Sticking to cellulose: exploiting Arabidopsis seed coat mucilage to understand cellulose biosynthesis and cell wall polysaccharide interactions

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1 Review Sticking to cellulose: exploiting Arabidopsis seed coat mucilage to understand cellulose biosynthesis and cell wall polysaccharide interactions Author for correspondence: Jonathan S. Griffiths Tel: Received: 7 October 2016 Accepted: 21 December 2016 Jonathan S. Griffiths 1,2 and Helen M. North 1 1 Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique (INRA), AgroParisTech, CNRS, Universite Paris-Saclay, Versailles F-78000, France; 2 Present address: National Research Council Canada (NRC), 110 Gymnasium Place, Saskatoon SK S7H 0J7, Canada doi: /nph Key words: Arabidopsis, arabinogalactan protein, cell wall, cellulose, hemicellulose, mucilage, pectin, receptor-like kinase. Summary The cell wall defines the shape of cells and ultimately plant architecture. It provides mechanical resistance to osmotic pressure while still being malleable and allowing cells to grow and divide. These properties are determined by the different components of the wall and the interactions between them. The major components of the cell wall are the polysaccharides cellulose, hemicellulose and pectin. Cellulose biosynthesis has been extensively studied in Arabidopsis hypocotyls, and more recently in the mucilage-producing epidermal cells of the seed coat. The latter has emerged as an excellent system to study cellulose biosynthesis and the interactions between cellulose and other cell wall polymers. Here we review some of the major advances in our understanding of cellulose biosynthesis in the seed coat, and how mucilage has aided our understanding of the interactions between cellulose and other cell wall components required for wall cohesion. Recently, 10 genes involved in cellulose or hemicellulose biosynthesis in mucilage have been identified. These discoveries have helped to demonstrate that xylan side-chains on rhamnogalacturonan I act to link this pectin directly to cellulose. We also examine other factors that, either directly or indirectly, influence cellulose organization or crystallization in mucilage. Introduction A major difference between animals and plants is the presence of an extracellular cellulosic cell wall matrix that encapsulates each plant cell. This wall is responsible for cell shape, cell adhesion and organ cohesion, and ultimately plant form and function. Cell walls are generally divided into two classes: the primary cell wall that is deposited during cell growth, and the secondary cell wall that can be generated once expansion has ceased. Common components of cell walls are proteins and three types of polysaccharides, cellulose, hemicellulose and pectin. Cellulose is the most abundant biopolymer found on earth and imparts immense tensile strength to the cell wall. Hemicelluloses are a more diverse group of polysaccharides, which include xylan, xyloglucan and glucomannan that can hydrogen bond to cellulose (Scheller & Ulvskov, 2010). Pectin is a heteropolysaccharide that contains galacturonic acid (GalA) residues. There are three types of pectin domains: homogalacturonan (HG), composed of linear a-d-1,4-gala residues, rhamnogalacturonan I (RG-I), composed of repeating a-d-gala 1,2-a-L-rhamnose (Rha)-1,4 residues that are frequently substituted at the O-4 position of Rha residues with complex side-chains of repeating a-l-araf residues (arabinan) and b-d-galp residues (galactan; Mohnen, 2008), and RG-II, composed of a-d-1,4-gala residues like HG, but with branched sidechains (Mohnen, 2008). The different components of the cell wall are ultimately connected to each other to form a cohesive network, through incompletely understood mechanisms and interactions. Cellulose is a central component of this network, and understanding how cellulose is synthesized and how it interacts with other wall components is a key objective in cell wall biology. Cellulose microfibrils form a structuring network in the cell wall Cellulose microfibrils are composed of individual parallel unbranched b-1,4-glucose polymers that form higher-order 959

2 960 Review New Phytologist microfibrils through intra- and intermolecular hydrogen bonds and van der Waals forces (Endler & Persson, 2011; Cosgrove, 2014; Hill et al., 2014; Nixon et al., 2016; Vandavasi et al., 2016). Cellulose microfibrils are arranged into highly ordered crystalline regions and more amorphous regions, depending on the alignment of the individual glucose molecules in the glucan polymer; variations in the degree of crystallinity can influence, or are influenced by, bonding with other cell wall polysaccharides (Endler & Persson, 2011; Cosgrove, 2014). Interactions between cellulose and other wall polymers can be severed and formed in muro, allowing for the selective growth of the plant cell wall (Cosgrove, 2014). Traditionally, hemicelluloses such as xyloglucans were thought to play a predominant role in forming direct interconnections with cellulose microfibrils, and transmitting the strength of the wall through a complex network of cell wall polymers (Cosgrove, 2014; Park & Cosgrove, 2015). Recent research has cast doubt on this hypothesis, suggesting that hemicelluloses share this role with pectin (Cavalier et al., 2008; Cosgrove, 2014; Park & Cosgrove, 2015). Evidence has begun to accumulate demonstrating that pectin can interact directly with cellulose microfibrils in vitro (Zykwinska et al., 2007; Ralet et al., 2016) and in vivo (Ralet et al., 2016; Wang & Hong, 2016). These new data suggest a prominent role for pectin in the modulation of cell wall properties through their interactions with cellulose. Cellulose biosynthesis is a complex process that requires multiple genes Cellulose is synthesized by a plasma membrane-bound protein complex called the cellulose synthase complex (CSC; Endler & Persson, 2011; McFarlane et al., 2014). This complex is thought to be composed of 18 or more subunits containing at least three different CELLULOSE SYNTHASE (CESA) isoforms (Gonneau et al., 2014; Hill et al., 2014; McFarlane et al., 2014). In growing primary cell walls, CESA1, CESA3 and CESA6 are the main components of the CSC, whereas CESA2, CESA5 and CESA9 are partially redundant with CESA6 (Desprez et al., 2007; Persson et al., 2007; Endler & Persson, 2011; McFarlane et al., 2014). Although double cesa5 cesa6 mutants are unviable, demonstrating their redundancy, CESA5 appears to be functionally distinct from CESA6, as its activity is regulated by phosphorylation in response to light (Bischoff et al., 2011). Aside from CESAs, a number of accessory proteins have been implicated in cellulose biosynthesis. COBRA (COB), CHITINASE-LIKE 1 (CTL1)/POM-POM1, KORRIGAN (KOR1) and CELLULOSE SYNTHASE INTE RACTING PROTEIN 1/POM-POM2 (CSI1) all play a major role in cellulose production (Lane et al., 2001; Bringmann et al., 2012; Sanchez-Rodriguez et al., 2012; McFarlane et al., 2014; Li et al., 2015). These proteins can influence the crystallinity of cellulose and interact with the CSC through mechanisms that remain to be defined. In addition, STELLO1 and STELLO2, pfam domain of unknown function 288 proteins, are required for CSC assembly and trafficking (Zhang et al., 2016). Many of these proteins were discovered through mutant screens for stunted plants, or plants with altered vasculature. Understanding their role in whole plants can be challenging, as tissues are often extremely heterogeneous and contain multiple types of cell walls at different developmental stages. Diversifying mutant screens and coexpression studies can help to identify new genes and new phenotypes that aid our understanding of cellulose biosynthesis. Seed coat epidermal cells are an excellent system to study cellulose biosynthesis One model system that is gaining increasing traction for the study of cellulose and cell wall polysaccharide interactions is the Arabidopsis seed coat epidermis (SCE). During seed development, the SCE produces a large quantity of polysaccharides that form mucilage when the mature seed is imbibed (Fig. 1a; Haughn & Western, 2012; North et al., 2014). Mucilage polysaccharides are deposited after cell expansion has ceased and are primarily composed of RG-I, with small amounts of cellulose, hemicellulose and homogalacturonan (North et al., 2014). Following mucilage deposition, secondary cell wall material including cellulose and callose is accumulated, forming a structure in the centre of the cell known as the columella (Fig. 1b; Western et al., 2000; Macquet et al., 2007; Stork et al., 2010; Mendu et al., 2011). Radial walls that abut each epidermal cell also undergo secondary thickening at the base of the wall (Fig. 1b; Stork et al., 2010; Mendu et al., 2011). The columella ultimately fills the area of the cell underneath the mucilage pocket, and the cell undergoes apoptosis (Mendu et al., 2011). The polarized deposition of mucilage and the shape of the mucilage pocket determine the location of columella formation, and their final shape (Western et al., 2004; Griffiths et al., 2014). CESA2, CESA5 and CESA9 are involved in radial cell wall reinforcement, but other factors must also be involved as columella deposition is only delayed in a cesa2 cesa5 cesa9 triple mutant (Stork et al., 2010; Mendu et al., 2011). The SCE is an excellent model system for identifying genes involved in cell wall biosynthesis as large amounts of cell wall polysaccharides are produced from a single cell type in a short timeframe. The polysaccharide composition of mucilage is relatively simple, being composed primarily of the pectin RG-I. Mucilage is easily accessible and extracted, facilitating analyses of composition and interactions between components (Macquet et al., 2007). Mucilage has proved to be a simple visual trait in genetic screens for both induced and natural mutants (Arsovski et al., 2009; Haughn & Western, 2012; Saez-Aguayo et al., 2014). The ease of use of mucilage and the large amounts of pectins in mucilage have facilitated our understanding of polysaccharide interactions. Cellulose plays a crucial role in mucilage structure and adherence Upon hydration of a mature Arabidopsis seed, mucilage polysaccharides expand rapidly, burst out of the apoplastic pocket where they have been stored and form a transparent halo that surrounds the seed. Arabidopsis mucilage is formed of two distinct layers: a nonadherent layer that dissociates from the seed, and an adherent layer that is tightly attached to the seed coat (Fig. 1a). Both layers are composed primarily of unbranched RG-I, while the adherent layer also contains small amounts of other types of pectin,

3 New Phytologist Review 961 (a) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (b) (m) (n) (o) (p) (q) Fig. 1 Arabidopsis seed coat mucilage is a useful system to study cell wall biosynthesis and interactions between cellulose and other polysaccharides. (a) Wildtype Col-0 seeds stained for pectin with ruthenium red showing the nonadherent (arrows) and adherent (arrowheads) mucilage layer. Bar, 100 lm. (b) Scanning electron microscopy of wild-type Col-0 seed epidermal cells, demonstrating radial walls (arrowheads), mucilage pocket (asterisks), and the columella (arrows). Bar, 10 lm. (c g) Ruthenium red stained wild-type, cesa5-2, sos5-2, cesa5 sos5 and csla2-3 seeds. Bars, 100 lm. (h q) Direct red stained seeds showing cellulose in the adherent mucilage layer. Arrows indicate rays, asterisks indicate columella, and arrowheads indicate the diffuse staining region. Bars: (h l) 100 lm; (m q) 25 lm. hemicellulose and cellulose (Macquet et al., 2007; North et al., 2014; Yu et al., 2014). The adherence of the pectin component of the adherent layer depends on cellulose, hemicellulose and other proteins (Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al., 2011; Griffiths et al., 2014, 2016; Yu et al., 2014; Voiniciuc et al., 2015a,b; Ralet et al., 2016). Another interesting aspect of mucilage is that distinct cellulosic structures can be easily visualized in extruded mucilage. In the adherent mucilage layer, when stained with cellulose-specific dyes like Pontamine Fast Scarlet S4B, or its equivalent Direct Red 23 (DR23; Griffiths et al., 2015; Voiniciuc et al., 2015a), a cellulosic ray that extends perpendicularly from the surface of the seed can be clearly observed above the central columella of each cell (Fig. 1h,m; Macquet et al., 2007; Mendu et al., 2011; Sullivan et al., 2011; Griffiths et al., 2014, 2015). Between the rays, another region is characterized by a more diffuse DR23 staining (Fig. 1m; Mendu et al., 2011; Griffiths et al., 2014). Both regions of the adherent layer are composed of pectin, hemicellulose and cellulose, but polysaccharides in the rays appear more densely packed (Griffiths et al., 2015, 2016; Voiniciuc et al., 2015a). Rays appear to be preformed in the apoplastic mucilage pocket, deposited in a helical fashion as a result of the unidirectional, circular movement of CESAs around the cytoplasmic column (Griffiths et al., 2015). Primary components involved in mucilage cellulose biosynthesis Formation of a functional CSC is believed to require multiple subunits composed of at least three different CESA isoforms. In primary cell walls, CESA1, CESA3 and CESA6-like components are required for cellulose biosynthesis; however, only two CESAs have been shown to be required for mucilage biosynthesis through mutant phenotypes. CESA5 is highly expressed during SCE differentiation, and mutation of this gene results in a loss of mucilage adherence and a reduction in seed crystalline cellulose amounts (Mendu et al., 2011; Sullivan et al., 2011; Griffiths et al., 2015). CESA3 is highly expressed during mucilage biosynthesis, and two missense mutations of cesa3 (isoxaben resistant 1; ixr1-1 and ixr1-2; Scheible et al., 2001) produce mucilage phenotypes (Fig. 2; Fig. 2 Diagram of the components involved in Arabidopsis mucilage cellulose biosynthesis. Pink represents the apoplastic mucilage pocket, purple indicates the cytoplasmic column, and orange lines correspond to microtubules. The white oval represents the Golgi. Components in blue are confirmed actors in mucilage biosynthesis whose mutation results in class 1 phenotypes, a loss of diffuse staining cellulose and the presence of cellulosic rays. Mutation of components in green result in class 2 phenotypes, characterized by the absence of cellulosic rays, and those in red give rise to class 3 phenotypes characterized by a general reduction in crystalline cellulose in both rays and diffuse regions. Components in yellow are unconfirmed actors in cellulose biosynthesis that are expressed during mucilage deposition (Table 1).

4 962 Review New Phytologist Griffiths et al., 2015). These mutations provide resistance in plants to the herbicide isoxaben and, in mucilage, result in altered ray structures, pectin repartitioning in the adherent mucilage layer and modified cellulose crystallinity (Griffiths et al., 2015). CESA2, CESA6 and CESA9 have no obvious role in mucilage biosynthesis, but CESA2 and CESA9 are active, along with CESA5, in secondary thickening of radial cell walls of seed coat epidermal cells (Mendu et al., 2011). It is still not clear how CESAs are selected for inclusion in the CSC, but mucilage and seed coat epidermal cells could help to unravel this mystery. While no major mucilage-related phenotype has been observed for any other CESA mutant, the participation of other CESAs could be masked by functional redundancy (Mendu et al., 2011; Sullivan et al., 2011; Griffiths et al., 2015). Other likely candidates for the third component are CESA1 and CESA10. Both genes are expressed during mucilage biosynthesis, and procesa10: Green Fluorescent Protein (GFP)-CESA10 is localized to the plasma membrane in a similar manner to GFP- CESA5 and GFP-CESA3 (Griffiths et al., 2015). While neither single cesa1 radial swelling1-10 or cesa10-1 mutants have a mucilage phenotype (Sullivan et al., 2011; Griffiths et al., 2015), CESA1 and CESA10 are highly similar and it is possible these two genes are redundant in the SCE. In addition to the prominent role played by CESA5 and CESA3 in mucilage cellulose biosynthesis, mutation of COBRA-LIKE 2 (COBL2) results in an almost identical phenotype to cesa5 seeds (Fig. 2; Ben-Tov et al., 2015). COBL2 is homologous to COBRA, which encodes an extracellular glycosyl-phosphatidylinositol (GPI) anchored protein that facilitates cellulose crystallization at the plasma membrane (Roudier et al., 2005). Mutant cobl2 seeds have reduced cellulose crystallinity, loss of mucilage adherence and reduced cellulose in the adherent mucilage layer (Ben-Tov et al., 2015). STELLO1 and STELLO2 have also been shown to affect mucilage adherence, cellulose ray structures and epidermal cell morphology, indicating that their reported influence on CSC assembly and cellulose biosynthesis in hypocotyls (Zhang et al., 2016) is conserved in seed coat epidermal cells (Fig. 2). Dynamics of cellulose biosynthesis in the seed coat epidermis In hypocotyls and roots, CSCs move bidirectionally in the plasma membrane, guided by cortical microtubules, at a velocity of c nm min 1 when actively synthesizing cellulose (Paradez et al., 2006; McFarlane et al., 2014; Li et al., 2015). By contrast, the velocity of GFP-CESA5, GFP-CESA3 and GFP-CESA10 in SCE cells during mucilage biosynthesis is substantially reduced compared with hypocotyls, with an average velocity of c nm min 1, and proceeds in a unidirectional manner around the central cytoplasmic column (Griffiths et al., 2015). Dephosphorylation of CESA5 in hypocotyls can reduce its velocity to a similar rate as that observed in the SCE (Bischoff et al., 2011), suggesting that CESA5 is not phosphorylated in the SCE. Mutation of CELLULOSE SYNTHASE INTERACTING 1 (CSI1) can also result in a strong decrease in CSC velocity in hypocotyls to 132 nm min 1 (Gu et al., 2010). This observation provides an alternative hypothesis, where the absence of CSI1 during mucilage biosynthesis, or interactions with similar proteins with altered functions, could be responsible for the unidirectional movement of the CSC and altered velocities observed in SCE. Understanding the differences between CSC velocity in the SCE compared with other tissues could provide key insights into how cellulose is synthesized. Interactions between cellulose and other cell wall components define adherent mucilage structure Three unique classes of cellulose-related phenotypes have been observed in mucilage mutants. These classes of phenotypes define three distinct pathways that participate in the adherence of mucilage RG-I to cellulose. The first class, typified by the cesa5 mutant, exhibits repartitioning of mucilage pectin from adherent to nonadherent, and the absence of diffuse cellulose-staining regions while cellulose rays are retained (Fig. 1d,i; Mendu et al., 2011; Sullivan et al., 2011). Multiple genes have similar mutant phenotypes, including MUCILAGE MODIFIED 5 (MUM5; also known as MUCILAGE RELATED 21; MUCI21), IRREGULAR XYLEM 7 (IRX7) and IRX14, CESA3 and COBL2 (Fig. 2; Ben-Tov et al., 2015; Griffiths et al., 2015; Voiniciuc et al., 2015b; Hu et al., 2016a,b; Ralet et al., 2016). The second class of cellulose-related mucilage phenotype is observed for two mutants that function in the same pathway and includes the fasciclin-like arabinogalactan protein SALT- OVERLY SENSITIVE 5 (SOS5) and the receptor-like kinase FEI2 (named for the Chinese word for fat) (Fig. 1e,j,o; Shi et al., 2003; Harpaz-Saad et al., 2011; Griffiths et al., 2014; Steinwand et al., 2014). This second class of mucilage phenotype is also characterized by the repartitioning of mucilage pectin from the adherent layer to the nonadherent layer (Fig. 1e), and is distinguished from the first class based on the different distribution of cellulose in the adherent layer, with a marked absence of cellulosic rays, while diffuse cellulose staining remains largely intact (Fig. 1o; Griffiths et al., 2014, 2016). When both cesa5 and sos5 mutants are combined together, an enhanced phenotype is observed with no cellulose evident in the adherent mucilage layer, demonstrating that these mutants are affected in two different pathways that mediate mucilage adherence independently (Figs 1f,k,p, 2; Griffiths et al., 2014, 2016). The size of mucilage polymer aggregates in nonadherent mucilage also differs between the cesa5 and sos5 mutant classes, with cesa5-like mutants lacking a population of large polymer aggregates while this fraction is retained in the sos5 class (Macquet et al., 2007; Sullivan et al., 2011; Griffiths et al., 2016; Hu et al., 2016a,b). The third class of cellulose-related mucilage phenotype is characterized by mutants affecting galactoglucomannan (GGM) production. GGM is a hemicellulose formed of a backbone of b-1-4-linked D-mannose and D-glucose residues, decorated with a-1-6- D-galactan side branches (Pauly et al., 2013). Like other mannan containing hemicelluloses it can bind tightly to cellulose microfibrils (Eronen et al., 2011), and was recently identified in mucilage (Voiniciuc et al., 2015a). GGM is synthesized in part by CELLULOSE-SYNTHASE-LIKE A2 (CSLA2) and MUCILA GE-RELATED 10 (MUCI10; Fig. 1g; Yu et al., 2014; Voiniciuc

5 New Phytologist Review 963 et al., 2015a). The mucilage of these mutants is characterized by a more compact adherent layer and reduced cellulose staining (Yu et al., 2014; Voiniciuc et al., 2015a). While the amount of seed crystalline cellulose and mucilage birefringence is reduced in these mutants, both rays and diffuse stained cellulose are still present (Fig. 1l,q). CSLA2 and MUCI10 thus appear to have a more general effect on crystalline cellulose, but how they interact with the CESA5- and SOS5-mediated pathways of mucilage adherence remains unclear (Fig. 2). The physicochemical basis of the three classes of cellulose related phenotypes CESA5-dependent cellulose biosynthesis is a major factor determining mucilage adherence, while hemicelluloses are likely to participate directly in linking pectin to cellulose. Analysis of cesa5 mucilage and other mutants with similar phenotypes has allowed us to understand how the pectin domain RG-I is linked to cellulose, a mechanism likely to be employed in other plant cell walls. MUM5 encodes a putative xylosyl transferase required for the addition of xylosyl side-chains to RG-I; these xylosyl side-chains have a strong affinity for cellulose, effectively binding RG-I to cellulose (Voiniciuc et al., 2015b; Ralet et al., 2016). IRX7 and IRX14 are also required for xylan synthesis in mucilage, and mutation of either gene results in an almost complete loss of adherent mucilage (Voiniciuc et al., 2015b; Hu et al., 2016a,b). The phenotype of irx7 and irx14 mutants is more severe than cesa5 or mum5 mutants, with greater reductions in cellulose staining (Voiniciuc et al., 2015b; Hu et al., 2016a,b): both rays and diffuse cellulose are lost on agitation, indicating another population of xylan in these mutants with a different function. Crystalline cellulose concentrations in irx7 and irx14 seeds and adherent mucilage birefringence are also reduced, suggesting that xylan can influence the formation of crystalline cellulose microfibrils (Voiniciuc et al., 2015b; Hu et al., 2016a,b). Identification of the function of these genes, and the characterization of mutants in the SCE model have allowed us to understand key linkages between RG-I, xylan and cellulose and contributed to our understanding of cell wall assembly. The mechanism behind the second class of mucilage phenotype, including the function of SOS5 and FEI2, is not as clear as the first class. Two competing hypotheses have been proposed to explain the function of SOS5 and FEI2. First, SOS5 could act as an extracellular sensor that initiates a signalling cascade through FEI2, which can then influence ACC SYNTHASE 5 (ACS5) and alter cellulose biosynthesis through an unknown mechanism (Xu et al., 2008; Steinwand et al., 2014; Showalter & Basu, 2016). A second hypothesis is that SOS5 and FEI2 act independently of cellulose biosynthesis and signalling, instead influencing cellulose microfibril organization in the apoplast through pectin (Shi et al., 2003; Griffiths et al., 2014, 2016). Multiple lines of evidence argue against a role for signalling and effects on cellulose biosynthesis. Combining mutations in sos5 or fei2 with cellulose synthesis mutants, including cesa5, cesa6 and cob1, results in enhanced phenotypes, suggesting they function independently (Xu et al., 2008; Griffiths et al., 2014). Cellulose biosynthesis is reduced in fei2 roots treated with salt, but this could be a general response of reduced cell elongation, also observed in the wild-type, which is exacerbated in the mutant (Xu et al., 2008). Furthermore, the phenotypes of sos5 and fei2 do not appear to be a specific response to salt. Both sos5 and fei2 enhance the phenotypes of cesa6 and cob1 without salt treatment (Xu et al., 2008), and reactive oxygen species are elevated in sos5 roots before salt treatment (Xue & Seifert, 2015). Finally, the mucilage phenotype of sos5 and fei2 is independent of salt, further demonstrating the value of the SCE model in revealing novel phenotypes and clues to gene function (Griffiths et al., 2014, 2016). SOS5 and FEI2 could play a structural role in the cell wall, with mutation of either gene inducing structural defects that are not detrimental for anisotropic cell expansion, but become limiting in high-salt or -sucrose conditions. The function of SOS5 and FEI2 could be linked to the effects of salt and/or sucrose in the cell wall, but do not result from osmotic stress signalling as mannitol did not have the same effect (Xu et al., 2008). SOS5 could organize cellulose microfibrils through interconnections with pectin or hemicellulose that would require the specific localization of SOS5 in the plasma membrane in a FEI2-dependent manner. Nevertheless, the function of both SOS5 and FEI2 has been linked to hormones, including auxin, ethylene and ABA, although the precise link to these pathways remains unclear (Xu et al., 2008; Steinwand et al., 2014; Xue & Seifert, 2015). The localization of SOS5 and FEI2 in the SCE, the structure of SOS5 glycosyl sidechains, and whether SOS5 or FEI2 interacts with other proteins will be key to understanding their function. Finally, GGM has been proposed to affect mucilage polymer spacing (Voiniciuc et al., 2015a), but most of the phenotypes observed in GGM-deficient mutants could be explained by GGM directly affecting cellulose crystallinity (Yu et al., 2014). Reduced cellulose crystallinity could increase interactions with xylan and thereby the amount of RG-I in the adherent layer, which would also be coherent with cesa5 having a reduced adherent layer, as cellulose production is reduced. GGM labelling with antibodies has a considerably increased signal in cesa5 mucilage (Griffiths et al., 2016). Furthermore, the compact mucilage phenotypes of clsa2 and muci10 closely resemble that of cesa3 ixr1-2, which exhibits similar alterations to cellulose crystallinity and increased pectin adherence (Griffiths et al., 2015). Testing the ability of GGM to bind to cellulose with differing degrees of crystallinity, and in competition with xylan would be informative. It will also be important to determine if GGM is directly linked to RG-I or to other hemicellulose polysaccharides. Analysis of double mutant mucilage phenotypes should also help to unravel the complex interactions between these polysaccharides. Future possibilities for characterizing cell wall polysaccharides using the seed coat epidermal cell model The SCE represents an excellent system to study the transcriptional regulation of cellulose biosynthesis. Mucilage polysaccharides are deposited in a short time-frame, which requires the synthesis and secretion of vast amounts of cell wall material, and the upregulation of the genes coding carbohydrate active enzymes and

6 964 Review New Phytologist Table 1 Relative expression of cellulose biosynthesis-related genes in the seed coat during seed development (Winter et al., 2007) Relative expression Gene Locus Preglobular Globular Heart Linear cotyledon Mature green CESA5 AT5G (19.51) (16.18) (1.33) (123.81) (52.13) COBL2 AT3G (250.79) (45.61) (272.47) (320.89) (163.12) STELLO1 AT2G (19.25) (11.19) (29.67) (1.01) (10.02) STELLO2 AT3G (1.12) (13.89) (20.45) (11.12) (6.6) CSI1 AT2G (76.07) (32.06) (28.97) (20.36) (7.12) CSI2 AT1G (5.1) 1.5 (0.44) 7.46 (5.29) 9.42 (6.69) 4.6 (1.91) CSI3 AT1G (1.22) (31.07) (18.61) (0.24) (4.87) CTL1 AT1G (143.24) (42.44) (115.99) (735.95) (779.43) CTL2 AT3G (0.05) 1.81 (0.07) 3.32 (0.95) 2.01 (0.18) (15.82) KOR1 AT5G (16.97) (53.72) (52.67) (229.75) (186.41) KOR2 AT1G (5.21) (15.99) (0.91) 1.19 (0.52) 1.72 (0.54) KOR3 AT4G (226.43) (69.53) (36.03) (1.52) (2.46) Values in brackets represent the standard deviation. Values in bold represent the highest level of expression for each gene during seed development. Mucilage biosynthesis reaches a maximum between the heart and linear cotyledon stage. proteins. A number of recent studies have used publicly available gene expression data and coexpression analysis to identify genes involved in mucilage biosynthesis (Ben-Tov et al., 2015; Francoz et al., 2015; Griffiths et al., 2015; Voiniciuc et al., 2015a,b). Many transcription factors that regulate SCE differentiation and mucilage biosynthesis are known and could be used to identify key regulatory regions in primary cell wall CESAs (North et al., 2014; Francoz et al., 2015). While the characterization of the role of CESA3, CESA5, COBL2 and STELLO1 and 2 in mucilage has contributed to our understanding of cellulose biosynthesis, the implication of other CSC proteins has yet to be investigated. Comparing the expression profiles of these genes with other cellulose biosynthesis accessory proteins can pinpoint further proteins potentially involved in cellulose biosynthesis in the SCE. For example, the endoglucanase KOR1 that influences the degree of cellulose crystallinity in the apoplast and its homologue KOR3 appear to be seed coatexpressed, as is CTL1, another protein involved in determining cellulose crystallinity (Table 1). CSI1, which is required for the interaction between CSCs and cortical microtubules, is also strongly expressed in the seed coat (Table 1). Studying these proteins during mucilage biosynthesis could enrich our understanding of their contribution to optimizing cellulose production (Fig. 2). Some exciting new mutants involved in cellulose and hemicellulose biosynthesis in the seed coat have been isolated in screens of natural variants of Arabidopsis. Four natural variants termed floating mucilage releasing (FMR) have a stronger phenotype than cesa5 seeds with almost no adherent mucilage layer, similar to irx7 and irx14, and could provide further insights into cellulose biosynthesis (Saez-Aguayo et al., 2014). Other accessions have been identified with a reduced adherent layer, cellulose staining, birefringence and altered mucilage composition indicative of GGM modification that could also provide an insight into cellulose and hemicellulose biosynthesis (Voiniciuc et al., 2015a). Besides Arabidopsis, many other species show interesting variations in amount, composition and structure of mucilage (Western, 2012; North et al., 2014). Certain species have unique ray structures, such as exaggerated spiral or hellicoidal rays (Western, 2012; North et al., 2014). For example, Aethionema spp. have very large and dense rays that are observable without staining and quince (Cydonia oblonga) seed mucilage has large hellicoidal rays (Abeysekera & Willison, 1988; Western, 2012). Closer examination of cellulose in these species could be informative with regard to how cellulose biosynthesis can modulate microfibril structure and interactions with other matrix polymers. Conclusion Arabidopsis seed coat mucilage has emerged as a powerful tool to understand cell wall assembly and biosynthesis, which provides unique advantages compared with other plant tissues. The function of many CAZy genes has been elucidated using mucilage, often where their mutation does not generate mutant phenotypes in other tissues. Key genes involved in the synthesis of all of the different components of the primary cell wall (pectin, hemicellulose, cellulose and protein) have now been characterized, and a critical link between cellulose and pectin through xylan side-chains has been established. Further genetic studies that incorporate double mutants, as well as the analysis of GGM binding to cellulose, would greatly improve our understanding of both mucilage structure and assembly and cell wall interactions. Combining studies of different classes of celluloserelated mucilage mutants with the recent advances in NMR analysis of cell wall assembly should provide particularly pertinent information about the assembly of all cell walls. Acknowledgements The authors wish to thank Catalin Voiniciuc for the gift of csla2-3 seeds and Adeline Berger for providing the SEM image of mature Arabidopsis seeds. The authors also thank Herman H ofte for critical reading of this article. J.S.G. received the support of the EU in the framework of the Marie-Curie FP7 COFUND People Programme, through the award of an Agreenskills fellowship under grant agreement no We wish to apologize for any research not included in this review owing to space and citation limitations.

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