Seed mucilage evolution: diverse molecular mechanisms generate versatile
ecological functionS for particular environmentS
Sébastien Viudes, Vincent Burlat*, Christophe Dunand*
Laboratoire de Recherche en Sciences Végétales, CNRS, UPS, Université de
Toulouse, 31326 Castanet‐Tolosan, France
* corresponding authors (burlat@lrsv.ups-tlse.fr;
dunand@lrsv.ups-tlse.fr)
Abstract
The polysaccharidic mucilage is a widespread plant trait with diverse
features, often present around plant structures in contact with the
environment, providing numerous functions including protection and
adhesion. In myxodiasporous species, a mucilage is released upon the
imbibition of the seed (myxospermy) or the fruit (myxocarpy), and
therefore can play roles in the early seedling stages. It is unclear
whether myxodiaspory has one or multiple evolutionary origins and why it
disappeared in several species. Here, we summarize the recent advances
on (i) the mucilage and mucilage secretory cell diversity, (ii) the
evolution of the molecular actors involved in myxospermy underlying the
observed inter- and intra-species natural diversity and (iii) the
recently identified ecological functions. At the intra-species level, a
high polymorphism was detected for a few genes in relation to the
observed morphological diversity. Well characterized transcriptions
factors interact in master regulatory complexes to balance carbon
partitioning in Arabidopsis thaliana seeds. These transcription
factors were sequentially recruited during seed plant evolution to
control diverse traits including myxospermy, and their functions in
seeds seem to be conserved across Rosids. Historically, the ecological
functions of seed mucilage were mostly related to germination and seed
dissemination but recently some exosystemic functions were uncovered
such as soil micro-organism control and plant establishment support.
These recent studies have advanced the understanding of seed mucilage
diversity and part of its evolution as well as its ecological functions.
Keys words: seed
mucilage, inter-species natural variability, intra-species natural
variability, ecological roles, myxospermy, myxocarpy, myxodiaspory, MBW
master regulator, MSC toolbox gene evolution
Running head : A radiative evolution of myxospermy for
diversified ecological functions
Introduction
Mucopolysaccharides, also called mucilage, are found to be produced in
early diverging non-vascular plant groups such as hornworts (e.g.
Anthoceros sp.) which extrude it around organs for various functions
such as dehydration protection during growth and reproduction
(Renzaglia, Duff, Nickrent & Garbary 2000). In flowering plants,
several kinds of mucilage with cell wall-like compositions can be
secreted by a wide range of organs such as seeds, fruits, roots, leaves,
and stems conferring an impressive diversity of physical properties
(Galloway, Knox & Krause 2020). The term myxodiaspory designates the
ability to extrude mucilage upon imbibition from the seed coat or the
fruit pericarp (Ryding 2001; Figure 1). The ability of species to
release seed mucilage from the seed coat epidermis is called myxospermy,
while the same ability coming from the fruit epicarp outermost cell
layer is called myxocarpy (Figure 1). Seed mucilage presence was
reported early in a Charles Darwin’s letter (Weitbrecht, Müller &
Leubner-Metzger 2011). The description of its morphology in several
species and its putative ecological functions has drawn the interest of
the scientific community for over a century. In the last 50 years,
myxodiaspory was described in a majority of angiosperm orders associated
with the diversity of mucilage secretory cells (MSCs) and their released
polysaccharidic mixtures (reviewed in Phan & Burton 2018). Recent
reviews have explored the biochemical composition and cell wall dynamics
of MSCs during seed development, and the molecular, biochemical and
structural characterization of mucilage, in particular inArabidopsis thaliana (Griffiths & North 2017; Golz et al.2018; Phan & Burton 2018; Šola, Dean & Haughn 2019a). Seed mucilage
can represent a significant metabolic cost as, for instance, it accounts
for 2-3% of the A. thaliana seed mass (Macquet, Ralet,
Kronenberger, Marion-Poll & North 2007b). In the large array of studied
species, this metabolic investment was documented to fulfill numerous
ecological functions such as seed protection and seed dispersal by
direct physical modification of the local conditions (reviewed in
Western 2012; Yang, Baskin, Baskin & Huang 2012c; North, Berger,
Saez-Aguayo & Ralet 2014). As mucilage has specific physical and
chemical properties and can be easily extracted, several applications
were developed in pharmaceutical and food industry, as dietary
supplement and biopolymer respectively (Mirhosseini & Amid 2012;
Soukoulis, Gaiani & Hoffmann 2018).
Even if myxodiaspory has been described all along the angiosperm
phylogenetic tree (Phan & Burton 2018), several species within the
studied plant orders do not have mucilage or do not extrude it. It is
unclear whether myxodiaspory has one or multiple evolutionary origins
and for which reason it was, supposedly, lost in several species. Full
answer to these evolutionary questions will require comparative and
integrative studies connecting seed mucilage function(s) to precise and
standardized morphological descriptions, as well as characterization of
the involved molecular actors on species spread across angiosperms. Such
rare studies concern a few species and are restricted to a single
research field (e.g. genetic characterization, physiology and
development or ecology). The overall aim of this review is to focus on
the under-studied field of myxodiaspory evolution. So far, a deep
characterization of the molecular actors implicated in seed mucilage
establishment was performed primarily in A. thaliana constituting
the so-called MSC tool box (Francoz, Ranocha, Burlat & Dunand 2015).
Computational analyses should identify orthologous relationships betweenA. thaliana MSC toolbox genes and genes in other species, in turn
clarifying the origin of this toolbox, and when it could have started to
be associated with myxospermy. However, numerous MSC toolbox genes form
a complex regulatory network that has unequal contributions to
myxospermy and individual genes can display pleiotropic functions (Golzet al. 2018). Therefore, it is very difficult to clearly identify
genes committed with myxospermy, merely from computational analyses.
Indeed, in A. thaliana seeds, a regulatory complex dictates
carbon partitioning between storage lipids, flavonoid pigments and the
seed mucilage (Song et al. 2017). Lipids accumulating in the
embryo and the endosperm will provide the required nutriments for proper
embryo development, while pigments will confer impermeability and
radiation protection (Baroux & Grossniklaus 2019), and the mucilage
will bring additional ecological advantages depending on the species.
The objective of this review is to integrate multiple levels of
information in a survey on (i) the occurrence and diversity of
myxospermy (and more widely of myxodiaspory), (ii) the evolution of the
molecular actors involved in this complex trait and (iii) the various
ecological functions of seed mucilage. First, we briefly expose seed
mucilage and MSC structural diversity mainly illustrated by comparison
between the pioneer A. thaliana model and the emerging flax
model. In the second part, we present the current status of the
literature concerning the evolutionary origin of the underlying genes,
initially identified in A. thaliana . Their integration with other
seed traits such as storage lipids and protective pigments are developed
focusing on their diversity at both the intra- and inter-species level.
In the last part, we discuss recent insights on seed mucilage numerous
ecological functions facing abiotic and biotic constraints, as well as
its impact on plant development obtained since the last reviewing in
2012 (Western 2012; Yang et al. 2012c).
Diversity of myxodiaspory
A convenient method to attest the presence of mucilage and to study
mucilage secretory cells (MSCs) is mucilaginous pectin staining
performed on hydrated seeds with ruthenium red, or section staining with
a generic and polychromatic stain such as toluidine blue (Western 2001).
Further characterization is obtained by immunofluorescence with cell
wall epitope antibodies directly performed on whole hydrated seeds or on
sections (Ben-Tov et al. 2018). Polysaccharide chemical analysis
can be performed, facilitated by the easy extraction of mucilage (Zhao,
Qiao & Wu 2017; Poulain, Botran, North & Ralet 2019). Ultrastructural
analysis by scanning electron microscopy and atomic force microscopy was
also reported (Kreitschitz & Gorb 2018; Williams et al. 2020).
MSCs correspond to the outermost seed coat epidermal cell layer enabling
mucilage extrusion to the environment (Figures 1, 2A). As exemplified inA. thaliana and Linum usitatissimum (flax), this specific
cell layer is differentiated during seed development to become a dead
layer at the end of the seed maturation (Figure 2; Western 2001; Miartet al. 2019). These two model species illustrate the diversity in
the MSC cell wall dynamics during seed development as well as in the
various mucilage organization patterns and extrusion modes.
During the MSC development, the A. thaliana seed mucilage is
trapped, with no apparent sub-layering, between the outer periclinal
primary wall and a volcano-shaped polarized secondary wall called
columella (Figure 2). In flax, a complex multilayered mucilage is
sequentially deposited in the MSCs and becomes visible after extrusion
(Figure 2). Beyond these two models, the MSC morphological diversity is
also illustrated in the various species studied over the years (Phan &
Burton 2018), as exemplified by the pioneer morphological survey of
mature MSCs conducted on 200 Brassicaceae species covering 90 genera
(Vaughan & Whitehouse 1971).
Upon seed imbibition, sequential events occur within seconds in all
species. First, the hydrophilic nature of the polysaccharide mixture
constituting the mucilage allows water absorption. This induces a
mucilage swelling pressure breaking the MSC primary cell wall (Figure
2). Finally, this leads to mucilage extrusion outwards the seed. As a
result, the seed mucilage volume and mass increase up to 75-fold inCapsella bursa-pastoris (Deng, Jeng, Toorop, Squire & Iannetta
2012) and 5.5-fold in Henophyton deserti (Gorai, El Aloui, Yang
& Neffati 2014), respectively. However, subtle differences occur in
seed mucilage extrusion modes contributing to the diversity of the trait
(Figure 2). Indeed, the rupture of peculiar primary wall domains occurs
either simultaneously in all Arabidopsis MSCs or sequentially in
adjacent flax MSCs (Figure 2). This organized explosion is carefully
prepared earlier during MSC seed development by differential cell wall
polysaccharide deposition and localized modifications. The
polysaccharidic-proteinaceous molecular scaffold enabling the A.
thaliana primary wall domain loosening starts to be uncovered (Kuniedaet al. 2013; Saez-Aguayo et al. 2013; Francoz et
al. 2019). In flax, the MSC polysaccharidic composition and its
internal organization are proposed to play a role in proper MSC opening
(Miart et al. 2019).
The structure and polysaccharidic composition of seed mucilage directly
contribute to the observable diversity of myxospermy. In Arabidopsis,
the released mucilage is separated between an adherent layer bound to
the seed and a non-adherent layer, both enriched in poorly branched type
I rhamnogalacturonan (RGI) pectin (Figure 2; Macquet et al.2007a; Poulain et al. 2019). In flax, the mucilage is composed by
four contrasted layers enriched in RGI, arabinoxylans and
xyloglucans/cellulose, respectively (Figure 2; Kreitschitz & Gorb 2017;
Miart et al. 2019). This type of seed mucilage variable layering
and composition also exists in other species such as for example,Lepidium perfoliatum (Huang, Wang, Yuan, Cao & Lan 2015),Neopallasia pectinata (Kreitschitz & Gorb 2017) orPlantago ovata (Tucker et al. 2017; Yu et al.2017). The comparison among multiple species has demonstrated the
diversity of seed mucilage microstructure observed by scanning electron
microscopy (Kreitschitz & Gorb 2018). Studying seed mucilage allowed
characterization of polysaccharide-polysaccharide specific interactions
(Yu et al. 2018) making seed mucilage an excellent model for cell
wall dynamics understanding (Arsovski, Haughn & Western 2010).
Interestingly, for myxocarpous species such as Salvia andArtemisia species, the mucilage is extruded by the outermost
fruit cell layer, named the achene (non-dehiscent fruit) pericarp, and
not by the seed integument (Ryding 2001). These are clear examples of
evolutionary convergences leading to the similar differentiation into
MSCs of different types of outer cells facing the environment. Indeed,
the Salvia hispanica (chia) achene mucilage and MSCs show
interesting parallels with myxospermous species such as A.
thaliana and L. usitatissimum . Mucilage accumulates in the outer
pericarp epidermal cells during chia seed development. After extrusion,
the mucilage remains indirectly attached to the seed via the inner
pericarp-seed tegument contact (Geneve, Hildebrand, Phillips, Al-Amery
& Kester 2017). Finally, an additional peculiarity may exist inMedicago truncatula and M. orbicularis , the cell wall of
the endosperm forms a mucilage gel between the seed coat and the embryo
(Song et al. 2017). Therefore, myxodiaspory is a trait that
encompasses multiple levels of diversity, including MSC structure,
mucilage extrusion mode, and mucilage polysaccharidic composition and
structural organization. However, there is no simple clear-cut
distribution of the myxodiasporous/non myxodiasporous traits along the
angiosperm families and even within families as exemplified for
Brassicaceae (Vaughan & Whitehouse 1971). For this reason, in the
following part, we will shed light on molecular mechanisms underlying
this morphological diversity through intra-species and inter-species
comparative studies for seed mucilage evolution understanding.
Evolution of the molecular actors underlying the intra- and
inter species myxospermy natural
variability
3.1. The MSC tool box genes may be separated between
upstream regulators and downstream
actors
Twenty years of forward and reverse genetics together with more global
approaches have allowed functional characterization of numerous genes
involved in seed mucilage and MSC physiology in A. thaliana.Recent reviews reported 54 genes (Francoz et al. 2015) and 82
genes (Phan & Burton 2018). Since then, 12 additional genes have been
characterized (Li, Zhang, Chen, Ji & Yu 2018; Shimada et al.2018; Takenaka et al. 2018; Voiniciuc et al. 2018; van
Wijk et al. 2018; Kunieda, Hara-Nishimura, Demura & Haughn 2019;
Šola et al. 2019b; Yang et al. 2019; Wang et al.2019; Fabrissin et al. 2019) bringing the list to 94 genes so
far. They constitute the continuously growing MSC toolbox that
participate to a proper seed mucilage production and release in
Arabidopsis (Francoz et al. 2015; Voiniciuc, Yang, Schmidt, Günl
& Usadel 2015). A majority of these genes are transcription factors
including well characterized upstream master regulators that will be
further discussed hereafter, and less characterized regulatory genes
whose position in the gene regulatory network is still puzzling (Golzet al. 2018). The other downstream genes of the toolbox mostly
encode direct actors responsible of seed mucilage synthesis, assembly
and secretion (Francoz et al. 2015).
3.2. The intra-species myxospermy natural variability
reveals a strong selection pressure targeted on a few downstream MSC
tool box genes directly involved in mucilage synthesis and
release
Detecting and understanding the effects of selection pressure on the MSC
toolbox genes should be easier when considering the intra-species rather
than the inter-species natural variability, because changes are still
relatively recent on the evolutionary scale time and are scarcer.
Indeed, the natural diversity occurring in L. usitatissimumcultivars (Liu et al. 2016; Miart et al. 2019), or inA. thaliana natural ecotypes (Saez-Aguayo et al. 2014;
Voiniciuc et al. 2016) shows a gradient of mucilage abundance and
myxospermy efficiency. This can reach a complete loss of mucilage
extrusion for A. thaliana natural populations such as Sha
(Macquet et al. 2007a) or the loss of adherent mucilage extrusion
for Rak-1 (Saez-Aguayo et al. 2014). Interestingly, the
absence of adherent mucilage extrusion does not necessarily mean a lack
of mucilage synthesis since Rak-1 releases even more non adherent
mucilage than Col-0 (Saez-Aguayo et al. 2014). All characterized
mutation in natural A. thaliana populations related to myxospermy
converge to three downstream genes of the MSC tool box, namelyPECTINMETHYLESTERASE INHIBITOR6 (PMEI6 ) for Dja,MUCILAGE-MODIFIED2/BETA-GALACTOSIDASE6 (MUM2/BGAL6 ) for
Sha, and PEROXIDASE36 (PRX36 ) and MUM2/BGAL6 for
Sk-1. This represents a low number of genes as compared to the MSC
toolbox size and considering the nearly 300 studied natural populations
(Saez-Aguayo et al. 2014; Voiniciuc et al. 2016). This
strongly suggests that downstream MSC toolbox genes undergo strong
selection pressure, but also that myxospermy is a fast-evolving trait as
exemplified by the seven independent MUM2/BGAL6 natural mutants
found in only two different geographical area (Saez-Aguayo et al.2014). PMEI6 , MUM2/BGAL6 and PRX36 encode enzymes
necessary for proper seed mucilage hydration and extrusion (Deanet al. 2007; Kunieda et al. 2013; Saez-Aguayo et
al. 2013) and PMEI6 and PRX36 functions are tightly and sequentially
related (Francoz et al. 2019). As non-myxospermous seeds have a
much better buyoancy efficiency and since Sha habitat is close to a
river, seed dispersal by water run-off is one of the seed mucilage
functions proposed to explain the loss of myxospermy (Macquet et
al. 2007a; Saez-Aguayo et al. 2014). Unfortunately, no clear
association can be established between the natural population habitats
and their mucilage phenotypes (Voiniciuc et al. 2016) but the
selection pressure toward a mucilage disappearance highlights its
ambivalent function for myxospermous species.
More recently, genome wide association studies (GWAS) conducted onA. thaliana (Fabrissin et al. 2019) and L.
usitatissimum (Soto-Cerda et al. 2018) allowed the
identification of the statistically most relevant single nucleotide
polymorphisms (SNPs) explaining the observed seed mucilage phenotype. InL. usitatissimum, out of the seven loci implicated in mucilage
content, five are orthologs of previously characterized A.
thaliana MSC tool box genes that are downstream direct actors of seed
mucilage synthesis or modification (Soto-Cerda et al. 2018). InA. thaliana , through a very precise and molecular phenotyping,
GWAS revealed only 8 peaks significantly above the high background of
less implicated positions, pointing to seed mucilage being a polygenic
trait (Fabrissin et al. 2019). Upon the 8 candidates, two genes
were identified, one already known to belong to the MSC toolbox and
another one characterized as implicated in the production of seed
mucilage pectin content (Fabrissin et al. 2019). These results
highlight the fact that the MSC toolbox starts to be well characterized
in A. thaliana and that it can be used to investigate whether
functional orthologs are present in other species. It also suggests that
at the intra-species level, the myxospermy selective pressure mostly
conserved the upstream master regulators but altered their downstream
target genes of the MSC toolbox. Hopefully, this conservation of the
upstream genes of the toolbox regulation network will allow to study the
evolutionary origin of part of the MSC toolbox genes across angiosperms.
3.3. The angiosperm inter-species myxospermy variability
involves a conserved master regulatory
complex
In A. thaliana , some of the master regulators belonging to the
MSC toolbox also regulate the formation of trichomes and root hairs
(Jones & Dolan 2012), the flavonoid biosynthesis (anthocyanidins and
proanthocyanidins) (Lloyd et al. 2017), and the seed carbon
partitioning (Golz et al. 2018; Li et al. 2018; Chen &
Wang 2019). The combinations of specific MYB and bHLH transcription
factors together with TRANSPARENT TESTA GLABRA 1 (TTG1), a WD40 domain
repeats (WDR) transcription factor allows the regulation of each of
these traits. They constitute the MYB-bHLH-WD40 repeat (MBW) regulatory
complexes (Figure 3). It is important to note that, to our knowledge,
(i) TTG1 is common to all aforementioned traits, (ii) each bHLH protein
is involved in the regulation of two or more traits and (iii) each MYB
mostly controls only one trait (Chen & Wang 2019; Zhang, Chopra,
Schrader & Hülskamp 2019; Figure 3). Accordingly, the A.
thaliana ttg1 mutant lacks root hairs and trichomes, has a
reduced level of anthocyanins and proanthocyanins and does not
accumulate mucilage in the MSCs (Galway et al. 1994; Walkeret al. 1999; Western 2001). In wild type A. thaliana , the
phosphorylation of TTG1 by SHAGGY-like kinases 11/12 (SK11:12) prevents
its interaction with TRANSPARENT TESTA2/MYB123 (TT2/MYB123), a
MYB member of the MBW complex, decreasing the transcription of the
downstream regulator GLABRA2 (GL2 ) (Li et al.2018); Figure 3). The consequence for the seeds is the promotion
of lipid storage in the embryo at the expense of mucilage and flavonoid
pigment production in the seed coat (Li et al. 2018; Figure 3).
This differential regulation of GL2 is probably responsible for
the differential balance between seed lipid and pigment/mucilage
contents in two natural Medicago species that correlates with GL2expression level (Song et al. 2017; Figure 3). However,
additional regulation mechanisms through interactions, competitions,
post-translational or epigenetic modifications may also occur (Xu, Dubos
& Lepiniec 2015; Nguyen, Tran & Nguyen 2019). Altogether, this
probably explains how a few molecular actors control several functions.
Land plant phylogenetic analysis of the WD40 domain repeats (WDR)
transcription factor family reveals that TTG1 appeared in the common
ancestor of angiosperms and gymnosperms (seed plants). It has undergone
a duplication at this node and acquired new functions for control of
epidermal cells differentiation in essentially all organs of plants, in
addition to its circadian clock ancestral function (Airoldi, Hearn,
Brockington, Webb & Glover 2019, Figure 4). Interestingly, the serine
215 that can be phosphorylated by SK11/12 is conserved across seed plant
TTG1 orthologs, suggesting that this ancestral regulation for this
master regulator allowing switches in carbon flow between the seed coat
and the embryo is independent of the presence of seed mucilage (Liet al. 2018; Airoldi et al. 2019).
The bHLH and MYB proteins have been subjected to numerous and recent
duplication events (Sullivan et al. 2019; Doroshkov et al.2019). Their different combinations within several MBW complexes have
probably been co-opted to control the emergence of new biological
process often linked to epidermal cells such as seed mucilage
establishment (Figure 4). Indeed, the A. thaliana MBW regulatory
complex controlling seed mucilage and seed coat pigments involves at
least two bHLHs (TT8-EGL3), one MYB (TT2) together with one WDR (TTG1)
(reviewed in (Golz et al. 2018). For the proper establishment of
myxospermy, there is a need of at least three additional MYBs
(MYB5-MYB23-MYB61) (Penfield 2001; Matsui, Hiratsu, Koyama, Tanaka &
Ohme-Takagi 2005; Li et al . 2009, Figure 3). The functional
conservation of TTG1 in the mucilage production and release has been
demonstrated in the two Brassicaceae species Matthiola incana(Dressel & Hemleben 2009) and Arabis alpina (Chopra et
al. 2014), and in M. truncatula (Pang et al. 2009).
Orthologs of TTG1 from Camellia sinensis (Liu et al.2018b) or even from the monocotyledonous species Setaria italicacan restore mucilage wild type phenotype of A. thalianattg1 mutant through the recovery of GL2 and MUCILAGE
MODIFIED4/RHAMNOSE BIOSYNTHESIS 2 (MUM4/RHM2) gene expression
(Liu et al. 2017). This functional conservation together with
clear orthologous relationships give more confidence for the presence of
TTG1 in the most recent common ancestor of seed plants (Figure 4). Using
a similar trans-complementation approach, the functional conservation of
the two bHLH proteins EGL3 and TT8 have also been demonstrated in all
tested angiosperms for all MBW complex-associated traits (root hair,
trichome, anthocyanin and proanthocyanin pigments and mucilage) (Zhang
& Hülskamp 2019). Additionally, the phylogeny of these two genes is
well resolved at the angiosperm level, with the Amborella
trichopoda ortholog gene branching early for each gene (Doroshkovet al. 2019, Figure 4). In A. thaliana, the three bHLH
genes, EGL3 , GL3 and MYC1 (root hair regulators)
originated from an ancestral bHLH gene after a triplication within
Brassicaceae (Doroshkov et al. 2019, Figure 4). Brassicaceae, as
many other plant clades have undergone a whole-genome duplication (Mabryet al. 2019). Thus, it will be not surprising to find several
duplication events at the Brassicaceae node for other genes belonging to
the MSC toolbox, especially for multigene family such as bHLHs and MYBs.
These duplicated genes then functionally diverged as shown by the
unexpected partial rescue of seed mucilage by the transcomplementation
of A. alpina GL3 in the A. thaliana
gl3 /egl3 /tt8 triple mutant (Zhang & Hülskamp 2019).
Since the Arabidopsis GL3 is not able to complement the mucilage
phenotype in the same triple mutant, this indicates an
intra-Brassicaceae divergence between both GL3 genes from their
last common ancestor, in agreement with the contrasted morphology ofA. alpina MSCs as compared to those of A. thaliana (Chopraet al. 2014). Similarly, in each Rosid family, few changes can be
expected in the myxospermy-related MBW complex, and the more the genes
are in the downstream part of the network (e.g. bHLHs), the
higher the divergence will be responsible of the observed morphological
diversity of MSCs, such as in Brassicaceae (Vaughan & Whitehouse 1971).
Concerning MYB genes, MYB5 and TT2 ortholog genes inM. truncatula have a conserved function because they positively
regulate seed coat pigment and mucilage (Liu, Jun & Dixon 2014),
suggesting that the MBW complexes dedicated to seed mucilage and seed
coat pigments are conserved among the Rosids species (Figure 4).
Furthermore, the identification by genome wide association study (GWAS)
of seven genes implicated in the seed mucilage content in flax, all
orthologous to A. thaliana MSC toolbox genes (Soto-Cerda et
al. 2018), show a conservation in sequences and functions between the
two species, even for some downstream genes. As their last common
ancestor is at the basis of the Rosid clade, this reinforces the
hypothesis of shared ancestral origin of myxospermy in the entire Rosid
clade (Figure 4). However, in its current form, the MYB family phylogeny
is not sufficiently well resolved to comfort this hypothesis, due to the
small size and the great variability of MYB genes (Doroshkov et
al. 2019). Since the phylogeny of this multigene family is difficult to
solve and since bHLH and MYB association in MBW complexes depend of
non-binary competitive interaction (Zhang et al. 2019) and of
post translational modifications (Li et al. 2018), more studies
will be helpful to fully characterize the evolution of the MBW
complexes.
These results suggest that during seed plant evolution, TTG1 first
appeared to balance carbon flow in seed tissues. It progressively
interacted with bHLH members allowing more regulatory functions through
a ternary complex modularity, with numerous and versatile recruitment of
MYB members for deeper specialization to control each trait in different
seed zones such as seed mucilage in the MSCs. A remaining dark area
concerning the mucilage evolution is located between the Rosids andA. thaliana . This could be solved by comparative studies between
Brassicaceae species closely related to A. thaliana such asCamelina sativa , or at a larger scale by comparing the two
emerging models for MSCs flax and A. thaliana .
Altogether, the A. thaliana MSC toolbox genes seem to have
undergone an asymmetrical selection pressure comparing the great
conservation of the MBW complex during evolution, to the strong and
rapid changes of downstream direct actors existing in intra-species
variability. In the following part, we present the newly identified
putative ecological functions of myxodiaspory that can be helpful to
understand why it can confer a selective advantage and to which kind of
constraints.
New insights in ecological function of seed
mucilage
Considering that seed mucilage establishment is costly for the mother
plant metabolism, its presence implies that it probably displays major
functions and that this trait is under a positive selection pressure in
the myxodiasporous species. According to mucilage adherent and
hydrophilic properties, the scientific community first investigated its
influence on seed dispersal and germination (reviewed in Western 2012;
Yang et al. 2012c), and more recently looked for potential
interactions between seed mucilage and the abiotic and biotic
constraints.
4.1. Influence on seed dispersal and
germination
A seed adaptation such as myxospermy is expected to influence seed
dispersal and germination (Figure 5A). However, these roles can be
completely different between closely related species, making it
difficult to extend the concept to all myxodiasporous species.
Counter-intuitively, mucilage can be a negative regulator of seed
germination in Leptocereus scopulophilus (Barrios, Flores,
González-Torres & Palmarola 2015) and also for the achene germination
of Artemisia monosperma (Huang & Gutterman 1999a). InBlepharis persica , the seed mucilage could block oxygen transfer
under water excess and then prevent germination (Witztum, Gutterman &
Evenari 1969). This function was regularly re-emphasized (last time in
Gorai et al. 2014) though never fully demonstrated. However,
mucilage can also improve germination (Figure 5A-1). The pioneer most
cited seed mucilage function in A. thaliana was a positive role
during germination under osmotic stress conditions, considering the
polyethylene glycol (PEG)-dependent decrease of germination rate
observed for myb61 , gl2 and ttg1 mutants (Penfield
2001). On another MSC toolbox downstream gene mutant, a defect in seed
mucilage extrusion induce a delayed germination, suggesting a positive
effect of mucilage on germination efficiency rather than on germination
rate (Arsovski et al. 2009). However, more recently no such
phenotypes were obtained in the mum2 or myb61 mutants
(Saez-Aguayo et al. 2014). Thus, the role of seed mucilage inA. thaliana germination deserves to be deeply explored. ForS. hispanica , the intact myxocarpous achenes germinate better
than the achenes without mucilage (Geneve et al. 2017).
Interestingly, seed mucilage-dependent better germination phenotypes are
obtained with PEG application and not with salt at equal osmotic
potential (Geneve et al. 2017). Upon five desert species
(Lavandula subnuda, Lepidium aucheri, Boerhavia elegans, Plantago
ciliata and Plantago amplexicaulis), the seed mucilage presence
increased water uptake but its removal led to contrasted germination
effects (Bhatt, Santo & Gallacher 2016). Therefore, the mucilage
function in germination seems to be related to water uptake and/or seed
permeability to water and possibly to gazes.
Some studies took advantage of the fact that two seed morphotypes occur
within the same species (myxospermous and non-myxospermous seeds). These
species use these dimorphic seeds to improve species persistence and
dispersion (Liu, Wang, Tanveer & Song 2018a). For three Brassicaceae
species having characterized dimorphic seeds for myxospermy, namelyDiptyocharpus strictus, Capsella bursa-pastorisi and Aethionema
arabicum, the seed morphotype with the higher dormancy are not
myxospermous (Lu, Tan, Baskin & Baskin 2010; Toorop et al. 2012;
Arshad et al. 2019) suggesting that myxospermous seeds should
germinate without delay. The co-occurrence of seed mucilage and wings on
seeds of D. strictus (Lu et al. 2010) and Henophyton
deserti (Gorai et al. 2014) questions whether antitelochory and
anemochory are opposite or can be complementary. Antitelochory prevents
seed dispersion far from the mother plant while anemochory favors
wind-driven dissemination under dry conditions until the seed encounters
water and stops its dispersion. Combination of both traits can give a
powerful advantage by an efficient dispersal until an optimal place for
hydric conditions is reached (Figure 5A-2). Interestingly, Lunaria
annua shows a surprising use of mucilage by secreting it from the inner
surface of the fruit to keep the four seeds sticked to it, even after
its dehiscence, allowing a differential dispersion between the two
halves of the fruit and their two attached seeds by the wind (Leins,
Fligge & Erbar 2018).
4.2. Influence facing abiotic
constraints
As seed mucilage constitutes highly hydrophilic gels, it is tempting to
propose that it may provide water for the embryo. For A.
thaliana , the seed mucilage takes a large amount of water from the
environment but sequesters it through ionic linkages with galacturonic
acid residues (Figure 5B-3; Saez-Aguayo et al. 2014). Indeed,
mutants that have a lack of mucilage, imbibed their seed faster than the
wild type or mutants with un-released mucilage (Saez-Aguayo et
al. 2014). However, the seed mucilage allows fast seed sinking in water
as compared to non myxospermous seeds that can float on water surface
for longer time and even germinate on it. Achenes of Artemisia
sphaerocephala germinate and float better when their mucilage was
removed (Huang & Gutterman 1999b). Interestingly, adhesive and
frictional properties of the seed mucilage can change according to its
hydration level influencing at least its dispersal properties
(Kreitschitz, Kovalev & Gorb 2015, 2016). In A. arabicum , the
seed mucilage thick fibers emerging upon imbibition are able to conserve
their structure and their size upon dehydration (Lenser et al.2016) allowing a dispersal efficiency compromise, for the re-dried
seeds, for wind, water run-off and buoyancy dissemination ways, as
compared to never imbibed seeds and fully imbibed seeds (Arshad et
al. 2019). A more complete understanding of the complex roles of seed
mucilage in water management would necessitate investigating its role in
more natural situations such as succession of wetting and drying cycles,
or flooding following a long drying period.
Soil physical properties can have a major impact on water availability
and root penetration (Figure 5B-4). The myxocarpy of Artemisia
sphaerocephala enhances seedling emergence in its sandy environment and
reduces plant mortality (Yang, Baskin, Baskin, Liu & Huang 2012a). By
adding seed mucilage extract from C. bursa pastoris , soil
rheological properties are modified particularly for hydraulic
conductivity retaining water for longer time (Deng et al. 2014).
A similar effect is provoked by S. hispanica seed mucilage
addition, which links soil particles to increase aggregate stability for
at least 30 days in diverse kinds of soil (Figure 5B-4; Di Marsicoet al. 2018). Therefore, seed mucilage could locally improve the
soil rheological in agreement with the non-disseminating lifestyle of
species such as A. thaliana excreting non-adherent mucilage.
4.3. Biotic interactions
Similar to root border cell mucilage (Knee et al. 2001),
non-adherent seed mucilage constitutes a significant amount of
polysaccharides released in the environment. Taking into consideration
the importance of micro-organisms in plant physiology along their
development and their omnipresence around plant organs, this metabolic
investment could indicate an involvement of seed mucilage in dealing
with biotic constraints (Figure 5C). Its influence on microbial
community for the plant was shown in A. thaliana using the
bacteria Streptomyces lividans that inhibits germination and
growth of the pathogenic fungus Verticillium dahlia that causes
the verticillium wilt. When both micro-organisms are co-inoculated on
seeds, S. lividans has a better proliferation within the seed
mucilage in comparison to V. dahlia and considerably reduced the
plant disease symptoms (Meschke & Schrempf 2010). This “selective
media effect” (Figure 5C-5) promoting microbial hyphae development was
further illustrated with Salvia hispanica achene mucilage andColletotrichum graminicola fungi (Geneve et al. 2017). The
desert plant Artemisia sphaerocephala achene mucilage was shown
to be degraded by micro-organisms, providing CO2 and
soluble sugars to promote seedling establishment (Yang, Baskin, Baskin,
Zhang & Huang 2012b). This beneficial effect was recently explained in
the same species through the mucilage positive effect on soil microbial
community composition and diversity to favor fungal-bacterial
interaction and soil enzyme activities, protecting young seedlings from
drought and pathogens (Hu et al. 2019b). Glomeromycota is one of
the groups of fungal species positively impacted by achene mucilage.
However, the positive effect on seedling growth of these fungi
responsible for arbuscular mycorrhizae (mutualistic symbiosis with
plants) do not show significant combined positive effect with mucilage,
acting probably independently to enhance seedling establishment (Huet al. 2019a). Active stress-associated enzymes, such as
nuclease, protease, and chitinase, are secreted from the seed coat of
several species even in seeds several decades old (Raviv et al.2017). However, this ability to secrete proteins is conserved in the non
myxospermous species Raphanus sativus or in the A.
thaliana gl2 mutant deprived of seed mucilage suggesting that
protein secretion and seed mucilage are independent (Raviv et al.2017).
Nematodes are in close interaction with plants and can have major
pathogenic impact on plant development. A. thaliana seed mucilage
contributes to attract root-knot nematodes (Figure 5C-6) with the
additional requirement of seed-surface carbohydrates and proteins (Tsaiet al. 2019). Considering the parasitic nature of those
nematodes, this attraction is more probably due to the nematode
adaptation rather than a plant adaptation. Conversely, nematodes could
be predators of even more dangerous organisms for plants. Capsella
bursa-pastoris, a closely relative species of A. thaliana, has
myxospermous seeds that are also able to attract nematodes (Roberts,
Warren & Provan 2018). Surprisingly, it seems to be a case of
protocarnivory because the massive death of trapped nematodes in seed
mucilage increases plant development from germination to young seedling
establishment, especially under low nutrient level (Roberts et
al. 2018). Thus, seed mucilage involvement in biotic interactions
starts to be uncovered revealing astonishing functions that could affect
the plant development in unexpected manners as compared to the
previously characterized functions
Conclusions and
perspectives
Myxodiaspory is a macroscopic seed trait that results from a surprising
diversity of microscopic features among angiosperms, at both the
intra-species and inter-species levels. This striking microscopic
morphological diversity makes it difficult to trace back the
evolutionary origin of seed mucilage based only on morphology. The
available deeply characterized A. thaliana mucilage secretory
cell (MSC) toolbox genes now allow comparison of these molecular actors
within a species or between species, which starts to shed light on this
mysterious evolutionary story. On the one hand, the intense selection
pressure that mucilage establishment undergoes appears to be mainly
applied on a few downstream genes of the MSC toolbox as illustrated in
non myxospermous A. thaliana natural populations. On the other
hand, the seed mucilage-related MBW upstream master regulatory complex
appeared sequentially during seed plants evolution and is notably
conserved across Rosids. From this regulatory complex, mucilage may have
evolved several times independently as a combination of highly diverse
traits including two MSC epidermal origins, various patterns of MSC cell
wall dynamics, various mucilage polysaccharidic composition and
sub-layering patterns or several modes of mucilage release. In turn,
this diversity of traits probably contributes to the wide range of
ecological functions observed in each species that face contrasted
environments. The biotic and abiotic constraints are the least studied
points in this field and probably the most promising tracks to uncover
new seed mucilage integrated functions related to particular
environments.
ACKNOWLEDGMENTS
The authors are thankful to Université Paul Sabatier-Toulouse III
(France) and CNRS for supporting their research work. S.V. benefited
from a PhD scholarship funded by the University Paul Sabatier-Toulouse
III. This work was also supported by the French Laboratory of Excellence
project “TULIP” (ANR-10-LABX-41; ANR-11-IDEX-0002-02) and the French
National Research Agency project “MicroWall” (ANR-18-CE20-0007). We
would like to thank Charles Uhlmann for improving the english language.
FIGURE LEGENDS:
Figure 1: Developmental scheme comparing the two epidermal
origins of mucilage secretory cells enabling mucilage release in
myxospermous vs myxocarpous species. Note that the different layers are
not drawn to scale and that more than one seed per fruit is commonly
found in myxospermous species.
Figure 2: Arabidopsis and flax as model species for seed
mucilage secretory cell (MSC) development illustrating the diversity of
cell wall dynamics, intracellular polysaccharidic mucilage organization,
and extrusion mechanisms upon water imbibition. (A) conceptual
seed cross section wide view and magnification illustrating the
localization of seed mucilage secretory cells (MSCs) (B)Kinetics of MSC development in the historical model Arabidopsis
thaliana on the left (adapted from (Western 2001; Francoz et al.2019), and in the emerging model Linum usitatissimum on the right
(adapted from (Miart et al. 2019). The 30-50 µm wide cells from
both species are drawn to similar scale, the released mucilage layers
are not drawn to scale. Note the numerous differences between both
models: Major features in A. thaliana : highly complex cell wall
dynamics with the presence of a volcano-shaped columella, the
simultaneous rupture of primary cell wall domain in each MSC cells and
the distinction between adherent mucilage (am) and non-adherent mucilage
layers (nam). Major features in L. usitatissimum : sequential
synthesis of four highly different mucilage layers (m1 to m4) and
sequential rupture of primary wall domains expending from cell to cell.
DAP=day after pollination
Figure 3: The MYB-bHLH-WDR (MBW) complexes regulate the
spatiotemporal carbon partitioning in Arabidopsis thaliana seeds
through a complex modularity. The conserved ancestral master regulator
WDR member (TTG1) and the phosphorylation status of a conserved serine
(P), together with a bHLH and MYB modularity of competitive interaction
enables the regulation of the spatiotemporal specificity of various seed
traits including mucilage production and release (Golz et al.2018; Li et al. 2018; Chen & Wang 2019).
Figure 4: Simplified phylogenetic tree of land plants
giving an overview of the sequential evolution of the A. thalianaMSC toolbox using published phylogenomic, transcomplementation and
functional characterization studies . Note that the members of the MBW
complex controlling the myxospermy trait in A. thalianasequentially evolved and were complete at the Rosid node, and that the
downstream enzymatic actors of the MSC tool box appear to be specific toA. thaliana . Species names on the tree leaves are the ones use in
the reviewed studies. EGL3* is the ancestor gene of AtEGL3, AtGL3 and
AtMYC1 that were later duplicated and diverged within the Brassicaceae
family. TTG1 phosphorylation site (P) seems to be conserved together
with the gene itself. The phylogenomic, transcomplementations and
functional characterizations studies used to build this Figure come from
Dressel & Hemleben 2009; Pang et al. 2009; Chopra et al.2014; Liu et al. 2014, 2017, 2018b; Li et al. 2018;
Airoldi et al. 2019; Zhang et al. 2019; Doroshkov et
al. 2019; Zhang & Hülskamp 2019.
Figure 5: Global overview of seed (or fruit) mucilage major
ecological functions facing environmental constraints. (A) Seed
mucilage may influence plant development through (1) positive or
negative impact on germination upon inappropriate condition, depending
on the species, or (2) seed dispersal in relation to conferred seed
physical properties such as sinking ability or soil anchoring(B) Seed mucilage is modified by abiotic conditions in close
environment through (3) regulation of water flux and water availability
as well as (4) soil rheological remodelling properties. (C)Seed mucilage is involved in biotic interactions though (5) direct or
indirect influence on microbial community establishment around the seed
and the future plant and (6) attraction ability of nematodes.
ORCID
Vincent Burlat https://orcid.org/0000-0002-0897-6011
Christophe Dunand https://orcid.org/0000-0003-1637-4042
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