Download Peptide signalling during angiosperm seed

Journal of Experimental Botany, Vol. 66, No. 17 pp. 5151–5159, 2015
doi:10.1093/jxb/erv336 Advance Access publication 20 July 2015
REVIEW PAPER
Peptide signalling during angiosperm seed development
Gwyneth Ingram1,* and Jose Gutierrez-Marcos2,*
1 Laboratoire Reproduction et Développement des Plantes, UMR 5667 CNRS/UMR 0879 INRA, ENS de Lyon, 46 Allée d’Italie, 69364
Lyon Cedex 07, France
2 School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK
* To whom correspondence should be addressed. E-mail: [email protected] or [email protected]
Received 28 April 2015; Revised 9 June 2015; Accepted 11 June 2015
Editor: Thomas Dresselhaus
Abstract
Cell–cell communication is pivotal for the coordination of various features of plant development. Recent studies in
plants have revealed that, as in animals, secreted signal peptides play critical roles during reproduction. However, the
precise signalling mechanisms in plants are not well understood. In this review, we discuss the known and putative
roles of secreted peptides present in the seeds of angiosperms as key signalling factors involved in coordinating different aspects of seed development.
Key words: Cysteine-rich protein, fertilization, receptor-like kinase, seed development.
Introduction
The angiosperm seed is a complex structure composed of
three genetically distinct components: the maternally derived
testa, and the zygotic endosperm and embryo. These three tissues are arranged like Russian dolls, one inside the other, and
develop synchronously post-fertilization to allow the formation of a viable seed, which, through a wide variety of strategies, including environmentally controlled dormancy and the
presence of specific morphological/physiological features,
ensures the dissemination and survival of the next generation. How the developmental coordination of seed development is achieved, however, remains poorly understood.
Plant cells communicate chemically with their neighbours
either symplastically (via plasmodesmata) or apoplastically,
by means of the transfer of information through the cell wall
and across membranes. In order to consider inter-compartmental communication in the seed, it is therefore necessary
first to consider the origins and structure of the boundaries
between each of the relevant compartments. This question
has recently been reviewed in some detail (Bencivenga et al.,
2011; Colombo et al., 2008; Ingram, 2010; Kelley and Gasser,
2009, amongst many), but is worth summarizing. The unfertilized ovule is a complete structure (Kelley and Gasser, 2009)
composed of organs and tissues of sporophytic origin (the
nucellus and the enveloping integuments), which enclose the
female gametophyte. In angiosperms, the female gametophyte
arises from the megaspore, one of four meiotic descendants
of the megasporocyte, which differentiates from an archesporial cell within the nucellus. In Arabidopsis and many other
angiosperms, after the degeneration of its three siblings, the
megaspore undergoes three rounds of mitosis to give the
embryo sac, containing eight nuclei repartitioned into seven
cells. Two of these cells, the haploid egg cell and the double
haploid central cell, are fertilization competent, and will give
rise to the embryo and the endosperm, respectively, upon
fusion with the two sperm cells delivered by the pollen tube.
During female gametophyte development, the initiation
and outgrowth of the integuments from the base of the nucellus leads to a partial or complete sheathing of the nucellus
in further layers of maternally derived tissue. In Arabidopsis,
this process is succeeded by the progressive degeneration of
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5152 | Ingram and Gutierrez-Marcos
the nucellus cells surrounding the distal regions of the female
gametophyte, so that by the time the ovule reaches maturity
much of the female gametophyte is surrounded by a composite wall deposited by both the nucellus and the innermost cell layer of the inner integument (the endothelium).
Unsurprisingly, there is no evidence for the presence of symplastic connections, in the form of plasmodesmata, crossing this complex wall. Furthermore, even in more proximal
zones where the antipodal cells of the female gametophyte
meet the surviving nucellar tissue, symplastic connections
that may have persisted during much of ovule development
are progressively lost. Although evidence is fragmentary, it
seems likely that even in this distal zone symplastic isolation
occurs at or soon after ovule maturity, and is subsequently
assured by the death of the antipodal cells (Song et al., 2014).
The situation in maize is more complex, since the nucellus is
highly proliferated and persists during early post-fertilization
development, and the antipodal cells can also proliferate and
persist. Nonetheless, there is little or any evidence for symplastic communication either between the nucellus and the
endosperm, or between endosperm cells and residual antipodal cells post-fertilization in maize (Diboll and Larson, 1966).
Interestingly, although symplastic connections between the
gametophyte and surrounding maternal tissues are progressively broken during ovule development, they appear to be
maintained between the egg cell and surrounding cells of the
female gametophyte until fertilization. Thereafter, a rapid loss
of symplastic connections occurs between the zygote and surrounding cells (Han et al., 2000; Stadler et al., 2005), although
residual plasmodesmata-like structures of unknown functionality have been observed between the suspensor and surrounding endosperm in a few species (Kozieradzka-Kiszkurno and
Bohdanowicz, 2010). Thus, from an early point in post-fertilization development, the three genetically distinct components of
the seed also represent three distinct symplastic fields, meaning
that all movement of water, solutes, and signalling molecules
between compartments must involve crossing the apoplast.
Given the symplastic isolation of the seed compartments,
it would be logical to assume that peptide-mediated (apoplastic) signalling might play a key role in inter-compartmental
communication during seed development. Any such communication is likely to involve the endosperm because of its
central position. Before developing this idea further, it may
therefore also be useful to consider the likely evolutionary
origin of the process of seed development, with a particular emphasis on the endosperm. Again, this subject has been
widely reviewed (Baroux et al., 2002; Becraft and GutierrezMarcos, 2012; Friedman, 2001). The endosperm plays a key
role as a conduit, transferring nutrients from maternal tissues
to the developing embryo (Berger et al., 2006). This function
is thought to be analogous to that of nutritive tissues formed
in gymnosperm seeds through the proliferation of the unfertilized female gametophyte, and into which gymnosperm the
embryo grows invasively, absorbing nutrients. However, whilst
in gymnosperms the transformation of the gametophyte into
a nutrient sink occurs independently of fertilization (and thus
independently of the presence of an embryo), in angiosperms
this transformation has become linked to fertilization by the
sexualization of the central cell. This innovation, and associated reductions in energy wastage, may have provided a key
evolutionary advantage to the angiosperms. Furthermore, by
rendering both a maternal and a paternal genome necessary
for endosperm development (and thus nutrient allocation), it
has transformed the endosperm into a zone of intense parental conflict (Baroux et al., 2002).
It is in this complex morphological, genetic, and evolutionary context that we wish to consider the role of peptide-mediated signalling in regulating post-fertilization seed
development. In this review we have specifically chosen to
place a strong emphasis on peptide-mediated signalling,
which we consider to be involved in controlling seed-specific,
inter-compartmental communication and, importantly—to
avoid in-depth discussion of signalling pathways whose roles
are maintained (and thus studied)—during post-embryonic
plant development. We would particularly like to investigate
two predictions, which we feel could emerge from consideration of the situation outlined above. First, we propose that
the fact that the concomitant development of the embryo and
endosperm evolved from a situation in which their development was effectively independent (as in gymnosperms) may
mean that any apoplastic signalling pathways coordinating
the development of these compartments in angiosperms may
either have evolved de novo and be angiosperm seed specific,
or have, at the very least, involved the requisitioning/adaptation of existing signalling pathways with the potential incorporation of seed-specific components. Secondly, we propose
that because communication between all three compartments of the seed should involve components expressed in
the endosperm, and could thus fundamentally affect nutrient allocation, these pathways may be specifically targeted for
parental control during seed development.
It is becoming increasingly evident that the coordination
of plant development is largely mediated by peptide signalling. Consistent with this, the genome of Arabidopsis contains
genes coding for over 600 receptor-like proteins, more than
400 peptides, and a plethora of secreted proteases that may
have the capacity to participate in the processing of bioactive
peptides (De Smet et al., 2009; Huang et al., 2015). In addition, plant genomes encode large numbers of genes encoding
enzymes potentially involved in the post-translational modification of receptors and peptide precursors. However, despite
the recent advances in genomic analysis, we still remain ignorant of the detailed transcriptomic complexity of these genes
during reproductive development, and only fragmentary
functional data are available for a handful of signalling peptides involved in seed development. We review this evidence
and discuss its implications in the context of the structural,
functional, and evolutionary peculiarities of the developing
angiosperm seed.
The role of signalling peptides in seed
development
Secreted peptides are highly abundant during seed development. This highlights their potential role in novel or modified
signalling pathways; however, we have only sparse information
Peptide signalling in seeds | 5153
the function of these peptides remains elusive, some of them
are actively expressed during microspore embryogenesis
(Balandin et al., 2005; Magnard et al., 2000). These findings
support the view that ESR peptides form part of an as yet
unknown signalling pathway (or pathways) that regulates
ESR cell fate, differentiation, and/or communication between
endosperm and embryo.
One distinct class of peptides found in maize ESR cells has
been used to define the clavata/esr-like (CLE) class of peptides. These peptides are widely expressed in different organs
(Jun et al., 2010) but some of them are restricted to reproductive tissues (Fiume et al., 2011). One of these peptides in
Arabidopsis, termed CLE8, is confined to the early developing
embryo and endosperm and plays a key role in the developmental regulation of both through the modulation of WOX8
expression (Fiume and Fletcher, 2012). The link between
CLE8 peptides and the WOX8 homeobox gene is particularly
relevant because it reveals that a peptide-transcriptional regulatory module has been co-opted for the regulation not only
of apical–basal embryo formation (Breuninger et al., 2008)
but also for later stages of embryo development.
Another class of secreted peptides expressed in seeds is
KISS OF DEATH (KOD) (Blanvillain et al., 2011). These
peptides are expressed in embryos and are involved in the
positive regulation of programmed cell death of suspensor
cells. Although the precise function of KOD peptides is not
fully known, it is likely that they form part of a signalling
B
1.0
0.8
0.6
Frequency
M
6
12
24
I
Post-fertilization
n= 166
M
6
12
24
Maternal contribution
n= 54
0.8
Frequency
0.8
0.6
0.0
0.2
0.4
0.0
0.2
Frequency
0.4
0.6
0.4
I
1.0
Post-fertilization
0.2
Egg cell
Embryo
Synergid?
Integument
0.0
Central cell
Endosperm
Synergid?
Integument
Fertilization
n= 153
1.0
Endosperm
0.0
Embryo
0.2
Frequency
0.8
1.0
Pre-fertilization
Pre-fertilization
n= 109
0.6
A
0.4
about their precise functions. Intriguingly, most of these peptides are exclusively expressed in specific gametophyte cells
and/or seed compartments and potentially play a role in the
coordination of seed development (Fig. 1A). The earliestacting peptides in seed development belong to the cysteinerich group and are expressed both in the gametes within the
female gametophyte and in the early products of fertilization
(Costa et al., 2014; Sprunck et al., 2012). For instance, in
Arabidopsis, ESF1 peptides are expressed in the central cell
and endosperm embryo-surrounding region (ESR) and act to
positively regulate zygote elongation and the development of
the suspensor (Costa et al., 2014). Intriguingly, these peptides
are related to maize MATERNAL EXPRESSED GENE1
(MEG1) peptides, which are expressed from a small gene
family that is exclusively expressed in basal endosperm transfer layer (BETL) cells (Gutierrez-Marcos et al., 2004). MEG1
peptides act as positive developmental regulators and, because
they control transfer cell development, they play a major
role in nutrient translocation in seeds (Costa et al., 2012).
In maize, several secreted peptides are expressed in the BETL
(Balandin et al., 2005; Magnard et al., 2000; Magnard et al.,
2003; Serna et al., 2001). Although their functions remain
unknown, they have been associated with pathogen defence
and nutrient translocation. In addition, it is possible that they
play regulatory roles to coordinate seed growth and development. Similarly, the ESR cells express cysteine-rich proteins
(CRPs) related to those expressed in transfer cells. Although
I
M
6
12
24
I
M
6
12
24
Fig. 1. Schematic representation showing the contribution of cysteine-rich proteins (CRPs) to early stages of seed development. (A) Peptides derived
from different cell types are potentially implicated in the regulation of seed development. (B) Discrete expression of secreted peptides during three critical
stages of sexual reproduction (pre-fertilization, fertilization, and post-fertilization) and evidence for the maternal cytoplasmic contribution of a small group
of CRPs (data re-analysed from: Costa et al., 2014; Huang et al., 2015). The x axis indicates samples analysed; the y axis indicates expression frequency.
n: Number of peptides identified for each expression group; I: immature ovules; M: mature ovules; 6: 6 hours after fertilization; 12: 12 hours after
fertilization; 24: 24 hours after fertilization.
5154 | Ingram and Gutierrez-Marcos
cascade that regulates the activation of cell death of the suspensor cell lineage during seed maturation (Zhao et al., 2013).
Not all peptides expressed in seeds are confined to embryo
and/or endosperm tissues. For instance, JEKYLL peptides in
barley are expressed primarily in the ovule and in seed integuments, and are involved in the regulation of nucellar development (Radchuk et al., 2006). JEKYLL is required not only
for regulating the cell fate of the nucellar tissue but also for
endosperm development, which highlights the importance of
peptide signalling in maternal/filial interactions during seed
development. The integuments and nucellus of potato ovules
accumulate rapid alkalinization factor (RALF)-like peptides
that regulate multiple aspects of ovule and seed development
(Chevalier et al., 2013). Although the exact role of these peptides is currently unknown, their expression coincides with an
increase of auxin in developing ovules, thus implying a crosstalk between phytohormones and signalling peptides in the
regulation of the initial stages of seed development.
A major limitation in the analysis of seed-specific secreted
peptides is the incomplete transcriptional information currently available. Although recent work has revealed much
of the transcriptome of different seed compartments in
Arabidopsis by coupling laser-capture microdissection (LCM)
and microarray analysis (Le et al., 2010), the expression of
signalling peptide encoding genes is rather incomplete as they
are not represented in the current expression platforms. This
problem is compounded for seed-specific signalling peptide
encoding genes, which are particularly poorly represented.
To overcome this caveat, Tesfaye et al. (2013) generated an
array including most known CRPs for Arabidopsis and
Medicago; however, due to the large number of duplications
in some CRP-encoding families, not all individual gene members were represented in this platform. Two recent studies in
Arabidopsis have shown that coupling manual dissection and
RNA sequencing is a suitable choice for the transcriptomic
analysis of signalling peptides in reproductive tissues (Huang
et al., 2015) (Fig. 1B). Perhaps the most suitable strategy
is the coupling of LCM to next-generation transcriptome
sequencing, as has been accomplished for maize seeds (Zhan
et al., 2015). Although these technologies should aid determination of the transcriptional profile of all signalling peptides in seeds, the computational prediction of these peptides
in recently sequenced plant genomes continues to be limited
(Zhou et al., 2013).
The role of receptors and potential
downstream components in seed
development
Because very few peptide/receptor pairs have been conclusively identified in plants, particularly in the context of intercompartmental communication in seeds, almost as much
can be learnt from considering developmentally important
receptors as from considering the potential peptide signals
themselves. Several receptors and/or receptor like molecules,
as well as components potentially acting in downstream
signalling cascades, have been shown to be involved in seed
development. Perhaps one of the earliest identified is the
receptor kinase HAIKU2, mutants in which show a strongly
reduced seed size (Garcia et al., 2003). HAIKU2 (IKU2) is
a member of the LRR XI subfamily of Arabidopsis LRR
receptor protein kinases (Luo et al., 2005), a subfamily containing several developmentally important proteins, including receptors involved in meristem maintenance (CLV1 and
BAM1, 2 and 3) (Clark et al., 1997; DeYoung et al., 2006),
vascular development (PXY/TDR) (Fisher and Turner,
2007), and organ abscission (HAESA and HSL1 and 2) (Jinn
et al., 2000; Stenvik et al., 2008), as well as proteins involved
in innate immunity (PEPR1 and 2) (Yamaguchi et al., 2010).
The putative protein ligands of receptors from this family are
diverse and fall into at least three different categories. IKU2
is expressed during early seed development specifically in the
endosperm, and loss of iku2 function leads to the production of seeds of reduced size, due to a reduced growth of the
early endosperm and reduced integument elongation (Garcia
et al., 2003; Luo et al., 2005). IKU acts together with several
other proteins, including the VQ-domain-containing IKU1
protein (Wang et al., 2010) and the WRKY transcription
factor MINISEED3/WRKY10 (Luo et al., 2005), in a pathway that has recently been shown to regulate seed growth by
affecting cytokinin homeostasis though the regulation of the
CYTOKININ OXIDASE 2 (CKX2)-encoding gene (Li et al.,
2013). Because IKU2 is expressed early in seed development,
at a stage when the endosperm is composed of a single, large,
coenocytic cell, it seems likely that if, as expected, IKU2 is
inserted in the plasma membrane, it should be involved in the
perception of molecules present either in the apoplast between
the endosperm and the testa, or between the endosperm and
the embryo. Although potential IKU ligands have not yet
been identified, genetic studies suggest that the IKU pathway
could be involved in cross-talk between the sporophytic integuments and the developing endosperm (Garcia et al., 2005),
suggesting the possibility that maternal sporophytic tissues
could be involved in the IKU signalling pathway. Studies of
the subcellular localization of IKU2 protein might help to
resolve this question, but are likely to be hampered by its very
low levels of endogenous expression.
Interestingly, IKU2 is not the only LRR XI subfamily
LRR receptor protein kinase to play a key, and most likely
seed-specific, role in post-fertilization seed development.
Two other members of the same family, GASSHO1 and
GASSHO2 (GSO1 and GSO2) (Tsuwamoto et al., 2008),
play a critical role in cross-talk between the two zygotic compartments: the embryo and the endosperm (Xing et al., 2013).
The two closely related GSO proteins have been shown to be
expressed within the developing embryo and to act redundantly to ensure the formation of a functional embryonic
cuticle (Tsuwamoto et al., 2008). Double gso1/gso2 mutants
show a dramatic reduction in seedling viability owing to the
fact that their cuticles are defective, leading to increased permeability (and thus seedling desiccation sensitivity), as well
as the adhesion of cotyledons to each other and/or to the
seedling hypocotyl. The cotyledons of gso1/gso2 mutants
also adhere to the endosperm during seed development,
leading to an inversion in the direction of embryo bending
Peptide signalling in seeds | 5155
in the seed. This phenotype has also been described in the
seeds of plants lacking an endosperm-expressed subtilisinlike serine protease called ABNORMAL LEAF SHAPE1
(ALE1) (Tanaka et al., 2001; Xing et al., 2013). Consistent
with the similarity in their phenotypes, genetic analysis has
confirmed that this protein likely acts in the same pathway
as GSO1 and GSO2, strongly suggesting that these two
receptor-like kinases (RLKs) are involved in an inter-organismal signalling pathway which reinforces the formation of
a functional apoplastic barrier between the embryo and the
endosperm (Xing et al., 2013). Unfortunately, as is the case
for IKU2, the ligands of GSO1/GSO2 have not yet been
identified, making it difficult to comment on the regulation
of this process. Unlike IKU2, the expression of GSO1 and
GSO2 is not restricted to the developing embryo, with both
showing expression during post-germination development
(Tsuwamoto et al., 2008). Furthermore, GSO1 plays a role
in the formation of another apoplastic barrier, the Casparian
strip, during root development (Pfister et al., 2014). Since it
is rather difficult to draw parallels between embryonic cuticle
reinforcement (deposition of cutin polymers on the surface
of the embryonic epidermis) and Casparian strip formation
(deposition of lignin and then suberin in a band around the
root endodermis) (Geldner, 2013), it seems likely that the role
of GSO1/GSO2 in seeds is distinct from that in roots, and
that the signalling pathway involved may involve seed-specific
components (such as ALE1).
The third signalling component that we will consider in
this context is the SHORT SUSPENSOR (SSP) protein, a
member of the RLCKII family of receptor-like cytoplasmic
kinases (RLCKs) (Bayer et al., 2009). Mutant alleles of SSP
cause strong reductions in the length of the embryonic suspensor, a structure required for nutrient uptake by the early
embryo from the endosperm (Kawashima and Goldberg,
2010). Intriguingly, the progeny of plants heterozygous for
ssp mutant alleles segregate embryos with short suspensors
and normal embryos in a 1:1 ratio, and this has been shown
to be due to a parent-of-origin effect. Unusually, the phenotype of the embryo is dependent upon the genotype of the
pollen; this phenomenon has been shown to be due to the
fact that the SSP protein, although expressed in the embryonic suspensor after fertilization, is in fact translated from
RNA delivered to the zygote from the sperm cell at fertilization (Bayer et al., 2009; Lin et al., 2013).
Unlike RLK, RLCKs are not transmembrane proteins
but can be anchored to the cytoplasmic faces of membranes
via N-terminal modifications such as palmitoylation and/or
mirostylation. This type of stable association is predicted
for SSP, and indeed, mutation of the domains predicted to
mediate membrane association renders the protein cytoplasmic and non-functional. In plants, RLCKs have commonly
been shown to act in association with RLKs, to modify signalling (reviewed recently in Lin et al., 2013). Although SSP
contains a kinase domain, this is thought to be inactive due
to the lack of critical residues in the kinase catalytic sites.
Despite this, considerable genetic evidence has been accrued
to suggest that SSP acts at the head of a signalling cascade
involving the mitogen-activated protein kinase kinase kinase
(MAPKKK) YODA, and the MAPK3 and MAPK6 proteins, to regulate suspensor length (Lukowitz et al., 2004).
These observations suggest that SSP most likely functions via
interaction with an active protein kinase at the plasma membrane, although the identity of this molecule, and whether it
is an RLK, remains for the moment unknown. Interestingly,
SSP is thought to have evolved relatively recently, specifically
in the Brassicaceae, after duplication of the ancestor of the
BRASSINOSTEROID KINASE1 (BSK1) gene. This duplication is thought to have permitted rapid neo-functionalization of SSP1, associated with loss of kinase activity (Bayer
et al., 2009; Liu and Adams, 2010).
Intriguingly, the SSP protein, which is translated in the
embryo from paternally expressed mRNAs, has recently been
shown to act synergistically with maternally produced ESF1
peptides present in the central cell before fertilization and the
embryo-surrounding endosperm immediately post-fertilization, suggesting that the regulation of suspensor development
may be a key site for parental control of nutrient allocation
(Costa et al., 2014).
Potential modifying enzymes acting
upstream of signalling pathways
Apoplastic signalling pathways depend not only upon the
presence of peptides and ligands, but potentially are also subject to regulation at the level of peptide processing and modification. Peptide precursors from several classes are extensively
modified by protein cleavage (mediated by proteases present
either in the secretory system or in the apoplast) and by posttranslational modification of specific amino acids by sulfation, hydroxylation, glycosylation, and potentially by other as
yet unidentified modifications (reviewed extensively, including Matsubayashi, 2011, 2012). Again, the enzymes involved
in these modifications may be present in either the secretory
apparatus or the apoplast. Furthermore, it is entirely possible that the apoplastic domains of receptor molecules are
extensively modified either during or after their insertion in
the plasma membrane. In the context of inter-compartmental
communication, it could be envisaged that peptides secreted
by one compartment are post-translationally modified by
enzymes secreted by an adjacent compartment. However,
no concrete examples of such signalling control mechanisms
have yet been uncovered in plants, possibly because functional studies of the enzymes involved in peptide modification
remain technically challenging and thus are few in number.
Furthermore, although candidate proteins for involvement in
pro-peptide cleavage, peptide sulfation, and peptide hydroxylation have been identified, the enzymes involved in glycosylation remain unknown.
In this context, it is perhaps unsurprising that little if anything
is known about the mechanisms via which post-translational
modifications can affect the early stages of post-fertilization
seed development. Nonetheless, at least one credible candidate
for pro-peptide cleavage has a confirmed role in inter-compartmental communication in seeds. The subtilisin serine protease ALE1 has been implicated in the GSO1/GSO2 signalling
5156 | Ingram and Gutierrez-Marcos
pathway described in the previous section (Tanaka et al., 2001;
Xing et al., 2013). Unlike GSO1 and GSO2, the expression
of ALE1 appears to be entirely seed-specific, and specifically
endosperm-specific, suggesting that this protease could impart
a tissue specificity to this pathway. However, until the substrate
of ALE1 is unambiguously identified, this question will remain
unresolved. Interestingly, another subtilisin-like serine protease, SBT1.1, has also been shown to play a key role in regulating seed size in both Medicago truncatula and Pisum sativum
(D’Erfurth et al., 2012). In Medicago, the SBT1.1 gene shows
strong expression at the endosperm/testa interface and, furthermore, the most closely related Arabidopsis gene, AtSBT1.1,
shows an endosperm-specific expression pattern. Intriguingly,
AtSBT1.1 cleaves the AtPSK4 phytosulfokine (PSK) precursor
in vitro. Phytosulfokines are known to regulate growth and have
been shown to stimulate somatic embryogenesis (Hanai et al.,
2000), suggesting that this subtilase could potentially play a
conserved role in coordinating growth within developing seeds
(Srivastava et al., 2008). Interestingly, although AtSBT1.1 has
unambiguous orthologues in most eudicots, the same is not
true for ALE1, suggesting that the function of ALE1 in seed
development may have emerged later during the course of
angiosperm diversification (Rautengarten et al., 2005).
Transcriptional regulation of
post-germination peptide signalling
pathways
Our starting hypothesis—that apoplastic signalling pathways involved in the post-fertilization coordination of angiosperm seed development may have evolved through the de
novo acquisition of seed-specific components—implies the
presence of transcription factors potentially involved in the
regulation of the seed-specific expression of pathway components. Such transcription factors do undoubtedly exist.
A prime example is the ZHOUPI (ZOU)/RGE (Kondou
et al., 2008; Yang et al., 2008) transcription factor, a member of the bHLH family, which is expressed exclusively in the
Arabidopsis endosperm. ZOU function is required for the
expression of ALE1, and thus for inter-compartmental communication leading to the formation of an intact embryonic
cuticle. We have proposed that the apoplastic separation of
the embryo and endosperm became a major developmental
problem only after endosperm development was rendered
fertilization dependent, due to the concurrent development
of the two organisms (Moussu et al., 2013). The fact that the
ALE1 protein does not appear to be a particularly ancient
gene supports this view. However, ZOU plays a second role
in the endosperm, which leads to endosperm degradation.
Given the apparently ancient origins of the ZOU protein, this
function (or at least a function in mediating female gametophyte degradation) may well date back to gymnosperms and
even lycophytes (Yang et al., 2008). It is therefore possible
that ZOU acquired novel functions in regulating inter-compartmental apoplastic communication pathways during the
angiosperm radiation. The functional characterization of
ZOU in angiosperms other than Arabidopsis may help to
elucidate to what extent these functions differ in different
angiosperm groups.
More recently, a key regulator necessary for seed development has been described in the legume M. truncatula. DOF
Acting in Seed embryogenesis and Hormone accumulation
(DASH), a member of the DOF transcription factor family,
appears to be necessary for the establishment of post-fertilization seed development in M. truncatula (Noguero et al., 2015).
DASH, like ZOU, shows strictly endosperm-specific expression, but this expression is restricted to a subpopulation of
cells in the chalazal zone adjacent to the point of attachment
of the seed to the placental tissues of the pod. Loss of DASH
function leads to markedly reduced seed size and, in some
alleles, to early seed abortion. These phenotypes are accompanied by an apparent defect in auxin distribution, since the
zygotic tissues of dash mutants show a dramatic over-accumulation of auxin and embryo defects consistent with defects
in auxin-mediated morphogenesis, whereas the surrounding
pod tissues show reduced expression of auxin-induced genes
compared to wild-type tissues. A possible explanation for this
complex phenotype is that DASH is necessary to establish a
flow of auxin from the zygotic to the maternal tissues, which
is, in turn, necessary to stimulate the stable establishment of
the seed as a nutrient sink. Interestingly, amongst the most
strongly down-regulated genes in dash mutants are two genes
encoding CRPs. These genes show an identical expression
pattern to DASH, suggesting that they could be direct targets of DASH. It is entirely possible that these peptides play
a role in establishing the contact zone between the chalazal
endosperm and the maternal seed tissues, but this possibility
has not yet been investigated.
A more concrete example of CRP function in establishing
the zone of nutrient transfer between maternal and zygotic
tissues is, of course, provided by the MEG1/CRPs described
above. At the level of transcription, these peptides appear to be
necessary for the expression of a master transcriptional regulator of the BETL, Myb-Related Protein-1 (MRP1) (Gómez
et al., 2002; Gómez et al., 2009) which in turn appears to feed
back on the expression of MEG-encoding genes. It is interesting to note that MRP1 and DASH, despite belonging to
totally different families of transcription factors, both act in
the endosperm transfer layer and appear to control very similar processes, permitting the establishment of the endosperm
as a nutrient sink. It is tempting to speculate that the rapid
establishment of nutrient transfer from maternal to endosperm
tissues became necessary only upon the sexualization of the
endosperm, and that the regulation of this process could have
evolved separately in the monocot and legume lineages. To
date, similar regulatory modules have not been uncovered in
other species, although a transfer-cell-expressed CRP has been
identified in barley and wheat (Kovalchuk et al., 2009).
Parental control of seed development is
mediated by peptide signalling pathways
Over the past three decades, numerous studies have revealed
that the development of seeds is under firm parent-of-origin
regulation (Haig, 2013). A key component of this regulation
Peptide signalling in seeds | 5157
is the imbalanced contribution of transcripts to the two products of fertilization—the embryo and the endosperm. There
are several mechanisms implicated in this phenomenon, but
the best studied is associated with differences in the DNA
methylation of male and female genomes in seed tissues.
The asymmetry in DNA methylation associates with the
unequal expression of parental transcripts after fertilization,
a phenomenon known as genomic imprinting. Intriguingly,
genomic imprinting in angiosperms primarily affects the
endosperm. This phenomenon mostly involves an extensive
loss of DNA methylation, which takes place in the central cell
gamete, and this information is later transmitted, after fertilization, to the endosperm (Gehring, 2013). There are currently
over 200 imprinted genes identified in the endosperm, most
of which encode putative transcriptional regulators and metabolic enzymes, with only a few encoding signalling peptides
(Jiang and Kohler, 2012). One of these imprinted peptides,
MEG1, is expressed exclusively through the maternal alleles
during the early stages of endosperm development (GutierrezMarcos et al., 2004). MEG1 acts in a dose-dependent manner to regulate the development of transfer cells and therefore
acts as a regulatory step in the translocation of nutrients to
the embryo (Costa et al., 2012). Although these studies have
identified imprinting as one potential means of regulating
peptide dosage and signalling pathways in seeds, the precise
components of this pathway (or pathways) remain unknown.
A second mechanism involved in the parent-of-origin
regulation of seed development is the direct contribution of
transcripts present in gametes that are deposited in the two
products of fertilization (Fig. 1). Several studies have revealed
that in both the embryo and endosperm the activation of
parental genomes is delayed, and consequently the earliest
stages of seed development must rely on proteins or transcripts deposited from the gametes (Autran et al., 2011; Del
Toro-De Leon et al., 2014; Grimanelli et al., 2005). As highlighted above, one of these deposited transcripts corresponds
to SSP. Mutants defective in SSP expression show parent-oforigin defects in embryo development because SSP is exclusively expressed in sperm cells and delivered via fertilization
to the zygote, where the protein is translated and functionally
relevant (Bayer et al., 2009). However, the greatest contribution of transcripts and proteins to the early products of fertilization comes from maternal gametes, primarily because
of their significant cytoplasmic contribution. Molecular and
genetic analyses in Arabidopsis have shown that a significant
number of transcripts in embryos and endosperms are maternally derived (Autran et al., 2011). However, it is not known
whether they are derived from the female gametes and/or
newly transcribed after fertilization from only the maternally
inherited genome. Some of the genes involved encode signalling peptides, as is the case for ESF1, that are expressed in
the central cell and early endosperm. Furthermore, central
cell expression is required for ESF1 function and the correct
development of the zygote and early embryo (Costa et al.,
2014). Because the expression of CRP peptides is primarily
confined to mature ovules and early seed development (Costa
et al., 2014; Huang et al., 2015) (see Fig. 1B), it is likely that
they play maternal regulatory roles in seeds. If this is the
case, the functional characterization of these peptides should
reveal their exact roles and the complex relationship between
gametes and all three of the seed components. To reduce the
complexity of these functional analyses, studies could focus
on those signalling peptides that are conserved across all
angiosperms.
Conclusions
In recent years, molecular and genetic studies have collectively identified signalling peptides as critical components
of seed development in plants. Surprisingly, we have precise
functional information for only a handful of them. However,
even this relatively restricted view has highlighted the fact that
many of these peptides are highly seed-specific and, moreover,
may act via signalling cascades containing seed-specific components. Furthermore, it is clear that seed-specific apoplastic
signalling pathways are privileged targets for parental control of seed development. Improved genome annotations and
molecular techniques should reveal the precise expression pattern of these peptides in seeds, including basal angiosperms.
Such analysis could aid not only their functional analysis in
model species, but also contribute to an understanding of
how these peptides, and their perception, evolved during and
after the emergence of the angiosperm lineage. Secreted peptides act as ligand molecules in receptor-mediated complexes
and have a major role in host recognition signalling mechanisms, pollen tube growth in female reproductive tissues, and
plant development. Despite the large number of peptides and
receptors encoded in plant genomes, only a few have been
characterized, and future work will focus on addressing this
gap in our understanding. It seems certain that novel techniques to uncover receptor–peptide interactions and novel
genome editing technologies will further help to uncover the
hidden secrets of seed signalling peptides.
Acknowledgements
We kindly acknowledge funding from the Royal Society, ESF/RTD
Framework COST action (FA0903), BBSRC grants (BB/E008585/1 and BB/
F008082), and the ANR grant INASEED (ANR-13-BSV2-0002).
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