Signalling events regulating seed coat development

Ovule structure and development

Ovule development in angiosperms is initiated as the formation of a finger-like protrusion from the placenta [5]. The developing ovule can be divided into three main zones along the proximal–distal axis: the funiculus, which connects the ovule to the carpel and through which nutrients are delivered from the maternal plant to the ovule; the chalaza in the middle, which will give rise to the ovule integuments; and the most distal part, the nucellus. Within the nucellus, a cell will differentiate into the megaspore mother cell that will undergo meiosis to form a tetrad of haploid spores. In angiosperms, including Arabidopsis, three spores will undergo programmed cell death, whereas only the most proximal spore will survive and form the functional megaspore that proceeds into megagametogenesis. The functional megaspore will then undergo three rounds of mitotic divisions to form the female gametophyte (reviewed in [6,7]).

Along with the maturation of the female gametophyte, seven cells containing eight nuclei are formed: the two gametic cells, the haploid egg cell and the diploid central cell, and five haploid accessory cells, two synergids and three antipodals [8]. Upon fertilization, the pollen tube enters the ovule through an opening called the micropyle and bursts within one of the synergids, delivering the two sperm cells that will fertilize the egg cell and the central cell. The former fertilization event will give rise to the embryo, whereas the latter will result in the formation of the embryo-nourishing endosperm that supports the growing embryo by providing it with nutrients [9]. In Arabidopsis, as in other dicotyledons, the endosperm is in part or largely consumed as the embryo grows. Nonetheless, the endosperm also has a role in signalling with both the embryo and the seed coat, and is necessary for normal seed development (discussed below) [10,11].

The ovule integuments are derived from the chalazal region of the ovule and grow surrounding the nucellus, leaving only a small opening that allows the pollen tube to enter [5,10]. Ovules in angiosperms generally have two integuments (bitegmic), whereas unitegmic (with only one integument) and ategmic (no integument) ovules are considered as being derived from a previously bitegmic condition [12]. Each integument is composed initially of two layers, but the inner integument undergoes an additional periclinal division, resulting in a three-layered structure [13]. The asymmetrical growth of the outer integument will result in the bending of the female gametophyte, placing the micropyle near the funiculus. In the absence of the outer integument, the embryo sac does not bend, resulting in an elongated ovule phenotype [14]. Interestingly, the female gametophyte is not required for integument development, but the inverse is not true: if the integuments fail to form, so does the female gametophyte [15–17].

Seed coat structure

Upon initiation of embryo and endosperm development, the surrounding integuments also undergo a process of growth and differentiation that will lead to the formation of the seed coat (Figure 1). Seed coat growth is a process driven mostly by cell elongation. This is illustrated by the fact that cell elongation can compensate for a reduced cell division rate in the integuments, but the reverse does not occur [18]. Each seed coat layer will have a different fate along its development [19]. In the endothelium, the innermost layer of the inner integument, the biosynthesis of condensed tannins [PAs (protoanthocyanidins)] occurs that confer the characteristic brownish colour to Arabidopsis seeds [20]. PAs are believed to confer protection against predators and pathogens as well as to contribute to seed dormancy and longevity [21,22]. Nevertheless, the endothelium is not essential for viable seed development [23]. The two outer layers of the inner integument undergo programmed cell death and collapse [24]. Finally, the two outermost layers of the seed coat also show different fates: the cells in the subepidermal layer produce thick cell walls that will withstand the pressure of the growing embryo [19], whereas the outer layer (epidermis) accumulates the polysaccharide mucilage (pectin), which has a role in seed dormancy, as well as in the uptake of water before germination [25]. Following mucilage secretion, secondary cell wall material is deposited inside the cell, forming a structure known as the columella [26].

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Figure 1 Structure of unfertilized ovules and early developing seeds in A. thaliana

(A) Micrograph of an unfertilized ovule. c.c., central cell nucleus; e.c., egg cell nucleus; syn, synergid nucleus; o.i., outer integument; i.i., inner integument. (B) Micrograph of a developing seed. em, embryo (eight-cell stage); en, endosperm (arrows indicate endosperm nuclei); s.c., seed coat. (C) Detail of the five seed coat layers (boxed in B). Layers derived from the outer integument are depicted in green, and layers derived from the inner integument are in blue. Scale bars, 30 μm.

At later stages of seed development, the seed coat cells die, but the structure is maintained by the mucilage and the columella. At this point, the PAs are released from the endothelium cells and impregnate the remaining two seed coat layers.

Cross-talk between the endosperm and the seed coat

Seed development is a co-ordinated process between the maternal tissues that form the seed coat and the two fertilization products: the embryo and the endosperm. Their synchronized growth thus requires communication between these structures. Namely, co-ordination between the endosperm and the seed coat is necessary for successful seed development [2–4]. The development of the sexual endosperm is necessary and sufficient for the initiation of seed coat growth, whereas the embryo by itself, on the other hand, does not trigger seed coat development [3]. Genes of the HAIKU pathway (IKU1, IKU2 and MINI3) are specifically expressed in the endosperm and affect seed coat growth, supporting a role for the endosperm as a key determinant of seed size [27–29]. Additionally, the expression of MINI3 and IKU2 depends on the activity of SHB1 (SHORT HYPOCOTYL UNDER BLUE1), a positive regulator of seed development in Arabidopsis [30]. A dominant allele of SHB1, shb1-D, promotes endosperm proliferation and delays its cellularization, whereas the loss-of-function mutant has the opposite effect, as for iku and mini3 mutants [30].

Whereas mutants of the HAIKU pathway form smaller, but viable, seeds, loss of the endosperm-specific type I MADS-box transcription factor AGL62 causes early arrest of seed development. The agl62 mutant undergoes several rounds of endosperm division, but fails to develop a seed coat [3,31]. Lack of seed coat development in agl62 is unlikely to be a consequence of an endosperm-proliferation defect, as mutants of the titan class also suffer from severe defects in endosperm proliferation, but are not compromised in seed coat development [3,32]. This suggests that AGL62 is necessary to form an endosperm-driven signal triggering seed coat development. Furthermore, these data disfavour the idea that seed coat growth is a process purely enforced by mechanical pressure of the endosperm, as, in that case, agl62 and titan mutants should be similarly compromised in seed coat growth. Nevertheless, the nature of the signal remains to be investigated.

Cross-talk between endosperm and seed coat is a bidirectional process, thus seed coat development also affects endosperm development as revealed by several mutants that show a maternal effect on endosperm growth. Mutations in TTG2 (TRANSPARENT TESTA GLABRA2) cause restricted cell elongation in the seed coat, resulting in a limited endosperm proliferation rate and early cellularization [18]. TTG2 was initially described to be involved in PA and mucilage biosynthesis in the seed coat, and is expressed in all layers of the seed coat, albeit at different time points [33]. Double mutants of ttg2 and iku have an additive phenotype, suggesting that both genes work in different pathways, notwithstanding having similar seed mutant phenotypes [18]. Thus TTG2 seems to control seed size maternally (by regulating integument cell elongation), whereas the IKU pathway exerts its control at the endosperm level.

Additional support for a positive effect of the maternal tissues on endosperm proliferation has been derived from analysis of the ap2 (apetala2) mutant that produces seeds that are considerably larger than wild-type seeds [34,35]. Increased seed size of ap2 is caused by increased elongation of seed coat cells and prolonged proliferation of the endosperm. The effect of ap2 on endosperm development is non-cell-autonomous and a consequence of increased seed coat growth, as is evident by the fact that the phenotype is maintained in seeds derived from crosses of a homozygous ap2 maternal plant pollinated with wild-type pollen [34–36]. Functional analysis of the KLUH/CYP78A5 (KLU) gene suggests that increased seed coat growth is sufficient to promote endosperm proliferation [37]. KLU codes for a cytochrome P450 and was described as acting non-cell-autonomously driving organ growth [38]. Mutant klu plants form smaller organs, whereas KLU-overexpressing plants show an increased cell proliferation, resulting in increased organ size [38]. Expression of KLU specifically before fertilization in ovule integuments results in increased cell proliferation in the integuments and increased seed size, revealing that the growth potential of the integuments determines seed size [37]. Similarly to KLU overexpression, loss of function of the ARF2 (AUXIN-RESPONSIVE FACTOR 2) in the mnt (megaintegumenta) mutant causes increased proliferation of cells in the integuments and, consequently, increased seed size [39].

Together, there is substantial genetic evidence for a bidirectional cross-talk between endosperm and seed coat, allowing a fine-tuned development of both seed tissues.

Communication routes between endosperm and seed coat

As outlined above, communication between endosperm and seed coat is critical for seed development; however, the nature of the signalling pathways between both structures remains to be understood. Nevertheless, it is apparent that these tissues communicate via the apoplast. Whereas the female gametophyte and the surrounding integuments share symplastic connections [40], the endosperm and the two seed coat layers form three separate symplastic domains [11,41]. This was demonstrated in Arabidopsis through monitoring the movement of GFP and of the symplastic tracer HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid) in each of these domains [41]. When GFP is expressed specifically in the endothelium, it spreads to the three cell layers derived from the inner integument, but not to the endosperm or outer integument layers. Similarly, when HPTS is loaded into the phloem and transported to the outer integument, it stays confined to the two cell layers of the outer integument. Nevertheless, plasmodesmata have been proposed to exist between the peripheral endosperm/aleurone layer and the seed coat in Medicago truncatula [42], suggesting that the communication between the endosperm and the seed coat may vary among angiosperms.

The symplastic barrier between the endothelium and the endosperm in Arabidopsis is additionally enforced by the deposition of a cuticle between the two tissues [43]. This implies that the communication between the seed coat and the endosperm is limited to apolar compounds and/or to small polar molecules that could pass through gaps in the cuticle layer. Since there is no cuticle formed at the chalazal seed coat, the import of molecules occurs particularly active at this part of the seed [11]. Hexoses, for instance, are polar molecules that are well known to pass through the integuments to the endosperm. In several species, sucrose-hydrolysing activity in the maternal tissues has been linked to the increased hexose content in endosperm during early seed developmental stages [44–46]. Sucrose feeding experiments suggest that sucrose transported through the phloem is hydrolysed in the seed coat and imported by the developing endosperm, the active sink, through the apoplast [47]. It has also been proposed that RNAs synthesized in the maternal chalazal tissues are transported into the chalazal endosperm [48]; this hypothesis remains to be tested experimentally.

In summary, the seed coat and the endosperm communicate in a non-cell-autonomous manner to co-ordinate their growth. The exchange of signal molecules is probably limited to apoplastic transport routes, limiting the nature of the signals that can be exchanged. Nevertheless, it is well possible that mechanical expansion of the tissues also play a role in this co-ordinated development.

Epigenetic regulation of seed coat development

After completion of ovule formation, the growth of the ovule is arrested until fertilization occurs. Mutants in the central cell-specific FIS (FERTILIZATION-INDEPENDENT SEED) PRC2 (Polycomb repressive complex 2) can bypass the fertilization requirement and initiate divisions of the central cell, leading to the formation of a diploid autonomous endosperm [49]. PRC2 is a chromatin-modifying complex that ensures mitotically stable repression of specific target genes by applying trimethylation marks at Lys²⁷ of histone H3 [50]. The FIS–PRC2 complex is composed of subunits MEA (MEDEA), FIS2, FIE (FERTILIZATION-INDEPENDENT ENDOSPERM) and MSI1 (MULTICOPY SUPPRESSOR OF IRA1). MEA and FIS2 are specific subunits of the FIS–PRC2 complex, whereas FIE and MSI1 are shared among PRC2 complexes that have functional roles during vegetative plant development [50]. Whereas autonomous endosperm initiated in mea and fis2 mutants is not sufficient to initiate seed coat development, fie and msi1 mutants form seed-like structures containing endosperm and a developed seed coat [3]. This suggests that the autonomous asexual endosperm is not competent to form the signal (or sufficient amounts of the signal) that is required to initiate seed coat formation. However, reduced function of PRC2 in the integuments can bypass the signal requirement and initiate seed coat formation [3]. This suggests that the signal formed by the sexual endosperm removes a block established by PRC2 in the integuments that couples seed coat formation to fertilization (Figure 2). Apparently, however, the consequences of reduced PRC2 activity differ between the central cell and the integuments. Although in the central cell loss of PRC2 activity initiates cell proliferation, this is not the case in the integuments, where growth is mainly driven by cell elongation [3]. However, it is possible that cell elongation in the integuments is a consequence of endopolyploidization, as has been proposed recently in Medicago [42]. Endopolyploidy is a well-documented factor controlling cell size. It arises from variations of the canonical G₁–S–G₂–M cell cycle, leading to genome replication without cell division [51]. Thus one likely scenario is that signal perception in the integuments will remove PRC2 repression and activate the cell cycle while bypassing mitosis, leading to endopolyploidy and cell elongation.

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Figure 2 Regulation of seed development by Polycomb group (PcG) proteins

In an unfertilized ovule (left-hand panel), PcG proteins belonging to the FIS–PRC2 complex repress proliferation of the central cell, whereas PcG proteins belonging to the VERNALIZATION (VRN)/EMBRYONIC FLOWER (EMF) PRC2 repress development of the integuments. After fertilization of the central cell by one of the paternal sperm cells (right-hand panel), FIS–PRC2 function is removed, triggering the development of the sexual endosperm. Additionally, signalling pathways become active in the endosperm that suppress VRN/EMF–PRC2 function in the integuments. The nature of such signalling mechanisms is yet to be understood (‘Signal X’). The development of the seed coat, in its turn, positively feeds back to the endosperm, promoting its proliferation. Double black lines represent apoplastic barriers between central cell and integuments and endosperm and seed coat respectively.

Funding

This research was supported by a European Research Council Starting Independent Researcher grant (to C.K.).