The synthesis and origin of the pectic polysaccharide rhamnogalacturonan II – insights from nucleotide sugar formation and diversity

There is compelling evidence showing that the structurally complex pectic polysaccharide rhamnogalacturonan II (RG-II) exists in the primary cell wall as a borate cross-linked dimer and that this dimer is required for the assembly of a functional wall and for normal plant growth and development. The results of several studies have also established that RG-II structure and cross-linking is conserved in vascular plants and that RG-II likely appeared early in the evolution of land plants. Two features that distinguish RG-II from other plant polysaccharides are that RG-II contains 13 different glycoses linked to each other by 22 different glycosidic linkages and that RG-II is the only polysaccharide known to contain apiose and aceric acid. Thus, one key event in land plant evolution was the emergence of genes encoding nucleotide sugar-biosynthetic enzymes that generate the activated forms of apiose and aceric acid required for RG-II synthesis. Many of the genes involved in the generation of the nucleotide sugars used for RG-II synthesis have been functionally characterized. By contrast, only one putative glycosyltransferase involved in the assembly of RG-II has been identified. Here we provide an overview of the formation of the activated sugars required for RG-II synthesis and point to the possible cellular and metabolic processes that could be involved in assembling and controlling the formation of a borate cross-linked RG-II molecule. We discuss how nucleotide sugar synthesis is compartmentalized and how this may control the flux of precursors to facilitate and regulate the formation of RG-II

Insights into the xylan degradation system of Cellulomonas sp. B6 : biochemical characterization of rCsXyn10A and rCsAbf62A

Valorization of the hemicellulose fraction of plant biomass is crucial for the sustainability of lignocellulosic biorefineries. The Cellulomonas genus comprises Gram-positive Actinobacteria that degrade cellulose and other polysaccharides by secreting a complex array of enzymes. In this work, we studied the specificity and synergy of two enzymes, CsXyn10A and CsAbf62A, which were identified as highly abundant in the extracellular proteome of Cellulomonas sp. B6 when grown on wheat bran. To explore their potential for bioprocessing, the recombinant enzymes were expressed and their activities were thoroughly characterized. rCsXyn10A is a GH10 endo-xylanase (EC 3.2.1.8), active across a broad pH range (5 to 9), at temperatures up to 55 °C. rCsAbf62A is an α-L-arabinofuranosidase (ABF) (EC 3.2.1.55) that specifically removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides (AXOS), xylan, and arabinan backbones, but it cannot act on double-substituted residues. It also has activity on pNPA. No differences were observed regarding activity when CsAbf62A was expressed with its appended CBM13 module or only the catalytic domain. The amount of xylobiose released from either wheat arabinoxylan or arabino-xylo-oligosaccharides increased significantly when rCsXyn10A was supplemented with rCsAbf62A, indicating that the removal of arabinosyl residues by rCsAbf62A improved rCsXyn10A accessibility to β-1,4-xylose linkages, but no synergism was observed in the deconstruction of wheat bran. These results contribute to designing tailor-made, substrate-specific, enzymatic cocktails for xylan valorization.Instituto de BiotecnologíaFil: Garrido, Mercedes Maria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Garrido, Mercedes Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garrido, Mercedes Maria. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Piccinni, Florencia Elizabeth. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Piccinni, Florencia Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Piccinni, Florencia Elizabeth. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Landoni, Malena. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigación en Hidratos de Carbono; ArgentinaFil: Landoni, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Peña, María Jesús. University of Georgia. Complex Carbohydrate Research Center; Estados UnidosFil: Topalian, Juliana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Topalian, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Couto, Alicia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Investigación en Hidratos de Carbono; ArgentinaFil: Wirth, Sonia Alejandra. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Wirth, Sonia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Urbanowicz, Breeanna Rae. University of Georgia. Department of Biochemistry and Molecular Biology; Estados UnidosFil: Campos, Eleonora. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Campos, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Identification of an algal xylan synthase indicates that there is functional orthology between algal and plant cell wall biosynthesis.

Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure-function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-β-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-β-xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-β-xylan synthase activity, and 1,4-β-xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4-β-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls

Identification of an algal xylan synthase indicates that there is functional orthology between algal and plant cell wall biosynthesis.

Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure-function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-β-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-β-xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-β-xylan synthase activity, and 1,4-β-xylan occurs in the K. flaccidum cell wall. These data suggest that plant 1,4-β-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls