# Seed coat thickness in the evolution of angiosperms

Olivier Coen, Enrico Magnani

► To cite this version:

Olivier Coen, Enrico Magnani. Seed coat thickness in the evolution of angiosperms. Cellular and Molecular Life Sciences, 2018, 75 (14), pp.2509-2518. 10.1007/s00018-018-2816-x. hal-02626267

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# Seed coat thickness in the evolution of angiosperms

Olivier Coen<fnref n="1" />,<fnref n="2" /> · Enrico Magnani<fnref n="1" />

Received: 28 November 2017 / Revised: 10 April 2018 / Accepted: 13 April 2018 / Published online: 5 May 2018
© The Author(s) 2018

## Abstract

The seed habit represents a remarkable evolutionary advance in plant sexual reproduction. Since the Paleozoic, seeds carry a seed coat that protects, nourishes and facilitates the dispersal of the fertilization product(s). The seed coat architecture evolved to adapt to different environments and reproductive strategies in part by modifying its thickness. Here, we review the great natural diversity observed in seed coat thickness among angiosperms and its molecular regulation in Arabidopsis.

**Keywords** Seed coat · Integument · Fertilization · Ovule · Seed evolution · Seed maternal tissues · Seed coat thickness

## Introduction

The plant sexual reproduction cycle—meiosis, sex differentiation, and fertilization—times the alternation of haploid and diploid generations [1]. Mitosis intercalates the cycle phases and determines the predominance of one generation versus the other. Whereas most non-vascular plants display a predominant gametophytic phase, vascular plants tend to develop large sporophytes [1]. Non-seed plants develop gametophytes physically separated from the sporophyte through the dispersal of spores. By contrast, the evolution of the seed habit in gymnosperms and angiosperms marked the retention of the female gametophyte on the sporophyte and the dispersal of the zygotic embryo (next sporophytic generation) [63]. Seeds greatly contributed to the successful colonization of land by vascular plants. Compared to spores, seeds carry nutrients, rely less on water for germination, convey a higher degree of protection to physical stress, and can disperse in different ways. Such evolutionary advantages were, to a great degree, achieved through the evolution of the seed coat, cell layers that surround, protect, and facilitate the dispersal of the seed fertilization product(s) [52].

The seed coat is a maternal sporophytic tissue that originates from the ovule integument(s). A typical ovule (the seed precursor) comprises four sporophytic tissues: (1) the funiculus, which transports nutrients from the placental tissue, (2) the chalaza, which forms one or more protective (3) integuments, and (4) the nucellus, whose megaspore mother cell undergoes meiosis to originate the gametophyte (Fig. 1). Ovule integuments grow as primordia from the chalaza and are referred to as dermal, if initiated solely by chalazal dermal cells, or sub-dermal, if originated by chalazal dermal and sub-dermal tissue [18, 43]. The developmental patterning of integuments has been thoroughly analyzed in *Arabidopsis thaliana* [13, 24, 72]. Arabidopsis plants display an outer (oi) and an inner (ii) integument that develop from the chalaza of a finger-like protruding ovule primordium (Fig. 1). The ii primordium initiates as a ring-like structure from dermal chalazal cells (ii initials) at the boundary with the nucellus. ii initials become visible when they undergo a periclinal or oblique cell division followed by cell elongation. The oi primordium grows adjacent to the ii proximal side and extends to the proximal extremity of the chalaza. Whereas the distal part of the oi primordium is of dermal origin and follows ii ontogeny, the basal part is subtended by sub-dermal chalazal tissue undergoing periclinal cell divisions. ii and oi annular primordia grow by anticlinal cell divisions to surround and shape the nucellus and female gametophyte. The distal extremity of the integuments forms the micropyle, a small opening through which the pollen tube penetrates. After fertilization, integument cell layers follow different pathways of cell differentiation [34] and grow in coordination with the fertilization product(s).

<figure><img src="image_3.png" /></figure>

<footnote>Enrico Magnani
enrico.magnani@inra.fr</footnote>

<footnote n="1">¹ Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, University of Paris-Saclay, Route de St-Cyr (RD10), 78026 Versailles Cedex, France</footnote>

<footnote n="2">² Ecole Doctorale 567 Sciences du Végétal, University Paris-Sud, University of Paris-Saclay, bat 360, 91405 Orsay Cedex, France</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Arabidopsis ovule primordium at stage 2-V stained with Calcofluor M2R White. *F* funiculus (violet), *C* chalaza (red), *N* nucellus (orange), *oi* outer integument (blue), *ii* inner integument (green). Bar = 20 µm</figcaption></figure>

[40]. Finally, the seed coat undergoes programmed cell death and establishes a protective barrier in between fertilization product(s) and surrounding environment [41].

Seed coat structure and molecular composition deeply influence seed physiology and evolved to adapt to different environmental conditions [43]. Natural diversity has been observed in the thickness of the seed coat, which affects seed germination, dormancy, and mortality [27, 47, 60]. Overall, seed coat thickness is determined by both integument number and cell thickness. Such a character changes along the

seed polarity axes and varies among individuals and species. This review explores the evolutionary patterns of seed coat thickness development in angiosperms.

## Natural diversity in seed coat thickness

### Integument number

Fossil records of Paleozoic pre-ovules from the earliest seed plants revealed the presence of only one integument (unitegmic, Fig. 2) [28, 80]. The integument structure of such pre-ovules varied among species, but it was typically lobed, vascular bundled, and detached from the nucellus. Leaf-like structures, termed cupules, have been found to partially enclose the ovule and are commonly viewed by paleo-botanists as the precursor of a second integument. Extant gymnosperm ovules are also unitegmic and the integument is probably homologous to that of ancestral seed plants. By contrast, angiosperms evolved two integuments (bitegmic), the so-called oi and ii (Fig. 2). Phylogenetic analyses suggest that bitegmy is the ancestral condition in angiosperms, nevertheless unitegmy arose several times during angiosperm evolution [18].

The transition from bitegmic to unitegmic ovules happened several times during angiosperms evolution along two major ontogenic pathways: (1) developmental suppression or retardation of one integument, (2) congenital integument fusion. Morphological analyses in the *Piperaceae* family revealed that the single integument of *Peperomia* shows similarity to the *ii* of *Piper*, suggesting that the *oi* of the former has been lost [88]. Similarly,

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Scheme of bitegmic, unitegmic, and bifid ovules. Outer integument (blue); inner integument (green); tissue of unclear origin (yellow)</figcaption></figure>

<figure><img src="image_6.png" /></figure>
early arrest of ii has been described in *Populus* [81]. Con-
genital fusion of ii and oi probably arose through differ-
ent mechanisms. Genetic analyses in Arabidopsis (see
below) suggest that integumentary fusion can be achieved
through lack of boundary formation between oi and ii
[14, 44]. This interpretation is confirmed by the analysis
of molecular markers of boundary formation in *Prunus*
bitegmic and unitegmic ovules [53] and by morphologi-
cal studies in *Anacardiaceae* and *Balsaminaceae* [43]. By
contrast, a mechanism of integumentary shifting has been
proposed for the development of the single integument
of a number of *Ranunculaceae* [6]. According to Bou-
man and Calis, oi sub-dermal tissue subtending ii initials
undergoes periclinal cell divisions and, in conjunction
with oi, shifts the ii upward. An incision at the tip of the
single integument reveals oi and ii remnants. According
to the authors, this mechanism would create a bifid integ-
ument in between uni- and bitegmic and would only be
possible in ovules carrying a sub-dermal oi and dermal ii
(Fig. 2). The process of integumentary shifting has been
also invoked to explain the development of the unitegmic
*Gentiana* ovule, where no oi and ii remnants are visible [7]
(Fig. 2). According to this model the sequence and posi-
tion of dermal and sub-dermal cell divisions might have
led to a unitegmic ovule through integumentary shifting
of a sub-dermal oi and dermal ii. Interestingly, species
with bitegmic, bifid and unitegmic ovules are grouped
in the *Impatiens* gender. Boesewinkel and Bouman have
presented the development and evolution of *Impatiens*
bifid and unitegmic ovules as integumentary shifting [5].
More recently, McAbee et al. have revised this theory and
described a change of sub-dermal growth from beneath the
oi to the region beneath both oi and ii, without invoking an
upward shift of the ii [55]. According to this hypothesis,
unitegmy would be the result of ii-oi intercalary growth.
All considered, we believe that available data on integu-
ment congenital fusion in angiosperms point to two non-
exclusive mechanisms: (1) total or partial lack of boundary
formation and (2) intercalary growth between ii and oi
primordia, regardless of their dermal or sub-dermal origin.

Further ovule reduction has been observed in *Santalales* [74]. Members of this clade do not develop any ovule integument (ategmy). Expression analyses revealed that integument molecular markers are expressed at the distal end of *Santalales* ategmic ovules [10]. These data have been interpreted as the fusion of an integument remnant to the nucellus. By contrast, some angiosperms evolved a third cell sheath in addition to oi and ii. *Annonaceae* ovules develop a so-called third integument in between oi and ii [12], whereas Myristicaceae and other groups initiate a third envelop, referred to as aril (not to be confused with arillodes), later on in development in a region proximal to the oi that has been interpreted as the funiculus [17, 66, 77]. Interestingly,

arils can also be found in gymnosperms, such as *Taxus bac-
cata* [54]. Arils either work as attractive organs that facilitate
seed dispersal or are reduced to a tiny protuberance, as in a
number of basal angiosperms [39].

Finally, the number of integument cell layers can change
after ovule fertilization during early seed development. Rice
ovules display two-cell-layered oi and ii. Within 3 days after
fertilization, oi and ii of most rice cultivars are absorbed by
the pericarp leaving only the cuticle layer [50].

Integument cell thickness

Two-cell-layered dermal integuments originate from the cha-
lazal dermal tissue, whereas three-cell-layered sub-dermal
integuments display two dermal and one sub-dermal cell
layer. Furthermore, integuments can develop more cell lay-
ers through periclinal cell division of sub-dermal or der-
mal cell layers and are referred to as multiplicative [43].
Integument cell thickness is considered a stable character
worth using in macrosystematic analyses [18]. Nevertheless,
3D morphological analyses of Arabidopsis ovules suggest
the contrary. Arabidopsis ovules were thought to display a
regular two- and three-cell-layered oi and ii, respectively
[72]. The ii grows a third cell layer (ii1') by periclinal cell
divisions of the innermost ii cell layer (ii1). Coen et al. have
described the formation of a fourth cell layer (ii1") by peri-
clinal cell division of the ii1' in 70 and 35% of *Wassilews-
kija* and *Columbia* Arabidopsis ovules, respectively [13].
Furthermore, the oi has been recently shown to initiate sub-
dermal cell stripes (oi') in 10% of the *Columbia* Arabidopsis
ovules tested [24]. These data highlight the importance of
3D imaging for the analysis of integument cell patterning
and suggest that cell thickness is a less stable character than
previously thought.

Finally, integument thickness is affected by cell size. Dur-
ing ovule development, cell size does not vary considerably
among integument cell layers [72]. Nevertheless, integument
cell layers follow different fates and expand dishomogene-
ously after fertilization. In Arabidopsis, ii1' and ii1" undergo
a more dramatic cell expansion process compared to the
other integument cell layers. Coen et al. have revealed that
ii1' and ii1" growth is not tightly coordinated with the rest
of the integument cell layers and have suggested a role in
cushioning seed coat development and offsetting perturba-
tions in its developmental program [13].

Integument thickness along the ovule polarity axes

Both integument number and cell thickness can vary con-
siderably along the ovule polarity axes (Fig. 1). Integu-
ment growth leads to the formation of a micropyle at the
distal side of the ovule. The majority of bitegmic ovules
are amphistomic, as both oi and ii contribute to micropyle
formation. More rarely, angiosperm ovules can be endos-
tomic or exostomic if only the ii or the oi, respectively,
generate the micropyle [18]. Endostomic ovules show dis-
tal thickening of the ii at the micropyle where the ii is not
covered by the oi, suggesting that oi limits ii growth [38].
Thickening of the distal region of the integument might
be instrumental to enclose the nucellus apex and form a
micropylar pore. Proximal ii thickening is instead visible in
Arabidopsis where ii1' and ii1" do not develop all the way to
the micropyle. Ectopic ii1' growth in the micropylar region
interferes with embryo development by limiting its growth
space and is repressed by a tight regulatory mechanism (see
below) [13].

In several basal angiosperms, oi and/or ii radial patterning
shows discontinuous growth and leads to lobation. Lobed iis
have been considered remnants of ancestral integuments or a
sign of atavism as paleozoic ovules displayed a lobbed integ-
ument [83]. Nevertheless, it has been argued that lobation
might have evolved as a way to restrict the circumference of
the integument and allow the formation of a micropyle, as an
alternative mechanism to distal integument thickening [38].

Finally, integument number and cell thickness deeply
affect ovule curvature along the adaxial-abaxial polar-
ity axis. The micropyle of anatropous (curved) ovules is
placed side by side to the chalaza, whereas orthotropous
(non-curved) ovules develop micropyle and chalaza along
a straight line. Orthotropous ovules are either unitegmic or
display a thin or partially developed oi suggesting that the oi
plays a major role in ovule curvature [18]. This hypothesis
is supported by a number of Arabidopsis mutants that revert
from anatropous to orthotropous when the oi is missing or
partially developed (see below) [14, 44]. Nevertheless, gym-
nosperm *Podocarpaceae* develop unitegmic anatropous
ovules. In such ovules, the function of the oi is taken over
by the epimatium, an ovuliferous scale that resembles the oi
and is involved in ovule curvature [82]. Angiosperm anatro-
pous ovules have either annular or semi-annular oi primordia
that grow into cup-shaped or hooded ois [92]. Ovules with a
hooded oi display a difference in thickness along the ovule
adaxial-abaxial polarity axis: the abaxial side shows a fully
developed oi whereas the adaxial side misses partially or
entirely the oi. The development of a hooded or cup-shaped
oi morphology has been correlated to the speed of devel-
opmental curvature: pronounced semi-annular oi primordia
tend to curve faster than annular primordia [18].

Molecular regulation of seed coat thickness
in arabidopsis

Establishment of the ovule proximal–distal polarity axis

In Arabidopsis, the ovule proximal-distal polarity axis is
morphologically evident at stage 2 of ovule development

when the distal nucellus develops a megaspore mother cell,
the central chalaza initiates integument primordia, and the
proximal funiculus differentiates a vascular strand (Fig. 1)
[72]. Genetic and molecular evidences suggest that ovule
proximal–distal patterning occurs sequentially starting from
the distal nucellus toward the funiculus [73]. The establish-
ment of chalazal identity, which has obvious repercussions
on integument initiation, is therefore deeply influenced by
nucellus development. More than 25 years of genetic studies
on Arabidopsis ovule development have revealed a handful
of genes involved in ovule proximal–distal patterning.

Mutations in the *AINTEGUMENTA* (*ANT*) gene, encod-
ing for an APETALA2 domain transcription factor, lead to
bare ovules with no or reduced integument primordia [16,
49]. Starting at stage 2, *ANT* is expressed in the chalaza
and integuments primordia where it is speculated to regulate
cell proliferation underlying integument outgrowth [16, 49].
Furthermore, *ANT* and the mitochondrial ribosomal protein
HUELLENLOS (HLL) have redundant functions in estab-
lishing the ovule proximal–distal axis [73, 79]. Ipomorph *hll*
alleles display early arrest of integument development and,
in combination with *ant* mutations, lead to reduced chalaza
and funiculus regions [73]. Similarly, HLL works synergisti-
cally with the SHORT INTEGUMENTS 2 (SIN2) mitochon-
drial DAR GTPase [9, 36]. *sin2* ovules show reduced integu-
ment growth, whereas *sin2;hll* double mutants undergo early
arrest of ovule primordia [9]. The nature of HLL and SIN2
protein indicates that metabolic defects can affect develop-
mental patterning through mechanisms yet to be discovered.

Another early marker of the chalazal region, the BEL1
homeodomain transcription factor, has been implicated in
the acquisition of chalazal identity [59, 67, 69]. *bell* ovules
fail to develop an ii and the oi turns into a swollen collar-like
tissue or secondary ectopic carpeloid structure, depending
on the *bell* mutation. A similar phenotype has been observed
in ovules over-expressing the *AGAMOUS* (*AG*) MADS-box
gene [67] and in the triple MADS-Box genes mutant *seedstic
k;shatterproof1;shatterproof2* (*stk;shp1;shp2*) [65]. Interest-
ingly, the carpeloid outgrowths of *bell* ovules are converted
into green leaf-like structures in the double *apetala2;bell1*
and quadruple *stk;shp1;shp2;bell1* mutants [8, 59]. Bram-
billa and coworkers have proposed a model for cell identity
acquisition in ovule integuments based on genetic analyses
and in vitro MADS-box proteins interactions [8]. According
to it, BEL1 interacts in the chalaza with AG and SEPAL-
LATA3 MADS-box proteins to repress carpel identity in the
integuments; this protein complex is then stabilized by an
ovule identity complex comprising STK, SHP1, and SHP2
proteins [8].

Whereas BEL1 and ANT work in a cell-autonomous
fashion, the WUSCHEL (WUS) homeodomain transcrip-
tion factor promotes integument initiation from the nucel-
lus, non-cell autonomously [33]. *wus* ovules fail to develop

<figure><img src="image_7.png" /></figure>
integuments and show defects in megagametogenesis. WUS and ANT do not regulate each other's expression suggesting that they establish independent pathways from the nucellus and chalaza, respectively. These data indicate that ANT determines responsiveness of chalazal cells to a WUS-mediated signal from the nucellus that ultimately induces integument formation. WUS expression in the nucellus is tightly regulated by a plethora of transcription factors. Defects in integument development of *bell*, *sporocyteless/nozzle* (*spl/nzz*, see below), *stk;shp1;shp2*, and *phabulosa;phavoluta;corona* (*phb;phv;cna*, see below) mutants have been linked to ectopic WUS mRNA accumulation in the chalaza [8, 75, 93]. Furthermore, exogenous application of cytokinin on ovules leads to integument arrest and concomitant expression of WUS in the integuments [4]. Finally, the expression of WUS in the chalaza under the control of the ANT promoter region expands the chalazal domain and leads to the initiation of an ii and two or more proximal integuments, some of which express oi markers [33, 75]. Only the ii of *proANT:WUS* ovules grows around the nucellus, whereas more proximal integuments fail to develop further.

The adaptor-like transcriptional repressor SPL/NZZ, another master regulator of the ovule proximal-distal polarity axis, is expressed throughout the ovule at stage 2, but more strongly in the dermal cells of the chalaza and in integument primordia [2, 71, 90, 94]. *spl/nzz* ovules display reduced nucellus and integuments growth as well as impaired sporogenesis, a phenotype that is partially suppressed by the *bell* mutation [2, 71, 94]. SPL/NZZ negatively regulates the expression of *ANT* and *BEL1* in the nucellus while positively regulating *WUS* expression [2, 75]. By contrast, *SPL/NZZ* expression in the chalaza is activated by ANT and BEL1 and it is necessary for the establishment of the adaxial-abaxial polarity axis, thus working as a link between proximal-distal and adaxial-abaxial patterning [2, 3].

## Establishment of ovule and integuments adaxial-abaxial polarity axes

In Arabidopsis, ovule primordia acquire adaxial-abaxial polarity and transition from radial to bilateral symmetry at stage 2-III (Fig. 1) [72]. Whereas the ii primordium arises simultaneously on the adaxial and abaxial side of the ovule primordium, the oi primordium initiates on the abaxial face and its predominant abaxial growth is responsible for the Arabidopsis ovule curvature [18]. Adaxial-abaxial patterning characterizes then the laminar growth of each integument (Fig. 1). Here again, genetic studies have helped better understand regulatory pathways underlying such processes.

KANADI (KAN) and YABBY (YAB) transcription factors are known to promote abaxial identity in vegetative

organs and ovules [48, 76]. *kan1;kan2* mutant ovules display reduced oi growth [21]. Similarly, mutations in the *YAB* gene *INNER NO OUTER (INO)* lead to the development of an amorphous protuberance at the place of the oi [85]. *INO* expression in the proximal-abaxial side of the chalaza (stage 2-I) is tightly regulated in a spatial-temporal fashion. Whereas *BEL1* is a pre-requisite for *INO* expression, ANT and SPL/NZZ act antagonistically to time the onset of *INO* expression which in turn establishes a positive auto-regulatory loop and feedbacks on *ANT* expression [3, 58]. The SUPERMAN (SUP) zinc-finger protein interferes with *INO* positive regulatory loop in the adaxial domain of ovule primordia, promoting oi abaxial development and ovule bilateral symmetry [26, 58, 85]. Similarly, *WUS*, *SPL/NZZ*, *HLL*, and *KAN4/ABERANT TESTA SHAPE (ATS)* work redundantly in delimitating *INO* expression to the abaxial side of the oi [3, 75, 85]. Finally, *INO* has been shown to directly interact with the co-repressors LEUNIG and SEUSS and the co-activator ADA2b/PROPORZ1 (PRZ1) to promote oi growth [78]. Acquisition of ovule adaxial identity is instead promoted by the class III homeodomain-leucine zipper transcription factor PHB. *PHB* mRNA initially localizes in the adaxial side of ovule primordia (stage 1-I) [75] and *phb-1d/PHB* gain-of-function mutants, insensitive to miRNA negative regulation, exhibit orthotropous ovules with reduced oi growth [45, 57].

Abaxial-adaxial polarity appears also within integuments, with both oi and ii displaying an adaxial and abaxial side, necessary to establish the oi-ii boundary (Fig. 1). *KAN4/ATS* is expressed in the abaxial side of ii primordia and loss of *KAN4/ATS* function leads to the fusion of ii and oi into a single thicker integument [56]. *KAN4/ATS* is post-translationally repressed by the UNICORN (UCN) active AGC VIII kinase [19, 20]. In *ucn* mutants as in *sk21-D*, a *kan4/ats* dominant allele, integuments form protrusions that originate from periclinal or oblique divisions of dermal cells [19]. Moreover, *KAN4/ATS* interacts with DELLA proteins, repressors of gibberellin-dependent pathways, to regulate the expression of genes involved in integument growth [32]. In situ hybridization analyses of the class III homeodomain-leucine zipper transcription factors *PHB*, *PHV*, and *CNA* have revealed their ii adaxial pattern of expression [45, 75]. *phb;phv;cna* triple mutant displays a variety of phenotypes such as reduced ii or oi growth and the formation of amorphous integuments [45]. *PHB* expression in the ii is regulated by *WUS*, *ANT*, and *SPL/NZZ*, whereas it is independent of *INO* and *KAN4/ATS* [45, 75]. Overall, these genetic analyses suggest that a balanced expression of adaxial and abaxial polarity determinants is necessary for both ii and oi growth, similar to what observed in leaves [45, 56].
## Regulation of multiplicative integuments

The growth of Arabidopsis sub-dermal integument cell layers has been found to be tightly regulated. The development of ii sub-dermal cell layers (ii1' and ii1'') by periclinal cell divisions of the ii1 is promoted by STK and TRANSPAR-ENT TESTA 16 (TT16) MADS-Box transcription factors [13]. *tt16;stk* ovules lack ii sub-dermal cell layers almost completely. Furthermore, TT16 is a master regulator of ii1' and ii1'' cell patterning [13, 15]. Wild-type ii1' arrests before the micropylar region, thus creating a proximal-distal polarity in seed coat thickness. *tt16* ovules exhibit a distal shift of ii1' and ii1'' development, thus creating a thicker micropylar and thinner chalazal region, compared to the wild type. *TT16* expression pattern in the ii1 marks in advance the development of the ii1' and is responsible for the correct positioning and progression of ii1 periclinal cell divisions. In *tt16* seeds, embryo development is impaired by the mechanical action of the seed ii1' invading the micropylar pole, highlighting the importance of tightly regulating sub-dermal integument growth. Finally, TT16 has been involved in the formation of oi sub-dermal cell stripes (oi'), which arise by periclinal cell divisions of sub-dermal chalazal cells [24]. The chalaza of *tt16* ovules appears more extended along the proximal distal axis, compared to the wild type, thus favoring the growth of sub-dermal cells in between oi1 and oi2.

After fertilization, ii1' and ii1'' undergo dramatic cell expansion, compared to all other integument cells, thus affecting considerably seed coat thickness. The differentiation of all integument cell layers was thought to be repressed by FERTILIZATION INDEPENDENT SEED (FIS) Polycomb group (PcG) proteins (see below) and induced by a signaling pathway initiated by the endosperm [22, 70]. Nevertheless, genetic analyses indicate that ii1' and ii1'' cell expansion is not repressed by FIS PcG proteins and requires a signal from both embryo and endosperm [13, 23]. ii1' and ii1'' sub-dermal position is not responsible for such a unique regulatory mechanism as oi' cell expansion responds to the same molecular pathways underlying the development of integument dermal cell layers [24].

## Hormonal signaling

As described above, cytokinins and gibberellins regulate ovule proximal-distal [4] and adaxial/abaxial patterning [32], respectively. Brassinosteroids induce ANT and HLL while repressing AP2 expression during early ovule development [37]. Finally, auxin has been shown to play a fundamental role in Arabidopsis integument initiation. Mutations in the auxin efflux facilitator PIN-FORMED 1 (PIN1) as well as treatments with the auxin flux inhibitor NPA lead to finger-like ovule structures [4]. SPL/NZZ and BEL1 regulate *PIN1* expression by acting through the

cytokinin signaling pathway [4]. Furthermore, SPL/NZZ has a role in auxin homeostasis by repressing the expression of *YUCCA* genes, which are involved in the biosynthesis of auxin [51]. Likewise, NAC family transcription factors CUP-SHAPED COTYLEDON 1 (CUC1) and 2 promote integument formation by modulating auxin flux through a PIN1-dependent mechanism [42]. Auxin affects not only ovule proximal-distal, but also adaxial-abaxial patterning. KAN4/ATS interacts with ETTIN (ETT), also known as AUXIN RESPONSIVE FACTOR 3 (ARF3), on the abaxial side of the ii [46]. KAN4/ATS-ETT/ARF3 protein complex promotes ii laminar growth and oi-ii separation by regulating *PIN1* expression [46]. Finally, *microRNA167* has been shown to promote integument growth by preventing *ARF6* and *ARF8* expression in the ovule [91].

## Kinase signaling

In addition to UCN (see above), a number of other Arabidopsis kinase proteins, acting in different signaling pathways, have been involved in ovule integument outgrowth.

The *ERECTA* (ER) gene family, encoding for LRR-receptor-like kinases (LRR-RLKs), is expressed in the ovule integuments and is important for proper integument growth [64]. When the expression level of *ER* and *ER*-like genes was dramatically reduced, the integuments failed to completely surround the nucellus. The *ER* gene family genetically interacts with the *PRETTY FEW SEEDS 2* (*PFS2*) homeo-box gene, which also promotes integument outgrowth [62, 64]. Similarly, mutations in the MITOGEN-ACTIVATED PROTEIN KINASES 3 (MPK3) and 6 (MPK6) lead to premature arrest of ii and oi growth and have been speculated to work downstream of the ERECTA pathway [86].

The LRR-RLK *strubbelig* (*sub*) mutant leads to incomplete and irregular growth of the oi [11]. SUB trafficking is mediated by HAPLESS13 (HAP13), the μ subunit of adaptor protein 1 that regulates protein sorting at the trans-Golgi network/early endosome. *hap13* ovules display reduced oi development probably due to SUB mistargeting [87]. A *sub*-like ovule phenotype was observed in *pfs2;ino* and *pfs2;nzz* ovules and was compared to integument lobes of primitive ovules [61]. A forward genetic approach, aimed at finding mutations causing *sub*-like ovule phenotypes, allowed Fullton and coworkers to identify the *quirky* (*qry*), *detorqueo* and *zerzaust* mutants [25]. *QKY* encodes a predicted membrane-anchored C2-domain protein that co-localizes with SUB at the plasmodesmata, thus raising the possibility that both proteins influence symplastic trafficking of molecules [84].

Finally, *ARABIDOPSIS CRINKLY* 4 encodes another RLK protein localized at the plasma membrane of L1 cell layers [29]. In *acr4* mutants, ovule integuments display a wide range of phenotypes, including arrest of integument
<figure><img src="image_8.png" /><figcaption>**Fig. 3** List of Arabidopsis genes and hormones known to regulate seed coat thickness</figcaption></figure>

growth and lack of integument cell layer organization [29, 30, 89].

## Epigenetic determinants

dsRNA processing and epigenetic control of transcriptional activation have been shown to regulate ovule integument development in Arabidopsis. Mutations in *SHORT INTEGUMENTS 1 (SIN1)* (also known as *SUSPENSOR1* or *CARPEL FACTORY*), which encodes a Dicer protein, result in premature integument arrest [31]. Nevertheless, the RNA targets of *SIN1* are yet to be identified.

As mentioned above, PcG proteins have also been involved in integument development. Two FIS components of the Polycomb repressive complex 2, MULTICOPY SUPRESOR OF IRA1 (MSI1) and FERTILIZATION INDEPENDENT ENDOSPERM (FIE), act sporophytically to repress integument cell expansion and differentiation before fertilization [70]. The study of FIE and MSI1 is complicated

by the seed lethal phenotype of loss of function mutants. Nevertheless, FIE and MSI1 are haploinsufficient and emasculated *fie/+* and *msi1/+* flowers produce both wild-type-looking ovules and enlarged autonomous seeds having a partially differentiated seed coat. Furthermore, Hennig and coworkers discovered a positive role for MSI1 in integument outgrowth [35]. *MSI1* co-suppression lines exhibited orthotropous ovules with limited oi development, resembling *ino* mutants.

## Conclusive remarks

Genetic analyses of Arabidopsis ovule development revealed a number of master regulators of integument number and cell thickness that might well be responsible for the natural diversity observed in seed coat thickness (Fig. 3). Genes, such as *ANT*, *ATS*, *BEL1*, *INO*, and *ETT*, have been used as markers to better understand the evolution of unigamy
and ategmy in angiosperms [10, 53, 55]. Furthermore, the expression pattern of *AGL6-like* genes suggests that the angiosperm ii is homologous to the gymnosperm single integument [68]. By contrast, the evolution of the oi remains more controversial. It has been hypothesized that cupules might be ancestral ois. In line with this model, the Arabidopsis oi expresses *INO*, member of a gene family implicated in leaf development [85]. Moreover, Arabidopsis *ap2;bell1* and *stk;shp1;shp2;bell1* mutant ovules show a conversion of the oi into a leaf-like structure. Alternatively, the oi might have evolved de novo through the *WUS* or *UCN* pathway. Less attention has been given to arils while addressing the evolution of the oi. The study of gymnosperms B-sister MADS-box genes, orthologous to Arabidopsis *TT16* and *GORDITA*, revealed their high expression in the integument of *Ginkgo biloba* ovules and low expression in integument and aril of *T. baccata* ovules [54]. These data suggest, despite being interpreted differently by the authors of such study, that *T. baccata* arils express an integument molecular marker and might be seen as integuments. Expression analyses of other integument master regulator genes in angiosperms and gymnosperms arils would help better address nature and evolution of these integument-like structures.

**Acknowledgements** Funding was provided by Labex Saclay Plant Sciences-SPS (Grant no. ANR-10-LABX-0040-SPS) and FP7-PEOPLE-2012-CIG Marie Skłodowska (Grant no. SEEDNET EU-CIG no. 321710).

**Open Access** This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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