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 bothstructural and storage functions in the plant cell wall. However, it is not yet knownhow mannans function in Arabidopsis (Arabidopsis thaliana) seedmucilage. In this study, CELLULOSE SYNTHASE-LIKE A2(CSLA2; At5g22740) expression was observed in several seedtissues, including the epidermal cells of developing seed coats. Disruption ofCSLA2 resulted in thinner adherent mucilage halos, although thetotal 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 comparedwith that of the wild type. In accordance with the role of CSLA2 in glucomannansynthesis, csla2-1 mucilage contained 30% less mannosyl and glucosylcontent than did the wild type. No appreciable changes in the composition, structure,or macromolecular properties were observed for nonmannan polysaccharides in mutantmucilage. Biochemical analysis revealed that cellulose crystallinity wassubstantially reduced in csla2-1 mucilage; this was supported by theremoval of most mucilage cellulose through treatment of csla2-1seeds with endo-β-glucanase. Mutation in CSLA2 also resultedin altered spatial distribution of cellulose and an absence of birefringent cellulosemicrofibrils within the adherent mucilage. As with the observed changes incrystalline cellulose, the spatial distribution of pectin was also modified incsla2-1 mucilage. Taken together, our results demonstrate thatglucomannans synthesized by CSLA2 are involved in modulating the structure ofadherent mucilage, potentially through altering cellulose organization andcrystallization.Mannan polysaccharides are a complex set of hemicellulosic cell wall polymers that areconsidered to have both structural and storage functions. Based on the particular chemicalcomposition of the backbone and the side chains, mannan polysaccharides are classified intofour 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-linkedbackbone containing Man or a combination of Glc and Man residues. In addition, the mannanbackbone can be substituted with side chains of α-1,6-linked Gal residues. Mannanpolysaccharides have been proposed to cross link with cellulose and other hemicellulosesvia hydrogen bonds (Fry, 1986; Iiyama et al., 1994; Obel et al., 2007; Scheller andUlvskov, 2010). Furthermore, it has been reported that heteromannans withdifferent levels of substitution can interact with cellulose in diverse ways (Whitney et al., 1998). Together, these observationsindicate the complexity of mannan polysaccharides in the context of cell wallarchitecture.CELLULOSE SYNTHASE-LIKE A (CSLA) enzymes have been shown to have mannan synthase activityin vitro. These enzymes polymerize the β-1,4-linked backbone of mannans orglucomannans, 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 beenidentified; different CSLAs are responsible for the synthesis of differentmannan types (Liepman et al., 2005, 2007). CSLA7 has mannan synthase activity in vitro(Liepman et al., 2005) and has been shown tosynthesize stem glucomannan in vivo (Goubet et al.,2009). Disrupting the CSLA7 gene results in defective pollengrowth and embryo lethality phenotypes in Arabidopsis, indicating structural or signalingfunctions of mannan polysaccharides during plant embryo development (Goubet et al., 2003). A mutation in CSLA9 results inthe inhibition of Agrobacterium tumefaciens-mediated root transformationin the rat4 mutant (Zhu et al.,2003). CSLA2, CSLA3, and CSLA9 are proposed to play nonredundant roles in thebiosynthesis of stem glucomannans, although mutations in CSLA2,CSLA3, or CSLA9 have no effect on stem development orstrength (Goubet et al., 2009). All of theArabidopsis CSLA proteins have been shown to be involved in the biosynthesis of mannanpolysaccharides in the plant cell wall (Liepman et al.,2005, 2007), although the precisephysiological functions of only CSLA7 and CSLA9 have been conclusively demonstrated.In Arabidopsis, when mature dry seeds are hydrated, gel-like mucilage is extruded toenvelop the entire seed. Ruthenium red staining of Arabidopsis seeds reveals two differentmucilage layers, termed the nonadherent and the adherent mucilage layers (Western et al., 2000; Macquet et al., 2007a). The outer, nonadherent mucilage is looselyattached and can be easily extracted by shaking seeds in water. Compositional and linkageanalyses suggest that this layer is almost exclusively composed of unbranchedrhamnogalacturonan I (RG-I) (>80% to 90%), withsmall amounts of branched RG-I, arabinoxylan, andhigh methylesterified homogalacturonan (HG). Bycontrast, the inner, adherent mucilage layer is tightly attached to the seed and can onlybe 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 composedof unbranched RG-I, but with small numbers ofarabinan 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 pecticHG in the adherent mucilage, with highmethylesterified HG in the external domain comparedwith the internal domain of the adherent layer (Willatset al., 2001; Macquet et al., 2007a;Rautengarten et al., 2008; Sullivan et al., 2011; Saez-Aguayo et al., 2013). In addition, the adherent mucilagecontains cellulose (Blake et al., 2006; Macquet et al., 2007a), which is entangled with RG-I and is thought to anchor the pectin-rich mucilageonto seeds (Macquet et al., 2007a; Harpaz-Saad et al., 2011, 2012; Mendu et al., 2011;Sullivan et al., 2011). As such, Arabidopsisseed mucilage is considered to be a useful model for investigating the biosynthesis of cellwall 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 genesencoding enzymes that are involved in the biosynthesis or modification of mucilagecomponents. RHAMNOSE SYNTHASE2/MUCILAGE-MODIFIED4 (MUM4) is responsible for the synthesisof UDP-l-Rha (Usadel et al., 2004; Western et al., 2004; Oka et al., 2007). The putative GALACTURONSYLTRANSFERASE11 canpotentially synthesize mucilage RG-I or HG pectin from UDP-d-GalUA (Caffall et al., 2009). GALACTURONSYLTRANSFERASE-LIKE5appears to function in the regulation of the final size of the mucilage RG-I (Kong et al.,2011, 2013). Mutant seeds defective inthese genes display reduced thickness of the extruded mucilage layer compared withwild-type Arabidopsis seeds.RG-I deposited in the apoplast of seed coatepidermal cells appears to be synthesized in a branched form that is subsequently modifiedby enzymes in the apoplast. MUM2 encodes a β-galactosidase thatremoves Gal residues from RG-I side chains (Dean et al., 2007; Macquet et al., 2007b). β-XYLOSIDASE1 encodes anα-l-arabinfuranosidase that removes Ara residues from RG-I side chains (Arsovski et al., 2009). Disruptions of these genes lead to defective hydrationproperties and affect the extrusion of mucilage. Furthermore, correct methylesterificationof mucilage HG is also required for mucilageextrusion. HG is secreted into the wall in a highmethylesterified form that can then be enzymatically demethylesterified by pectinmethylesterases (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 eitherPMEI6 or SBT1.7 results in the delay of mucilagerelease.Although cellulose is present at low levels in adherent mucilage, it plays an importantadhesive role for the attachment of mucilage pectin to the seed coat epidermal cells. Theorientation and amount of pectin associated with the cellulose network is largelydetermined by cellulose conformation properties (Macquetet al., 2007a; Haughn and Western,2012). Previous studies have demonstrated that CELLULOSE SYNTHASE A5 (CESA5) isrequired for the production of seed mucilage cellulose and the adherent mucilage in thecesa5 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 thebiosynthesis and functions of mucilage polysaccharides in seed coats. In this study, weshow that CSLA2 is involved in the biosynthesis of mucilage glucomannan. Furthermore, weshow that CSLA2 functions in the maintenance of the normal structure of the adherentmucilage layer through modifying the mucilage cellulose ultrastructure.