Plants invest a lot of their resources into the production of an extracellular matrix built of polysaccharides. While the composition of the cell wall is relatively well characterized, the functions of the individual polymers and the enzymes that catalyze their biosynthesis remain poorly understood. We exploited the Arabidopsis (Arabidopsis thaliana) seed coat epidermis (SCE) to study cell wall synthesis. SCE cells produce mucilage, a specialized secondary wall that is rich in pectin, at a precise stage of development. A coexpression search for MUCILAGE-RELATED (MUCI) genes identified MUCI10 as a key determinant of mucilage properties. MUCI10 is closely related to a fenugreek (Trigonella foenumgraecum) enzyme that has in vitro galactomannan a-1,6-galactosyltransferase activity. Our detailed analysis of the muci10 mutants demonstrates that mucilage contains highly branched galactoglucomannan (GGM) rather than unbranched glucomannan. MUCI10 likely decorates glucomannan, synthesized by CELLULOSE SYNTHASE-LIKE A2, with galactose residues in vivo. The degree of galactosylation is essential for the synthesis of the GGM backbone, the structure of cellulose, mucilage density, as well as the adherence of pectin. We propose that GGM scaffolds control mucilage architecture along with cellulosic rays and show that Arabidopsis SCE cells represent an excellent model in which to study the synthesis and function of GGM. Arabidopsis natural varieties with defects similar to muci10 mutants may reveal additional genes involved in GGM synthesis. Since GGM is the most abundant hemicellulose in the secondary walls of gymnosperms, understanding its biosynthesis may facilitate improvements in the production of valuable commodities from softwoods.

Grass cell wall properties influence food, feed, and biofuel feedstock usage efficiency. The glucuronoarabinoxylan of grass cell walls is esterified with the phenylpropanoid-derived hydroxycinnamic acids ferulic acid (FA) and para-coumaric acid (p-CA). Feruloyl esters undergo oxidative coupling with neighboring phenylpropanoids on glucuronoarabinoxylan and lignin. Examination of rice (Oryza sativa) mutants in a grass-expanded and -diverged clade of BAHD acyl-coenzyme A-utilizing transferases identified four mutants with altered cell wall FA or p-CA contents. Here, we report on the effects of overexpressing one of these genes, OsAt10 (LOC_Os06g39390), in rice. An activation-tagged line, OsAT10-D1, shows a 60% reduction in matrix polysaccharide-bound FA and an approximately 300% increase in p-CA in young leaf tissue but no discernible phenotypic alterations in vegetative development, lignin content, or lignin composition. Two additional independent OsAt10 overexpression lines show similar changes in FA and p-CA content. Cell wall fractionation and liquid chromatography-mass spectrometry experiments isolate the cell wall alterations in the mutant to ester conjugates of a fivecarbon sugar with p-CA and FA. These results suggest that OsAT10 is a p-coumaroyl coenzyme A transferase involved in glucuronoarabinoxylan modification. Biomass from OsAT10-D1 exhibits a 20% to 40% increase in saccharification yield depending on the assay. Thus, OsAt10 is an attractive target for improving grass cell wall quality for fuel and animal feed.

Plant cells are surrounded by a cell wall that plays a key role in plant growth, structural integrity, and defense. The cell wall is a complex and diverse structure that is mainly composed of polysaccharides. The majority of noncellulosic cell wall polysaccharides are produced in the Golgi apparatus from nucleotide sugars that are predominantly synthesized in the cytosol. The transport of these nucleotide sugars from the cytosol into the Golgi lumen is a critical process for cell wall biosynthesis and is mediated by a family of nucleotide sugar transporters (NSTs). Numerous studies have sought to characterize substrate-specific transport by NSTs; however, the availability of certain substrates and a lack of robust methods have proven problematic. Consequently, we have developed a novel approach that combines reconstitution of NSTs into liposomes and the subsequent assessment of nucleotide sugar uptake by mass spectrometry. To address the limitation of substrate availability, we also developed a two-step reaction for the enzymatic synthesis of UDP-L-rhamnose (Rha) by expressing the two active domains of the Arabidopsis UDP-L-Rha synthase. The liposome approach and the newly synthesized substrates were used to analyze a clade of Arabidopsis NSTs, resulting in the identification and characterization of six bifunctional UDP-LRha/UDP-D-galactose (Gal) transporters (URGTs). Further analysis of loss-of-function and overexpression plants for two of these URGTs supported their roles in the transport of UDP-L-Rha and UDP-D-Gal for matrix polysaccharide biosynthesis. membrane transport | proteoliposomes | glycan biosynthesis | galactan

L-Ara, an important constituent of plant cell walls, is found predominantly in the furanose rather than in the thermodynamically more stable pyranose form. Nucleotide sugar mutases have been demonstrated to interconvert UDP-Larabinopyranose (UDP-Arap) and UDP-L-arabinofuranose (UDP-Araf) in rice (Oryza sativa). These enzymes belong to a small gene family encoding the previously named Reversibly Glycosylated Proteins (RGPs). RGPs are plant-specific cytosolic proteins that tend to associate with the endomembrane system. In Arabidopsis thaliana, the RGP protein family consists of five closely related members. We characterized all five RGPs regarding their expression pattern and subcellular localizations in transgenic Arabidopsis plants. Enzymatic activity assays of recombinant proteins expressed in Escherichia coli identified three of the Arabidopsis RGP protein family members as UDP-L-Ara mutases that catalyze the formation of UDP-Araf from UDP-Arap. Coimmunoprecipitation and subsequent liquid chromatography-electrospray ionization-tandem mass spectrometry analysis revealed a distinct interaction network between RGPs in different Arabidopsis organs. Examination of cell wall polysaccharide preparations from RGP1 and RGP2 knockout mutants showed a significant reduction in total L-Ara content (12-31%) compared with wild-type plants. Concomitant downregulation of RGP1 and RGP2 expression results in plants almost completely deficient in cell wall-derived L-Ara and exhibiting severe developmental defects.

b-1,4-Galactans are abundant polysaccharides in plant cell walls, which are generally found as side chains of rhamnogalacturonan I. Rhamnogalacturonan I is a major component of pectin with a backbone of alternating rhamnose and galacturonic acid residues and side chains that include a-1,5-arabinans, b-1,4-galactans, and arabinogalactans. Many enzymes are required to synthesize pectin, but few have been identified. Pectin is most abundant in primary walls of expanding cells, but b-1,4-galactan is relatively abundant in secondary walls, especially in tension wood that forms in response to mechanical stress. We investigated enzymes in glycosyltransferase family GT92, which has three members in Arabidopsis thaliana, which we designated GALACTAN SYNTHASE1, (GALS1), GALS2 and GALS3. Loss-of-function mutants in the corresponding genes had a decreased b-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher b-1,4-galactan content. The plants did not have an obvious growth phenotype. Heterologously expressed and affinity-purified GALS1 could transfer Gal residues from UDP-Gal onto b-1,4-galactopentaose. GALS1 specifically formed b-1,4-galactosyl linkages and could add successive b-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as b-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties.

BackgroundCost-efficient generation of second-generation biofuels requires plant biomass that can easily be degraded into sugars and further fermented into fuels. However, lignocellulosic biomass is inherently recalcitrant toward deconstruction technologies due to the abundant lignin and cross-linked hemicelluloses. Furthermore, lignocellulosic biomass has a high content of pentoses, which are more difficult to ferment into fuels than hexoses. Engineered plants with decreased amounts of xylan in their secondary walls have the potential to render plant biomass a more desirable feedstock for biofuel production.ResultsXylan is the major non-cellulosic polysaccharide in secondary cell walls, and the xylan deficient irregular xylem (irx) mutants irx7, irx8 and irx9 exhibit severe dwarf growth phenotypes. The main reason for the growth phenotype appears to be xylem vessel collapse and the resulting impaired transport of water and nutrients. We developed a xylan-engineering approach to reintroduce xylan biosynthesis specifically into the xylem vessels in the Arabidopsis irx7, irx8 and irx9 mutant backgrounds by driving the expression of the respective glycosyltransferases with the vessel-specific promoters of the VND6 and VND7 transcription factor genes. The growth phenotype, stem breaking strength, and irx morphology was recovered to varying degrees. Some of the plants even exhibited increased stem strength compared to the wild type. We obtained Arabidopsis plants with up to 23% reduction in xylose levels and 18% reduction in lignin content compared to wild-type plants, while exhibiting wild-type growth patterns and morphology, as well as normal xylem vessels. These plants showed a 42% increase in saccharification yield after hot water pretreatment. The VND7 promoter yielded a more complete complementation of the irx phenotype than the VND6 promoter.ConclusionsSpatial and temporal deposition of xylan in the secondary cell wall of Arabidopsis can be manipulated by using the promoter regions of vessel-specific genes to express xylan biosynthetic genes. The expression of xylan specifically in the xylem vessels is sufficient to complement the irx phenotype of xylan deficient mutants, while maintaining low overall amounts of xylan and lignin in the cell wall. This engineering approach has the potential to yield bioenergy crop plants that are more easily deconstructed and fermented into biofuels.

The cuticle is a complex aliphatic polymeric layer connected to the cell wall and covers surfaces of all aerial plant organs. The cuticle prevents nonstomatal water loss, regulates gas exchange, and acts as a barrier against pathogen infection. The cuticle is synthesized by epidermal cells and predominantly consists of an aliphatic polymer matrix (cutin) and intracuticular and epicuticular waxes. Cutin monomers are primarily C 16 and C 18 unsubstituted, v-hydroxy, and a,v-dicarboxylic fatty acids. Phenolics such as ferulate and p-coumarate esters also contribute to a minor extent to the cutin polymer. Here, we present the characterization of a novel acyl-coenzyme A (CoA)-dependent acyl-transferase that is encoded by a gene designated Deficient in Cutin Ferulate (DCF). The DCF protein is responsible for the feruloylation of v-hydroxy fatty acids incorporated into the cutin polymer of aerial Arabidopsis (Arabidopsis thaliana) organs. The enzyme specifically transfers hydroxycinnamic acids using v-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs, preferentially feruloyl-CoA and sinapoyl-CoA, as acyl donors in vitro. Arabidopsis mutant lines carrying DCF loss-of-function alleles are devoid of rosette leaf cutin ferulate and exhibit a 50% reduction in ferulic acid content in stem insoluble residues. DCF is specifically expressed in the epidermis throughout all green Arabidopsis organs. The DCF protein localizes to the cytosol, suggesting that the feruloylation of cutin monomers takes place in the cytoplasm.The cuticle, a complex polymeric layer connected to the cell wall of epidermal cells, covers the surfaces of all aerial plant organs. The cuticle prevents nonstomatal water loss, regulates gas exchange, acts as a barrier against pathogen infection, and prevents organ fusions (Lolle et al., 1998;Sieber et al., 2000). The plant cuticle is synthesized by epidermal cells and predominantly consists of a lipophilic polymer matrix (cutin) and intracuticular and epicuticular waxes. Cutin monomers are primarily aliphatic C 16 and C 18 unsubstituted, v-hydroxy, and a,v-dicarboxylic fatty acids. Polyhydroxy fatty acids, fatty alcohols, phenolic acids such as ferulic and p-coumaric acid, and glycerol may also contribute to cutin composition in different plant species (Baker and Martin, 1963;Kolattukudy, 1980;Pollard et al., 2008;Samuels et al., 2008;Schreiber, 2010).In Arabidopsis (Arabidopsis thaliana), several enzymes have been identified to be involved in cutin monomer synthesis and polymerization processes, such as glycerol-3-phosphate acyl-transferases 4 and 8 (Li et al., 2007), Bodyguard (Kurdyukov et al., 2006), and Defective in Cuticular Ridges (DCR), a BAHD family enzyme involved in cutin polyester synthesis in Arabidopsis roots, flowers, and seeds. DCR mutant lines are characterized by an almost complete lack of 9 (10),16-dihydroxy-hexadecanoic acid, a major component of the flower cutin polymer, accompanied by several cuticle-associated phenotypic alterations in response to abiotic stresses (e.g. impa...

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