Genetic analysis of seed coat
development in Arabidopsis
George Haughn1
and Abed Chaudhury2
1
Department of Botany, University of British Columbia, 6270 University Blvd, Vancouver, BC, Canada V7R 2T4
2
CSIRO Plant Industry, GPO Box 1600, ACT 2601, Australia
In the angiosperms, fertilization initiates the formation
of the seed from the ovule, including the differentiation
of the seed coat from the ovule integuments. Seed coat
differentiation includes some of the most dramatic
cellular changes of seed development and culminates
in the death of the seed coat cells. Recently, genetic
analyses in Arabidopsis have contributed substantially
to our understanding of many aspects of seed coat
biology and it might not be long before the entire
differentiation pathway is understood. Such an advance
would contribute substantially to our understanding of
many important cellular events, including secondary cell
wall synthesis, cell morphogenesis, vacuolar targeting
and cell death, and would provide tools for the
manipulation of seed dormancy and germination.
Seed coats
In the angiosperms, fertilization results in the formation
of the seed from the ovule (Figure 1). This remarkable
transformation involves the activation and coordination
of the distinct developmental pathways leading to an
embryo, endosperm and seed coat. The seed coat (testa)
consists of several layers of specialized maternal cell types
that provide an important interface between the embryo
and the external environment during embryogenesis,
dormancy and germination. Differentiation of the seed
coat from the ovule integuments includes some of the most
dramatic cellular changes observed during seed development
and culminates in the death of the seed coat cells.
Even in death, the specialized cell types impart protection,
improve dormancy and germination, and enhance seed
dispersal. In addition, the seed coat cells of some species,
such as the seed coat epidermal trichomes of cotton (which
we weave into textiles), are an important commercial
commodity.
Relative to those of many other tissues of the seed, the
molecular and cellular events underlying seed coat
differentiation have received little attention. However, in
recent years, the use of genetic analysis in Arabidopsis
has contributed substantially to our understanding of
many aspects of seed coat biology. In this article, we
outline this progress, highlighting some of the interesting
questions, including how seed coat development is
initiated and coordinated with other seed tissues, what
the structure and function of the various seed coat cell
layers are, and how the differentiation of the specialized
cell types is regulated. Owing to space limitations, we do
not discuss the genes required for ovule morphogenesis
that also impact seed coat development but instead refer
readers to reviews of the topic [1,2].
Overview of Arabidopsis seed coat development
In response to fertilization, the Arabidopsis seed coat
differentiates primarily from cells of the ovule integuments
(Figure 2a) over a period of 2–3 weeks [3–6]. Cells in
both layers of the outer integument (Figure 2a, layers 1,2)
and all three layers of the inner integument (Figure 2a,
layers 3–5) go through a dramatic period of growth in the
first few days after fertilization through both cell division
and expansion (Figure 2b). The five cell layers follow one
of four distinct fates (Figure 2c–e). Cells of the innermost
layer (the endothelium; Figure 2a, layer 5) synthesize
proanthocyanidin (PA) flavonoid compounds, also known
as condensed tannins [7], which accumulate in the central
vacuole during the first week after fertilization and later
oxidize, imparting a brown color to the seed coat. By
contrast, cells of the other two inner integument layers
(Figure 2a, layers 3,4) do not appear to differentiate
further and are crushed together as the seed develops
(Figure 2d,e). Cells of both outer integument layers
(Figure 2a, layers 1,2) accumulate starch-containing
TRENDS in Plant Science
Integuments
Seed coat
Endosperm
Embryo
Egg cell
Central cell
(a) (b)
Figure 1. The seed is derived from the ovule (a). The egg and sperm nuclei fuse to
form the diploid zygote that develops into the embryo (b). A second sperm cell
fuses with the diploid central cell, initiating triploid endosperm development (b).
These fertilization events induce the maternal diploid integument cells to
differentiate into a seed coat (b).
Corresponding author: Haughn, G. (haughn@interchange.ubc.ca).
Available online 8 September 2005
Review TRENDS in Plant Science Vol.10 No.10 October 2005
www.sciencedirect.com 1360-1385/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2005.08.005
amyloplasts during the growth phase (Figure 2b) before
diverging in fate. The subepidermal layer (Figure 2a,
layer 2) produces a thickened wall on the inner tangential
side of the cell (palisade, Figure 2c–e). The cells of the
epidermal layer (Figure 2a, layer 1) synthesize and secrete
a large quantity of mucilage (a pectinaceous carbohydrate)
into the apoplast specifically at the junction of the outer
tangential and radial cell walls (Figure 2c). As mucilage
deposition proceeds, the vacuole contracts, leaving a cytoplasmic
column in the center of the cell surrounded by a
donut-shaped apoplastic space filled with mucilage.
Following mucilage synthesis, a secondary cell wall is
deposited that completely fills the space occupied by the
cytoplasmic column, forming the columella (Figure 2d,e).
During the later stages of seed development, the cells of
all seed coat layers die. The structure of the epidermal
cells is preserved by the mucilage and columella, and the
remaining layers are crushed together by the end of seed
maturation. PAs are apparently released from the endothelial
cells and impregnate the inner three cell layers
during this period (Figure 2e).
Interactions between seed coat and endosperm during
early seed coat development
Seed coat growth and differentiation are initiated by
fertilization and proceed coordinately with those of the
embryo and endosperm [3–5]. Unlike the embryo and
endosperm, the seed coat is not directly involved in the
fertilization process. Therefore, one or more events during
or following fertilization must signal the seed coat to
develop coordinately with the other tissues. Two recent
studies have used mutants to provide evidence for the
influence of the endosperm on both the growth and the
differentiation of the seed coat.
The control of seed coat growth relative to that of the
embryo and endosperm appears to be complex [8,9]. Seed
coat growth occurs soon after fertilization and involves
both cell division and cell elongation (Figure 2b). Limitations
in seed coat cell division are compensated for by
an increase in cell elongation, but the converse is not
true. This indicates that cell elongation, but not division,
responds to seed size. Mutations in the HAIKU gene result
in limited growth of the syncytial endosperm [9]. This
defect in endosperm growth also has a non-cell-autonomous
effect on growth of the developing seed coat such
that cell elongation (but not cell division) in the expanding
seed coat is restricted. These results suggest that the
growing endosperm regulates the extent to which the
ovule integument cells elongate following the initiation
of seed coat development. Conversely, loss of function
mutations in the TRANSPARENT TESTA GLABRA2
(TTG2) gene [10] restrict cell elongation of the seed coat,
which limits endosperm growth in a non-cell-autonomous
manner [8]. This ‘cross-talk’ between the developing
endosperm and seed coat appears to coordinate growth
between the endosperm and seed coat, ultimately establishing
seed size. Although the mechanism for the
coordinate control of growth is not known, mechanical
force or restraint is consistent with all data, but
endosperm–seed-coat signaling cannot be ruled out.
Evidence for the involvement of the endosperm in the
initiation of seed coat development has come from the
analysis of seed coat development (G. Haughn et al.,
unpublished) in sporophytic (titan1 [11]) and gametophytic
(fertilization independent seed development1–fertilization
1
2
5
1
2
3
4
5
En
1
2
5 En
1
2
5
Al
Em
1
Al
Em
Oi
Ii Es
En
(a)
(c)
(d)
(e)
(b)
4
3
4
3
3/4
2–5
Figure 2. Development of the ovule integuments into a seed coat following
fertilization. This figure shows several stages (a–e) of seed development for the
whole seed (left) and a detail of the developing seed coat (right). The two cell layers
of the ovule outer integument [(a), right, 1,2] and three cell layers of the inner
integument [(a), right, 3–5] undergo a period of growth within the first 5 days after
fertilization (b). Cells of individual layers differentiate (c) into specialized cell types
including endothelium (5), palisade (2) and epidermis (1), a process that is almost
complete 10 days after fertilization (d). By seed maturity (15 days) (e), cells of all
layers are dead and have been crushed together, except for the epidermis, the
shape of which is maintained by the thick secondary cell wall of the columella.
Single arrows indicate starch-containing plastids (b), mucilage in the apoplast (c) or
secondary cell wall forming in the epidermis (d). The double arrows indicate the
secondary cell wall of the palisade (c,d). Abbreviations: Al, endosperm aleurone;
Em, embryo; En, endosperm; Es, embryo sac; Ii, inner integument; Oi, outer
integument. Scale barZ80 mm (b–d, left); 40 mm (a, left, a–d, right).
Review TRENDS in Plant Science Vol.10 No.10 October 2005 473
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independent seed development3 [12]) lethal mutants.
These studies suggest that a signal from the syncytial
endosperm is sufficient to initiate seed coat development
in cells of the ovule integument. Once initiated, all aspects
of seed coat differentiation, except growth (see below),
appear to proceed independently of both endosperm and
embryo development. The nature of the signal and the
mechanism through which it is enacted are key questions
that still need to be answered.
Endothelial development
The isolation of mutants with altered seed coat color
{transparent testa (tt), transparent testa glabra (ttg) and
banyuls (ban) [7,13,14]} or PA levels [tannin deficient seed
(tds) [15]] have enabled the identification of genes
required for the differentiation of the flavonoid-producing
endothelial cells. Many of these genes have been cloned
and can be grouped into two general classes based on the
function of their products. One class of genes encodes
proteins required for the biosynthesis and compartmentation
of the flavonoid compounds; these include TT3, TT4,
TT5, TT6, TT7, BAN, TT12, TT19, TDS4/TT18 and
AUTOINHIBITED HC
-ATPase ISOFORM 10 (AHA10)
[7,14–18]. These genes have been useful in elucidating the
PA biosynthetic pathway. A notable example was the
discovery of the ban mutant, which accumulates anthocyanin
instead of PAs in endothelial cells [19]. The cloning
of BAN and the characterization of its product identified a
previously unknown but pivotal enzymatic step, anthocyanidin
reductase, in the PA biosynthetic pathway [20–22].
Another important aspect of PA biosynthesis concerns the
targeting of the precursors to the vacuole. The genes TT12,
TT19 and AHA10 encode a putative multidrug and toxic
compound extrusion transporter, glutathione-S-transferase
and HC
-ATPase, respectively, and are believed to be
involved in transporting flavan-3-ols (PA precursors) to
the vacuole. This conclusion is based on the deduced
identity of the gene products and the phenotypes of the
loss of function mutants, which include a late block in PA
biosynthesis (tt12, tt19 and aha10) and disruption in
vacuolar biogenesis in endothelial cells (tt12 and aha10)
[17,18,23]. It is possible that one or more flavonoid compounds
produced by the PA pathway are bound by
glutathione-S-transferase in the cytoplasm and transported
to the vacuole via a vacuolar protein pump that
requires HC
-ATPase activity. However, considerably more
work is required to substantiate and characterize this
putative transport process.
The second class of TT genes encodes transcription
factors (TT1, TT2, TT8, TT16, TTG1, TTG2). TT2 is an
endothelial-specific regulatory protein (a MYB transcription
factor) that is necessary for PA biosynthesis. In
addition to controlling PA biosynthesis in the endothelium,
TT8, a basic helix–loop–helix (bHLH) transcription
factor, also functions in the seed coat epidermis (see
below). Both proteins act, at least in part, by directly
regulating transcription of flavonoid biosynthetic genes,
such as TT3 and BAN, 3–4 days after fertilization (late
globular stage of embryogenesis [6,16,24–26]). TTG1, a
WD40 repeat protein [27], and TTG2, a WRKY protein
[10], have roles in endothelial cells similar to those of TT8
and TT2 but, in addition, are required for the differentiation
of a range of other cells types in the plant,
including trichomes, root hairs and seed coat epidermal
cells [10,28–31] (see below). TTG1 forms an endotheliumspecific
transcription complex with TT8 and TT2 (Figure 3c)
[26] to activate transcription of the target genes. This
complex is analogous to those TTG1 forms with different
bHLH and MYB polypeptides to specify the differentiation
of several other plant cell types [32,33]. By contrast, TTG2
must function downstream of the TTG1 complex because
its transcription is dependent on TTG1 activity (Figure 3)
[10]. However, TTG2 is not required for BAN transcription
[6] and so it must play a different role in PA biosynthesis.
Two other transcription factors, TT1 (a WIP zinc-finger
protein [34]) and TT16 (a MADS protein [35]), are produced
specifically in the endothelium and are required for
PA biosynthesis. The probable role of TT16 is in specifying
endothelial cell type because it is required not only for
normal transcript levels of ban but also for normal endothelial
cell shape and vacuolization. Indeed, cells derived
from all three layers of the inner integument appear to
TRENDS in Plant Science
(a)
(a)
(b)
(b)
(c)
(c)
(d)
(d)
1
2
3
4
5
1
2
3
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5
1
23 4
5
1
2 3 4
5 Al
Ovule
Inner integument Outer integument
Fertilization
Cell growth Cell growth
Endosperm
TT1?, TT16 AP2
Testa epidermis and palisadeEndothelium
Mucilage and secondary
cell wall biosynthesis
Tannin biosynthesis
BAN, TT3
TTG2
MUM4
TTG1, TT8, TT2 TTG1, EGL3/TT8, MYB23?
GL2 TTG2 MYB61
Al
Figure 3. Seed coat differentiation is controlled by a series of regulatory steps
beginning with fertilization of the ovule (a). The developing endosperm influences
integument development into the seed coat (b) and layer-specific regulatory
cascades lead to differentiation (c,d), including an increase in the transcription of
genes encoding enzymes needed for PA and mucilage biosynthesis. 1–5 in (a–d)
indicate the cell layers of the seed coat. Abbreviation: Al, aleurone cells.
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divide and differentiate abnormally in the tt16 mutant,
suggesting that TT16 controls the differentiation of all
cells of the inner integument [6,35]. Thus, TT16 can be
considered to be a global regulator of endothelial
differentiation, lying upstream of the other transcription
factors (Figure 3). The specific role of TT1 is less clear. TT1
is not required for ban transcription [6] and there have
been conflicting reports concerning its role in establishing
endothelial cell shape [6,34]. TT1 could promote aspects of
endothelial differentiation required for PA biosynthesis
that are independent of the TT2–TT8–TTG1 complex and
its targets. Determinating the expression of tt1 and of tt2,
tt8, ttg1, ttg2 and tt16 in the tt1 mutant background might
shed some light on the role of TT1. It is interesting that
TT1 and TT16 are not required for the differentiation of a
small section of seed coat endothelium in the chalazal and
micropylar ends of the seed. The differentiation of such
cells must be under the control of a separate regulator(s).
Seed coat epidermal development
Mutants defective in seed coat epidermal development
have been identified primarily through a decrease in the
amount or chemical composition of the seed coat mucilage
extruded when the seed is exposed to water [mucilage
modified (mum), ttg, myb protein 61 (myb61), apetala2
(ap2), glabra2 (gl2), enhancer of glabra3 (egl3) and tt8]. As
in the case of endothelial differentiation, the epidermal
mutants define genes that fall into two general classes:
those apparently required for mucilage biosynthesis and
those regulating differentiation.
Mutations in five genes result in seed coats with an
altered amount (mum4) or composition (mum1, mum2,
mum3, mum5) of mucilage [31,36]. Mucilage is composed
primarily of pectin, the major component of which is
rhamnogalacturonan 1 (RG1) [4,36–39]. Because little is
known about the biosynthesis of this important carbohydrate,
the MUM genes represent a valuable resource.
Three of these genes have been cloned. Mutations in
mum4 result in a significant decrease in seed coat mucilage.
mum4 encodes an enzyme thought to be required for
the biosynthesis of rhamnose, a monosaccharide that is in
high abundance in mucilage [36,40]. mum4 transcript is
upregulated in the seed coat during the period when
mucilage is synthesized. Interestingly, MUM4 is required
not only for normal levels of mucilage but also for complete
formation of the columella and contraction of the vacuole.
These data suggest that normal mucilage biosynthesis has
a role in cell morphogenesis, an intriguing connection that
needs to be investigated. mum2, the loss of function
phenotype of which includes defects in the extrusion of
mucilage from the seed coat, has recently been shown to
encode a glycosyl hydrolase (G. Dean et al., unpublished).
Mutations in mum5 result in mucilage with altered
cohesive properties. mum5 encodes a pectin methyl
esterase (M. Facette and C.R. Somerville, unpublished).
More extensive biochemical and molecular analyses of
these genes, their products and their roles might shed
light on the biosynthesis and biological properties of
pectinaceous compounds.
Seven additional genes required for normal differentiation
of the seed coat epidermis encode transcription
factors (ttg1, ttg2, myb61, ap2, gl2, egl3 and tt8). A TTG1
regulatory complex, analogous to the one that controls
trichome and root hair differentiation and PA biosynthesis
in the endothelium [6,32], regulates the biosynthesis of
mucilage during seed coat epidermal differentiation.
EGL3 is a bHLH transcription factor that, like TT8,
interacts with TTG1 and is partially redundant with TT8
in promoting mucilage biosynthesis [41]. Thus, both EGL3
and TT8 act as the TTG1 complex bHLH transcription
factor that is involved in epidermal differentiation.
To date, no MYB protein has been clearly identified that
acts in a complex with TTG1 and EGL3/TT8. One possible
candidate is MYB23 because it is closely related in
sequence to other MYB proteins that are known to act
with TTG1 (GL1, WER and TT2) (Figure 3) [32,42–44]. In
addition, transgenic Arabidopsis plants that show ectopic
production of a chimeric MYB23 fused to a transcriptional
repressor domain from the SUPERMAN protein lacks
seed coat mucilage, suggesting that MYB23 could have a
role in seed coat epidermal development [45,46]. Because
loss-of-function mutations in myb23 have no phenotype
and expression in the seed coat has not been demonstrated,
strong conclusions concerning the role of myb23 in
seed coat development cannot be made. Another MYB
gene known to be required for seed coat epidermal differentiation
is myb61 [38]. However, MYB61 it is not a good
candidate for membership of a TTG1 complex because it
belongs to a MYB subfamily that is only distantly related
to GL1/WER and preliminary evidence suggests that
TTG1 and MYB61 do not regulate the same targets in
the seed coat epidermis (see below).
The transcription factors GL2 and TTG2 act downstream
of TTG1 (Figure 3) [10,47,48]. However, despite
the similarity in their loss of function phenotypes in
the seed coat epidermis, the downstream targets of the
two transcription factors do not appear to be identical
(see below). Based on loss of function phenotypes, TTG1,
GL2, MYB61 and TTG2 are all required for both mucilage
synthesis and columella formation. Expression analyses
have shown that the upregulation of mum4 requires both
TTG1 and GL2 but not MYB61 or TTG2 [36]. These data
suggest that MYB61 and TTG2 control different aspects of
mucilage biosynthesis from TTG1/GL2 (Figure 3). Targets
of MYB61 and TTG2 remain to be identified.
AP2 is distinct from the other regulators of the seed
coat epidermis in that it apparently acts earlier and affects
a wider array of seed coat epidermal characteristics.
Functional AP2 is required for all aspects of differentiation
of both the epidermis and palisade layers following
growth of the integument cells [31,49]. Consistent with
this hypothesis is the fact that AP2 is required for normal
levels of GL2, TTG2 and MUM4 transcript in the seed coat
[36]. Thus, AP2 could be the outer integument counterpart
of TT16 in being an early regulator of cell type (Figure 3).
Programmed cell death in the seed coat
By the time the seed coat is mature, the cells in all the
layers are dead. The various layers die at different times of
development and in a specific sequence, suggesting that
the cell death is programmed as part of the differentiation
process. The cell layers that undergo cell death earliest are
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the two outer layers of the inner integument, which do not
appear to develop specialized features (Figure 2, seed coat
layers 3 and 4). A proteinase with caspase-like activity
and sequence similarity to a family of vacuolar processing
enzymes (VPEs), termed dVPE, was found to appear
specifically in seed coat layers 3–4 early in seed coat
development (maximum at 4–5 days after fertilization)
[50] and this production of dVPE is correlated with
programmed cell death (PCD) in these layers. Indeed,
loss of function mutations in the gene encoding dVPE
significantly delayed the onset of PCD. Questions about
additional proteins involved in PCD, the manner in which
PCD is regulated and whether similar processes promote
cell death in the other layers remain to be addressed.
Functions of the seed coat
The roles typically ascribed to the seed coat include the
promotion of dormancy, protection and dispersal. In the
Arabidopsis seed coat, the special features attained
through differentiation include inner cell walls impregnated
with PA, two sets of thickened secondary cell walls
in the outer two layers, and mucilage in the apoplast of the
epidermis that is extruded upon exposure to water. In
other species, the presence of PA has been associated with
resistance to pathogens and herbivores. Secondary cell
walls are assumed to provide support, protection and
impermeability to water and oxygen. It has been proposed
that mucilage assists germination and protects the seed
against toxic chemicals. The availability of mutants defective
in the seed coat epidermis, palisade and endothelium
has been useful in testing some of these hypotheses concerning
seed coat function. Most seed coat mutants tested,
including those specifically defective in PA biosynthesis
(e.g. ban) and the secondary cells walls of the outer layers
(e.g. ap2), but not those specifically defective in mucilage
biosynthesis (e.g. gl2), had seed coats that were significantly
more permeable than those of the wild type. In
addition, most mutants produced seeds that were less
dormant and showed a decreased capacity for germination
following long-term storage. Furthermore, several
mutants (tt4, tt12 and ttg1) germinate more readily in
response to gibberellic acid treatments [28,51,52]. These
data support the hypothesis that one role of PA, thick
secondary cell walls and mucilage is to restrict the
exchange of gases and fluids between the environment
and the seed, prolonging the life of the dormant embryo as
well as protecting it and preventing germination until
conditions are favorable. Mutants with reduced mucilage
also have a decreased ability (relative to the wild type) to
germinate under conditions of limited water availability
[38], suggesting that hydrated mucilage provides an
aqueous environment that assists germination.
Future prospects
Seed coat development involves a fascinating interaction
between endosperm and maternal tissue, and several
unique differentiation pathways ending in cell death. The
dispensable nature of the seed coat and its accessibility
make it a good model genetic system for cell differentiation,
and have enabled researchers to identify a regulatory
cascade for two cell types, to define the biosynthetic
pathway of PA and to verify its role in seed dormancy,
germination and storage longevity. Further analysis of the
seed coat has considerable potential to make additional
significant contributions to our understanding of cell
biology. First, the manner by which the endosperm and
seed coat communicate has yet to be determined. Second,
because all the integument cells originate from the first
(epidermal) layer, it is unclear how the different cell types
in the seed coat are determined. The fact that ablation of
the endothelial layer did not influence adjacent cells of the
inner integument to differentiate as endothelial cells [6]
suggests that layer determination might not be based
primarily on position, although it is possible that early
cell–cell inductive processes occurred before ablation.
Third, the mechanisms for PA deposition in the vacuole
await investigation; the possibility of defining both
regulatory and enzymatic steps in pectin and secondary
cell wall biosynthesis holds promise. Fourth, mucilage is
secreted to a specific domain of the apoplast (at the
junction of the outer tangential and radial cell walls),
resulting in a donut-shaped apoplast and cytoplasmic
column. Thus, studying the seed coat epidermis could help
to elucidate targeted secretion and cell morphogenesis in
plants. Finally, the seed coat is a powerful model system
for studying PCD. These, and many other questions that
will arise in the future, should keep cell and developmental
biologists interested in the seed coat for many
years to come.
Acknowledgements
We apologize to those whose research we do not cite because of space
limitations. We thank Tamara Western for the conceptual design of
Figure 1 and Tamara Western, Sharon Abrahams and Gillian Dean for
comments on the manuscript. G.H. was supported by Killam and
MacMaster fellowships while on sabbatical leave at CSIRO Plant
Industry and an NSERC Discovery Grant for his research in Canada.
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