seed coat mucilage secretory cell development
in Arabidopsis. Plant Physiol. 127:998–1011; 2001.
Western, T. L.; Haughn, G. W. BELL1 and AGAMOUS genes promote ovule
identity in Arabidopsis thaliana. Plant J. 18:329–336; 1999.
Western, T. L.; Skinner, D. J.; Haughn, G. W. Differentiation of mucilage
secretory cells of the Arabidopsis seed coat. Plant Physiol.
122:345–355; 2000.
Western, T. L.; Young, D. S.; Dean, G. H.; Tan, W. L.; Samuels, A. L.;
Haughn, G. W. MUCILAGE-MODIFIED4 encodes a putative pectin
biosynthetic enzyme developmentally regulated by APETALA2
seed coat. Plant Physiol. 134:296– 306; 2004.
Wilcox, J. R. Performance and use of seedcoat mutants in soybean. Crop Sci.
28:30–32; 1988.
Windsor, J. B.; Symonds, V. V.; Mendenhall, J.; Lloyd, A. M. Arabidopsis
seed coat development: morphological differentiation of the outer
integument. Plant J. 22:483–493; 2000.
Wingender, R.; Rohrig, H.; Horicke, C.; Wing, D.; Schell, J. Differential
regulation of soybean chalcone synthase genes in plant defense,
symbiosis and upon environmental stimuli. Mol. Gen. Genet.
218:315–322; 1989.
Winkel-Shirley, B. Flavonoid biosynthesis. A colourful model for genetics,
biochemistry, cell biology, and biotechnology. Plant Physiol.
126:485–493; 2001.
Wisman, E.; Hartmann, U.; Sagasser, M.; Baumann, E.; Palme, K.; Hahlbrock,
K. Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana
population generate phenylpropanoid biosynthesis phenotypes. Proc.
Natl Acad. Sci. USA 95:12432–12437; 1998.
Wobus, U.; Weber, H. Sugars as signal molecules in plant seed
development. Biol. Chem. 380:937–944; 1999.
Wolf, W. J.; Baker, F. L. Scanning electron microscopy of soybeans. Cereal
Sci. Today 17:125–130; 1972.
Wu, S.; Druka, A.; Horvath, H.; Kleinhofs, A.; Kannangara, C. G.; von
Wettstein, D. Functional characterization of seed coat-specific
members of the barley germin gene family. Plant Physiol. Biochem.
38:685–698; 2000.
Xavier-Filho, J.; Oliveira, A. E. A.; da Silva, L. B.; Azevedo, C. R.;
Venâncio, T. M.; Machado, O. L. T.; Oliva, M. L.; Fernandes, K. V.
S.; Xavier-Neto, J. Plant insulin or glucokinin: a conflicting issue.
Bras. J. Plant Physiol. 15:67– 78; 2003.
Xie, D.-Y.; Sharma, S. B.; Paiva, N. L.; Ferreira, D.; Dixon, R. A. Role of
anthocyanidin reductase, encoded by BANYULS in plant flavonoid
biosynthesis. Science 299:396–399; 2003.
Xudong, Y.; Al-Babili, S.; Klöti, A.; Zhang, J.; Lucca, P.; Beyer, P.;
Potrykus, I. Engineering the provitamin A (b-carotene) biosynthetic
pathway into (carotenoid-free) rice endosperm. Science
287:303–305; 2000.
Yaklich, R. W.; Barla-Szabo, G. Seed coat cracking in soybean. Crop Sci.
33:1016–1019; 1993.
Yaklich, R. W.; Vigil, E. L.; Erbe, E. F.; Wergin, W. P. The fine structure of
aleurone cells in the soybean seed coat. Protoplasma 167:108–119;
Yamazaki, T.; Takaoka, M.; Katoh, E.; Hanada, K.; Sakita, M.; Sakata, K.;
Nishiuchi, Y.; Hirano, H. A possible physiological function and the
tertiary structure of a 4-kDa peptide in legumes. Eur. J. Biochem.
270:1269–1276; 2003.
Yeung, E. Adhesion of endosperm cells to the inner surface of the bean seed
coat. J. Struct. Biol. 105:103–110; 1990.
Young, N. D.; Mudge, J.; Ellis, T. H. N. Legume genomics: more than peas in
a pod. Curr. Opin. Plant Biol. 6:199–204; 2003.
Zeng, C.-L.; Wang, J.-B.; Liu, A.-L.; Wu, X.-M. Seed coat microsculpturing
changes during seed development in diploid and amphidiploid
Brassica species. Ann. Bot. 93:555–566; 2004.
Zhang, F.; Gonzalez, A.; Zhao, M.; Payne, C. T.; Lloyd, A. A network of
TTG1dependent pathways of Arabidopsis. Development 130:
4859–4869; 2003.