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
REFERENCES
Airoldi C.A., Hearn T.J., Brockington S.F., Webb A.A.R. & Glover B.J. (2019) TTG1 proteins regulate circadian activity as well as epidermal cell fate and pigmentation. Nature Plants 5 , 1145–1153.
Arshad W., Sperber K., Steinbrecher T., Nichols B., Jansen V.A.A., Leubner-Metzger G. & Mummenhoff K. (2019) Dispersal biophysics and adaptive significance of dimorphic diaspores in the annualAethionema arabicum (Brassicaceae). New Phytologist221 , 1434–1446.
Arsovski A. a., Haughn G.W. & Western T.L. (2010) As a model for plant cell wall research. Plant Signaling & Behavior 5 , 796–801.
Arsovski A.A., Popma T.M., Haughn G.W., Carpita N.C., McCann M.C. & Western T.L. (2009) AtBXL1 encodes a bifunctional -D-Xylosidase/ -L-Arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells. Plant Physiology150 , 1219–1234.
Baroux C. & Grossniklaus U. (2019) Seeds—An evolutionary innovation underlying reproductive success in flowering plants. Current Topics in Developmental Biology 131 , 605–642.
Barrios D., Flores J., González-Torres L.R. & Palmarola A. (2015) The role of mucilage in the germination of Leptocereus scopulophilus(Cactaceae) seeds from Pan de Matanzas, Cuba. Botany 93 , 251–255.
Ben-Tov D., Idan-Molakandov A., Hugger A., Ben-Shlush I., Günl M., Yang B., … Harpaz-Saad S. (2018) The role of COBRA-LIKE 2 function, as part of the complex network of interacting pathways regulating Arabidopsis seed mucilage polysaccharide matrix organization.Plant Journal 94 , 497–512.
Bhatt A., Santo A. & Gallacher D. (2016) Seed mucilage effect on water uptake and germination in five species from the hyper-arid Arabian desert. Journal of Arid Environments 128 , 73–79.
Chen S. & Wang S. (2019) GLABRA2, a common regulator for epidermal cell fate determination and anthocyanin biosynthesis in Arabidopsis.International Journal of Molecular Sciences 20 , 4997.
Chopra D., Wolff H., Span J., Schellmann S., Coupland G., Albani M.C., … Hülskamp M. (2014) Analysis of TTG1 function in Arabis alpina . BMC Plant Biology 14 , 16.
Dean G.H., Zheng H., Tewari J., Huang J., Young D.S., Hwang Y.T., … Haughn G.W. (2007) The Arabidopsis MUM2 gene encodes a -Galactosidase required for the production of seed coat mucilage with correct hydration properties. The Plant Cell 19 , 4007–4021.
Deng W., Hallett P.D., Jeng D.S., Squire G.R., Toorop P.E. & Iannetta P.P.M. (2014) The effect of natural seed coatings of Capsella bursa-pastoris L. Medik. (shepherd’s purse) on soil-water retention, stability and hydraulic conductivity. Plant and Soil387 , 167–176.
Deng W., Jeng D.S., Toorop P.E., Squire G.R. & Iannetta P.P.M. (2012) A mathematical model of mucilage expansion in myxospermous seeds ofCapsella bursa-pastoris (shepherds purse).
Di Marsico A., Scrano L., Labella R., Lanzotti V., Rossi R., Cox L., … Amato M. (2018) Mucilage from fruits/seeds of chia (Salvia hispanica L.) improves soil aggregate stability.Plant and Soil 425 , 57–69.
Annals of Botany 109 , 419–427.
Doroshkov A. V., Konstantinov D.K., Afonnikov D.A. & Gunbin K. V. (2019) The evolution of gene regulatory networks controllingArabidopsis thaliana L. trichome development. BMC Plant Biology 19 , 53.
Dressel A. & Hemleben V. (2009) Transparent Testa Glabra 1 (TTG1) and TTG1-like genes in Matthiola incana R. Br. and related Brassicaceae and mutation in the WD-40 motif. Plant Biology11 , 204–212.
Fabrissin I., Cueff G., Berger A., Granier F., Sallé C., Poulain D., … North H.M. (2019) Natural variation reveals a key role for rhamnogalacturonan I in seed outer mucilage and underlying genes.Plant Physiology 181 , 1498–1518.
Francoz E., Ranocha P., Burlat V. & Dunand C. (2015) Arabidopsis seed mucilage secretory cells: regulation and dynamics. Trends in Plant Science 20 , 515–524.
Francoz E., Ranocha P., Le Ru A., Martinez Y., Fourquaux I., Jauneau A., … Burlat V. (2019) Pectin demethylesterification generates platforms that anchor peroxidases to remodel plant cell wall domains.Developmental Cell 48 , 261-276.
Galloway A.F., Knox P. & Krause K. (2020) Sticky mucilages and exudates of plants: putative microenvironmental design elements with biotechnological value. New Phytologist 225 , 1461–1469.
Galway M.E., Masucci J.D., Lloyd A.M., Walbot V., Davis R.W. & Schiefelbein J.W. (1994) The TTG gene is required to specify epidermal cell fate and cell patterning in the Arabidopsis root.Developmental Biology 166 , 740–754.
Geneve R.L., Hildebrand D.F., Phillips T.D., Al-Amery M. & Kester S.T. (2017) Stress influences seed germination in mucilage-producing chia.Crop Science 57 , 2160–2169.
Golz J.F., Allen P.J., Li S.F., Parish R.W., Jayawardana N.U., Bacic A. & Doblin M.S. (2018) Layers of regulation – Insights into the role of transcription factors controlling mucilage production in the Arabidopsis seed coat. Plant Science 272 , 179–192.
Gorai M., El Aloui W., Yang X. & Neffati M. (2014) Toward understanding the ecological role of mucilage in seed germination of a desert shrubHenophyton deserti : interactive effects of temperature, salinity and osmotic stress. Plant and Soil 374 , 727–738.
Griffiths J.S. & North H.M. (2017) Sticking to cellulose: exploiting Arabidopsis seed coat mucilage to understand cellulose biosynthesis and cell wall polysaccharide interactions. New Phytologist214 , 959–966.
Hu D., Baskin J.M., Baskin C.C., Wang Z., Zhang S., Yang X. & Huang Z. (2019a) Arbuscular mycorrhizal symbiosis and achene mucilage have independent functions in seedling growth of a desert shrub.Journal of Plant Physiology 232 , 1–11.
Hu D., Zhang S., Baskin J.M., Baskin C.C., Wang Z., Liu R., … Huang Z. (2019b) Seed mucilage interacts with soil microbial community and physiochemical processes to affect seedling emergence on desert sand dunes. Plant Cell and Environment 42 , 591–605.
Huang D., Wang C., Yuan J., Cao J. & Lan H. (2015) Differentiation of the seed coat and composition of the mucilage of Lepidium perfoliatum L.: a desert annual with typical myxospermy. Acta Biochimica et Biophysica Sinica 47 , 775–787.
Huang Z. & Gutterman Y. (1999a) Water absorption by mucilaginous achenes of Artemisia monosperma : floating and germination as affected by salt concentrations. Israel Journal of Plant Sciences47 , 27–34.
Huang Z. & Gutterman Y. (1999b) Germination of Artemisia sphaerocephala (Asteraceae), occurring in the sandy desert areas of Northwest China. South African Journal of Botany 65 , 187–196.
Jones V.A.S. & Dolan L. (2012) The evolution of root hairs and rhizoids. Annals of botany 110 , 205–212.
Knee E.M., Gong F.C., Gao M., Teplitski M., Jones A.R., Foxworthy A., … Bauer W.D. (2001) Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source. Molecular Plant-Microbe Interactions 14 , 775–784.
Kreitschitz A. & Gorb S.N. (2017) How does the cell wall ‘stick’ in the mucilage? A detailed microstructural analysis of the seed coat mucilaginous cell wall. Flora: Morphology, Distribution, Functional Ecology of Plants 229 , 9–22.
Kreitschitz A. & Gorb S.N. (2018) The micro- and nanoscale spatial architecture of the seed mucilage—Comparative study of selected plant species. PLOS ONE 13 , e0200522.
Kreitschitz A., Kovalev A. & Gorb S.N. (2015) Slipping vs sticking: water-dependent adhesive and frictional properties of Linum usitatissimum L. seed mucilaginous envelope and its biological significance. Acta Biomaterialia 17 , 152–159.
Kreitschitz A., Kovalev A. & Gorb S.N. (2016) “Sticky invasion” – the physical properties of Plantago lanceolata L. seed mucilage.Beilstein Journal of Nanotechnology 7 , 1918–1927.
Kunieda T., Hara-Nishimura I., Demura T. & Haughn G.W. (2019) Arabidopsis FLYING SAUCER 2 functions redundantly with FLY1 to establish normal seed coat mucilage. Plant and Cell Physiology 61 , 308–317.
Kunieda T., Shimada T., Kondo M., Nishimura M., Nishitani K. & Hara-Nishimura I. (2013) Spatiotemporal secretion of PEROXIDASE36 is required for seed coat mucilage extrusion in Arabidopsis. The Plant Cell 25 , 1355–1367.
Leins P., Fligge K. & Erbar C. (2018) Silique valves as sails in anemochory of Lunaria (Brassicaceae). Plant Biology 20 , 238–243.
Lenser T., Graeber K., Cevik Ö.S., Adigüzel N., Dönmez A.A., Grosche C., … Leubner-Metzger G. (2016) Developmental control and plasticity of fruit and seed dimorphism in Aethionema arabicum . Plant Physiology 172 , 1691–1707.
Li C., Zhang B., Chen B., Ji L. & Yu H. (2018) Site-specific phosphorylation of TRANSPARENT TESTA GLABRA1 mediates carbon partitioning in Arabidopsis seeds. Nature Communications9 , 571.
Li S.F., Milliken O.N., Pham H., Seyit R., Napoli R., Preston J., … Parish R.W. (2009) The Arabidopsis MYB5 transcription factor regulates mucilage synthesis, seed coat development, and trichome morphogenesis. The Plant Cell 21 , 72–89.
Liu C., Jun J.H. & Dixon R.A. (2014) MYB5 and MYB14 play pivotal roles in seed coat polymer biosynthesis in Medicago truncatula .Plant Physiology 165 , 1424–1439.
Liu J., Shim Y.Y., Shen J., Wang Y., Ghosh S. & Reaney M.J.T. (2016) Variation of composition and functional properties of gum from six Canadian flaxseed (Linum usitatissimum L.) cultivars.International Journal of Food Science and Technology 51 , 2313–2326.
Liu K., Qi S., Li D., Jin C., Gao C., Duan S., … Chen M. (2017) TRANSPARENT TESTA GLABRA 1 ubiquitously regulates plant growth and development from Arabidopsis to foxtail millet (Setaria italica ).Plant Science 254 , 60–69.
Liu R., Wang L., Tanveer M. & Song J. (2018a) Seed heteromorphism: an important adaptation of halophytes for habitat heterogeneity.Frontiers in Plant Science 9 , 1515.
Liu Y., Hou H., Jiang X., Wang P., Dai X., Chen W., … Xia T. (2018b) A WD40 repeat protein from Camellia sinensis regulates anthocyanin and proanthocyanidin accumulation through the formation of MYB–bHLH–WD40 ternary complexes. International Journal of Molecular Sciences 19 , 1686.
Lloyd A., Brockman A., Aguirre L., Campbell A., Bean A., Cantero A. & Gonzalez A. (2017) Advances in the MYB-bHLH-WD Repeat (MBW) pigment regulatory model: addition of a WRKY factor and co-option of an anthocyanin MYB for betalain regulation. Plant and Cell Physiology 58 , 1431–1441.
Lu J., Tan D., Baskin J.M. & Baskin C.C. (2010) Fruit and seed heteromorphism in the cold desert annual ephemeral Diptychocarpus strictus (Brassicaceae) and possible adaptive significance.Annals of Botany 105 , 999–1014.
Mabry M., Brose J., Blischak P., Sutherland B., Dismukes W., Bottoms C., … Pires C. (2019) Phylogeny and multiple independent whole-genome duplication events in the Brassicales. bioRxiv preprint .
Macquet A., Ralet M.-C., Loudet O., Kronenberger J., Mouille G., Marion-Poll A. & North H.M. (2007a) A naturally occurring mutation in an Arabidopsis accession affects a β-d-Galactosidase that increases the hydrophilic potential of rhamnogalacturonan I in seed mucilage.The Plant Cell 19 , 3990–4006.
Macquet A., Ralet M.C., Kronenberger J., Marion-Poll A. & North H.M. (2007b) In situ, chemical and macromolecular study of the composition ofArabidopsis thaliana seed coat mucilage. Plant and Cell Physiology 48 , 984–999.
Matsui K., Hiratsu K., Koyama T., Tanaka H. & Ohme-Takagi M. (2005) A chimeric AtMYB23 repressor induces hairy roots, elongation of leaves and stems, and inhibition of the deposition of mucilage on seed coats in Arabidopsis. Plant and Cell Physiology 46 , 147–155.
Meschke H. & Schrempf H. (2010) Streptomyces lividans inhibits the proliferation of the fungus Verticillium dahliae on seeds and roots of Arabidopsis thaliana . Microbial Biotechnology3 , 428–443.
Miart F., Fournet F., Dubrulle N., Petit E., Demailly H., Dupont L., … Pageau K. (2019) Cytological approaches combined with chemical analysis reveals the layered nature of flax mucilage. Frontiers in Plant Science 10 , 684.
Mirhosseini H. & Amid B.T. (2012) A review study on chemical composition and molecular structure of newly plant gum exudates and seed gums. Food Research International 46 , 387–398.
Nguyen C.T., Tran G.B. & Nguyen N.H. (2019) The MYB–bHLH–WDR interferers (MBWi) epigenetically suppress the MBW’s targets.Biology of the Cell 111 , 284–291.
North H.M., Berger A., Saez-Aguayo S. & Ralet M.C. (2014) Understanding polysaccharide production and properties using seed coat mutants: future perspectives for the exploitation of natural variants. Annals of Botany 114 , 1251–1263.
Pang Y., Wenger J.P., Saathoff K., Peel G.J., Wen J., Huhman D., … Dixon R.A. (2009) A WD40 repeat protein from Medicago truncatula is necessary for tissue-specific anthocyanin and proanthocyanidin biosynthesis but not for trichome development.Plant Physiology 151 , 1114–1129.
Penfield S. (2001) MYB61 is required for mucilage deposition and extrusion in the Arabidopsis seed coat. The Plant Cell13 , 2777–2791.
Phan J.L. & Burton R.A. (2018) New insights into the composition and structure of seed mucilage. In Annual Plant Reviews . pp. 1–41. John Wiley & Sons, Ltd, Chichester, UK.
Poulain D., Botran L., North H.M. & Ralet M.-C. (2019) Composition and physicochemical properties of outer mucilage from seeds of Arabidopsis natural accessions. AoB PLANTS 11 , plz031.
Raviv B., Aghajanyan L., Granot G., Makover V., Frenkel O., Gutterman Y. & Grafi G. (2017) The dead seed coat functions as a long-term storage for active hydrolytic enzymes. PLOS ONE 12 , e0181102.
Renzaglia K.S., Duff R.J., Nickrent D.L. & Garbary D.J. (2000) Vegetative and reproductive innovations of early land plants: implications for a unified phylogeny. Philosophical Transactions of the Royal Society B: Biological Sciences 355 , 769–793.
Roberts H.R., Warren J.M. & Provan J. (2018) Evidence for facultative protocarnivory in Capsella bursa-pastoris seeds. Scientific Reports 8 , 10120.
Ryding O. (2001) Myxocarpy in the Nepetoideae (Lamiaceae) with notes on myxodiaspory in general. Systematics and Geography of Plants71 , 503–514.
Saez-Aguayo S., Ralet M.-C., Berger A., Botran L., Ropartz D., Marion-Poll A. & North H.M. (2013) PECTIN METHYLESTERASE INHIBITOR6 promotes Arabidopsis mucilage release by limiting methylesterification of homogalacturonan in seed coat epidermal cells. The Plant Cell25 , 308–323.
Saez-Aguayo S., Rondeau-Mouro C., Macquet A., Kronholm I., Ralet M.C., Berger A., … North H.M. (2014) Local evolution of seed flotation in Arabidopsis. PLoS Genetics 10 , e1004221.
Shimada T., Kunieda T., Sumi S., Koumoto Y., Tamura K., Hatano K., … Hara-Nishimura I. (2018) The AP-1 complex is required for proper mucilage formation in Arabidopsis seeds. Plant & cell physiology 59 , 2331–2338.
Šola K., Dean G.H. & Haughn G.W. (2019a) Arabidopsis seed mucilage: a specialised extracellular matrix that demonstrates the structure–function versatility of cell wall polysaccharides. InAnnual Plant Reviews online . pp. 1085–1116. Wiley.
Šola K., Gilchrist E.J., Ropartz D., Wang L., Feussner I., Mansfield S.D., … Haughn G.W. (2019b) RUBY, a putative galactose oxidase, influences pectin properties and promotes cell-to-cell adhesion in the seed coat epidermis of arabidopsis. Plant Cell 31 , 809–831.
Song Y., He L., Wang X.-D., Smith N., Wheeler S., Garg M.L. & Rose R.J. (2017) Regulation of carbon partitioning in the seed of the model legume Medicago truncatula and Medicago orbicularis: a comparative approach.Frontiers in Plant Science 8 , 2070.
Soto-Cerda B.J., Cloutier S., Quian R., Gajardo H.A., Olivos M. & You F.M. (2018) Genome-wide association analysis of mucilage and hull content in flax (Linum usitatissimum l.) seeds.International Journal of Molecular Sciences 19 , 2870.
Soukoulis C., Gaiani C. & Hoffmann L. (2018) Plant seed mucilage as emerging biopolymer in food industry applications. Current Opinion in Food Science 22 , 28–42.
Sullivan A.M., Arsovski A.A., Thompson A., Sandstrom R., Thurman R.E., Neph S., … Queitsch C. (2019) Mapping and dynamics of regulatory DNA in maturing Arabidopsis thaliana siliques. Frontiers in Plant Science 10 , 1434.
Takenaka Y., Kato K., Ogawa-Ohnishi M., Tsuruhama K., Kajiura H., Yagyu K., … Ishimizu T. (2018) Pectin RG-I rhamnosyltransferases represent a novel plant-specific glycosyltransferase family.Nature Plants 4 , 669–676.
Toorop P.E., Campos Cuerva R., Begg G.S., Locardi B., Squire G.R. & Iannetta P.P.M. (2012) Co-adaptation of seed dormancy and flowering time in the arable weed Capsella bursa-pastoris (shepherds purse).Annals of Botany 109 , 481–489.
Tsai A.Y.L., Higaki T., Nguyen C.N., Perfus-Barbeoch L., Favery B. & Sawa S. (2019) Regulation of root-knot nematode behavior by seed-coat mucilage-derived attractants. Molecular Plant 12 , 99–112.
Tucker M.R., Ma C., Phan J., Neumann K., Shirley N.J., Hahn M.G., … Burton R.A. (2017) Dissecting the genetic basis for seed coat mucilage heteroxylan biosynthesis in Plantago ovata using gamma irradiation and infrared spectroscopy. Frontiers in Plant Science8 , 328.
Vaughan J.G. & Whitehouse J.M. (1971) Seed structure and the taxonomy of the Cruciferae. Botanical Journal of the Linnean Society64 , 383–409.
Voiniciuc C., Engle K.A., Günl M., Dieluweit S., Schmidt M.H.-W., Yang J.-Y., … Usadel B. (2018) Identification of key enzymes for pectin synthesis in seed mucilage. Plant Physiology 178 , 1045–1064.
Voiniciuc C., Yang B., Schmidt M.H.W., Günl M. & Usadel B. (2015) Starting to gel: How Arabidopsis seed coat epidermal cells produce specialized secondary cell walls. International Journal of Molecular Sciences 16 , 3452–3473.
Voiniciuc C., Zimmermann E., Schmidt M.H.-W., Günl M., Fu L., North H.M. & Usadel B. (2016) Extensive natural variation in Arabidopsis seed mucilage structure. Frontiers in Plant Science 7 , 803.
Walker A.R., Davison P.A., Bolognesi-Winfield A.C., James C.M., Srinivasan N., Blundell T.L., … Gray J.C. (1999) The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in arabidopsis, encodes a WD40 repeat protein.The Plant Cell 11 , 1337–1349.
Wang M., Xu Z., Ahmed R.I., Wang Y., Hu R., Zhou G. & Kong Y. (2019) Tubby-like Protein 2 regulates homogalacturonan biosynthesis in Arabidopsis seed coat mucilage. Plant Molecular Biology99 , 421–436.
Weitbrecht K., Müller K. & Leubner-Metzger G. (2011) First off the mark: early seed germination. Journal of Experimental Botany62 , 3289–3309.
Western T.L. (2001) Isolation and characterization of mutants defective in seed coat mucilage secretory cell development in Arabidopsis.Plant Physiology 127 , 998–1011.
Western T.L. (2012) The sticky tale of seed coat mucilages: production, genetics, and role in seed germination and dispersal. Seed Science Research 22 , 1–25.
van Wijk R., Zhang Q., Zarza X., Lamers M., Marquez F.R., Guardia A., … Munnik T. (2018) Role for Arabidopsis PLC7 in stomatal movement, seed mucilage attachment, and leaf serration. Frontiers in Plant Science 9 , 1721.
Williams M.A.K., Cornuault V., Irani A.H., Symonds V.V., Malmstrom J., An Y., … North H.M. (2020) Polysaccharide structures in the outer mucilage of Arabidopsis seeds visualised by AFM.Biomacromolecules 21 , 1450–1459.
Witztum A., Gutterman Y. & Evenari M. (1969) Integumentary mucilage as an oxygen barrier during germination of Blepharis persica .Botanical Gazette 130 , 238–241.
Xu W., Dubos C. & Lepiniec L. (2015) Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends in Plant Science 20 , 176–185.
Yang B., Voiniciuc C., Fu L., Dieluweit S., Klose H. & Usadel B. (2019) TRM4 is essential for cellulose deposition in Arabidopsis seed mucilage by maintaining cortical microtubule organization and interacting with CESA3. New Phytologist 221 , 881–895.
Yang X., Baskin C.C., Baskin J.M., Liu G. & Huang Z. (2012a) Seed mucilage improves seedling emergence of a sand desert shrub. PloS one 7 , e34897.
Yang X., Baskin C.C., Baskin J.M., Zhang W. & Huang Z. (2012b) Degradation of seed mucilage by soil microflora promotes early seedling growth of a desert sand dune plant. Plant, Cell and Environment35 , 872–883.
Yang X., Baskin J.M., Baskin C.C. & Huang Z. (2012c) More than just a coating: ecological importance, taxonomic occurrence and phylogenetic relationships of seed coat mucilage. Perspectives in Plant Ecology, Evolution and Systematics 14 , 434–442.
Yu L., Lyczakowski J.J., Pereira C.S., Kotake T., Yu X., Li A., … Dupree P. (2018) The patterned structure of galactoglucomannan suggests it may bind to cellulose in seed mucilage. Plant Physiology178 , 1011–1026.
Yu L., Yakubov G.E., Zeng W., Xing X., Stenson J., Bulone V. & Stokes J.R. (2017) Multi-layer mucilage of Plantago ovata seeds: rheological differences arise from variations in arabinoxylan side chains. Carbohydrate Polymers 165 , 132–141.
Zhang B., Chopra D., Schrader A. & Hülskamp M. (2019) Evolutionary comparison of competitive protein-complex formation of MYB, bHLH, and WDR proteins in plants. Journal of Experimental Botany70 , 3197–3209.
Zhang B. & Hülskamp M. (2019) Evolutionary Analysis of MBW Function by Phenotypic Rescue in Arabidopsis thaliana . Frontiers in Plant Science 10 , 375.
Zhao X., Qiao L. & Wu A.-M. (2017) Effective extraction of Arabidopsis adherent seed mucilage by ultrasonic treatment. Scientific Reports 7 , 40672.