CELLULOSE SYNTHASE-LIKE A2, a Glucomannan Synthase,Is Involved in Maintaining Adherent Mucilage Structure in Arabidopsis Seed

Mannans are hemicellulosic polysaccharides that are considered to have both structural and storage functions in the plant cell wall. However, it is not yet known how mannans function in Arabidopsis (Arabidopsis thaliana) seed mucilage. In this study, CELLULOSE SYNTHASE-LIKE A2 (CSLA2; At5g22740) expression was observed in several seed tissues, including the epidermal cells of developing seed coats. Disruption of CSLA2 resulted in thinner adherent mucilage halos, although the total amount of the adherent mucilage did not change compared with the wild type. This suggested that the adherent mucilage in the mutant was more compact compared with that of the wild type. In accordance with the role of CSLA2 in glucomannan synthesis, csla2-1 mucilage contained 30% less mannosyl and glucosyl content than did the wild type. No appreciable changes in the composition, structure, or macromolecular properties were observed for nonmannan polysaccharides in mutant mucilage. Biochemical analysis revealed that cellulose crystallinity was substantially reduced in csla2-1 mucilage; this was supported by the removal of most mucilage cellulose through treatment of csla2-1 seeds with endo-β-glucanase. Mutation in CSLA2 also resulted in altered spatial distribution of cellulose and an absence of birefringent cellulose microfibrils within the adherent mucilage. As with the observed changes in crystalline cellulose, the spatial distribution of pectin was also modified in csla2-1 mucilage. Taken together, our results demonstrate that glucomannans synthesized by CSLA2 are involved in modulating the structure of adherent mucilage, potentially through altering cellulose organization and crystallization.Mannan polysaccharides are a complex set of hemicellulosic cell wall polymers that are considered to have both structural and storage functions. Based on the particular chemical composition of the backbone and the side chains, mannan polysaccharides are classified into four types: pure mannan, glucomannan, galactomannan, and galactoglucomannan (Moreira and Filho, 2008; Wang et al., 2012; Pauly et al., 2013). Each of these polysaccharides is composed of a β-1,4-linked backbone containing Man or a combination of Glc and Man residues. In addition, the mannan backbone can be substituted with side chains of α-1,6-linked Gal residues. Mannan polysaccharides have been proposed to cross link with cellulose and other hemicelluloses via hydrogen bonds (Fry, 1986; Iiyama et al., 1994; Obel et al., 2007; Scheller and Ulvskov, 2010). Furthermore, it has been reported that heteromannans with different levels of substitution can interact with cellulose in diverse ways (Whitney et al., 1998). Together, these observations indicate the complexity of mannan polysaccharides in the context of cell wall architecture.CELLULOSE SYNTHASE-LIKE A (CSLA) enzymes have been shown to have mannan synthase activity in vitro. These enzymes polymerize the β-1,4-linked backbone of mannans or glucomannans, depending on the substrates (GDP-Man and/or GDP-Glc) provided (Richmond and Somerville, 2000; Liepman et al., 2005, 2007; Pauly et al., 2013). In Arabidopsis (Arabidopsis thaliana), nine CSLA genes have been identified; different CSLAs are responsible for the synthesis of different mannan types (Liepman et al., 2005, 2007). CSLA7 has mannan synthase activity in vitro (Liepman et al., 2005) and has been shown to synthesize stem glucomannan in vivo (Goubet et al., 2009). Disrupting the CSLA7 gene results in defective pollen growth and embryo lethality phenotypes in Arabidopsis, indicating structural or signaling functions of mannan polysaccharides during plant embryo development (Goubet et al., 2003). A mutation in CSLA9 results in the inhibition of Agrobacterium tumefaciens-mediated root transformation in the rat4 mutant (Zhu et al., 2003). CSLA2, CSLA3, and CSLA9 are proposed to play nonredundant roles in the biosynthesis of stem glucomannans, although mutations in CSLA2, CSLA3, or CSLA9 have no effect on stem development or strength (Goubet et al., 2009). All of the Arabidopsis CSLA proteins have been shown to be involved in the biosynthesis of mannan polysaccharides in the plant cell wall (Liepman et al., 2005, 2007), although the precise physiological functions of only CSLA7 and CSLA9 have been conclusively demonstrated.In Arabidopsis, when mature dry seeds are hydrated, gel-like mucilage is extruded to envelop the entire seed. Ruthenium red staining of Arabidopsis seeds reveals two different mucilage layers, termed the nonadherent and the adherent mucilage layers (Western et al., 2000; Macquet et al., 2007a). The outer, nonadherent mucilage is loosely attached and can be easily extracted by shaking seeds in water. Compositional and linkage analyses suggest that this layer is almost exclusively composed of unbranched rhamnogalacturonan I (RG-I) (>80% to 90%), with small amounts of branched RG-I, arabinoxylan, and high methylesterified homogalacturonan (HG). By contrast, the inner, adherent mucilage layer is tightly attached to the seed and can only be removed by strong acid or base treatment, or by enzymatic digestion (Macquet et al., 2007a; Huang et al., 2011; Walker et al., 2011). As with the nonadherent layer, adherent mucilage is also mainly composed of unbranched RG-I, but with small numbers of arabinan and galactan ramifications (Penfield et al., 2001; Willats et al., 2001; Dean et al., 2007; Macquet et al., 2007a, 2007b; Arsovski et al., 2009; Haughn and Western, 2012). There are also minor amounts of pectic HG in the adherent mucilage, with high methylesterified HG in the external domain compared with the internal domain of the adherent layer (Willats et al., 2001; Macquet et al., 2007a; Rautengarten et al., 2008; Sullivan et al., 2011; Saez-Aguayo et al., 2013). In addition, the adherent mucilage contains cellulose (Blake et al., 2006; Macquet et al., 2007a), which is entangled with RG-I and is thought to anchor the pectin-rich mucilage onto seeds (Macquet et al., 2007a; Harpaz-Saad et al., 2011, 2012; Mendu et al., 2011; Sullivan et al., 2011). As such, Arabidopsis seed mucilage is considered to be a useful model for investigating the biosynthesis of cell wall polysaccharides and how this process is regulated in vivo (Haughn and Western, 2012).Screening for altered seed coat mucilage has led to the identification of several genes encoding enzymes that are involved in the biosynthesis or modification of mucilage components. RHAMNOSE SYNTHASE2/MUCILAGE-MODIFIED4 (MUM4) is responsible for the synthesis of UDP-l-Rha (Usadel et al., 2004; Western et al., 2004; Oka et al., 2007). The putative GALACTURONSYLTRANSFERASE11 can potentially synthesize mucilage RG-I or HG pectin from UDP-d-GalUA (Caffall et al., 2009). GALACTURONSYLTRANSFERASE-LIKE5 appears to function in the regulation of the final size of the mucilage RG-I (Kong et al., 2011, 2013). Mutant seeds defective in these genes display reduced thickness of the extruded mucilage layer compared with wild-type Arabidopsis seeds.RG-I deposited in the apoplast of seed coat epidermal cells appears to be synthesized in a branched form that is subsequently modified by enzymes in the apoplast. MUM2 encodes a β-galactosidase that removes Gal residues from RG-I side chains (Dean et al., 2007; Macquet et al., 2007b). β-XYLOSIDASE1 encodes an α-l-arabinfuranosidase that removes Ara residues from RG-I side chains (Arsovski et al., 2009). Disruptions of these genes lead to defective hydration properties and affect the extrusion of mucilage. Furthermore, correct methylesterification of mucilage HG is also required for mucilage extrusion. HG is secreted into the wall in a high methylesterified form that can then be enzymatically demethylesterified by pectin methylesterases (PMEs; Bosch and Hepler, 2005). PECTIN METHYLESTERASE INHIBITOR6 (PMEI6) inhibits PME activities (Saez-Aguayo et al., 2013). The subtilisin-like Ser protease (SBT1.7) can activate other PME inhibitors, but not PMEI6 (Rautengarten et al., 2008; Saez-Aguayo et al., 2013). Disruption of either PMEI6 or SBT1.7 results in the delay of mucilage release.Although cellulose is present at low levels in adherent mucilage, it plays an important adhesive role for the attachment of mucilage pectin to the seed coat epidermal cells. The orientation and amount of pectin associated with the cellulose network is largely determined by cellulose conformation properties (Macquet et al., 2007a; Haughn and Western, 2012). Previous studies have demonstrated that CELLULOSE SYNTHASE A5 (CESA5) is required for the production of seed mucilage cellulose and the adherent mucilage in the cesa5 mutant can be easily extracted with water (Harpaz-Saad et al., 2011, 2012; Mendu et al., 2011; Sullivan et al., 2011).Despite all of these discoveries, large gaps remain in the current knowledge of the biosynthesis and functions of mucilage polysaccharides in seed coats. In this study, we show that CSLA2 is involved in the biosynthesis of mucilage glucomannan. Furthermore, we show that CSLA2 functions in the maintenance of the normal structure of the adherent mucilage layer through modifying the mucilage cellulose ultrastructure.