Site-Directed Mutagenesis of IRX9, IRX9L and IRX14 Proteins Involved in Xylan Biosynthesis:Glycosyltransferase Activity Is Not Required for IRX9 Function in Arabidopsis

Xylans constitute the main non-cellulosic polysaccharide in the secondary cell walls of plants. Several genes predicted to encode glycosyltransferases are required for the synthesis of the xylan backbone even though it is a homopolymer consisting entirely of β-1,4-linked xylose residues. The putative glycosyltransferases IRX9, IRX14, and IRX10 (or the paralogs IRX9L, IRX14L, and IRX10L) are required for xylan backbone synthesis in Arabidopsis. To investigate the function of IRX9, IRX9L, and IRX14, we identified amino acid residues known to be essential for catalytic function in homologous mammalian proteins and generated modified cDNA clones encoding proteins where these residues would be mutated. The mutated gene constructs were used to transform wild-type Arabidopsis plants and the irx9 and irx14 mutants, which are deficient in xylan synthesis. The ability of the mutated proteins to complement the mutants was investigated by measuring growth, determining cell wall composition, and microscopic analysis of stem cross-sections of the transgenic plants. The six different mutated versions of IRX9 and IRX9-L were all able to complement the irx9 mutant phenotype, indicating that residues known to be essential for glycosyltransferases function in homologous proteins are not essential for the biological function of IRX9/IRX9L. Two out of three mutated IRX14 complemented the irx14 mutant, including a mutant in the predicted catalytic amino acid. A IRX14 protein mutated in the substrate-binding DxD motif did not complement the irx14 mutant. Thus, substrate binding is important for IRX14 function but catalytic activity may not be essential for the function of the protein. The data indicate that IRX9/IRX9L have an essential structural function, most likely by interacting with the IRX10/IRX10L proteins, but do not have an essential catalytic function. Most likely IRX14 also has primarily a structural role, but it cannot be excluded that the protein has an important enzymatic activity

Plant Glycosyltransferases Beyond CAZy: A Perspective on DUF Families

The carbohydrate active enzyme (CAZy) database is an invaluable resource for glycobiology and currently contains 45 glycosyltransferase families that are represented in plants. Glycosyltransferases (GTs) have many functions in plants, but the majority are likely to be involved in biosynthesis of polysaccharides and glycoproteins in the plant cell wall. Bioinformatic approaches and structural modeling suggest that a number of protein families in plants include GTs that have not yet been identified as such and are therefore not included in CAZy. These families include proteins with domain of unknown function (DUF) DUF23, DUF246, and DUF266. The evidence for these proteins being GTs and their possible roles in cell wall biosynthesis is discussed

Identification et caractérisation de gènes de glycosyltranférases impliqués dans la biosynthèse des polysaccharides pariétaux

Dans la Nature, le cycle du carbone commence avec la fixation du C02par les plantes photosynthétiques. Une partie se retrouve sous forme de molécules glucidiques qui seront utilisées par la cellule végétale pour construire le réseau de polysaccharides pariétaux. En raison de leur importance économique connue source d'énergie renouvelable, il apparaît essentiel de mieux comprendre les mécanismes cellulaires responsables de leur biosynthèse et donc d'identifier les glycosyltransférases (GTs), les enzymes clé de cette machinerie cellulaire. L'objectif principal de ce projet de recherche est d'identifier et caractériser la fonction de gènes potentiellement impliqués dans la biogenèse des polysaccharides pariétaux. Une approche bioinformatique innovante a permis d'identifier plus de 150 nouvelles séquences candidates dans le génome de Arabidopsis, dont une vingtaine présentent des signatures peptidiques les apparentant à des GTs. Pour certaines familles de GTs, la modélisation moléculaire a permis de progresser dans la connaissance des relations structure-fonction des GTs. Cette approche a été utilisée pour déterminer les bases moléculaires responsables de la spécificité de substrats des enzymes connues de la famille GT8, ceci afin de faciliter l'annotation des nombreux gènes d'Arabidopsis de fonction inconnue présents dans cette famille. L'effort a porté également sur l'analyse fonctionnelle d'un petit nombre de gènes, à travers l'étude du phénotype et de la composition pariétale de mutants d'Arabidopsis et leur expression en système hétérologue.The carbon cycle in nature starts with fixation of carbon dioxide by photosynthetic plants. Part of the sugar generated is used by the plant cell to synthesize cell wall polysaccharides through the activity of biosynthetic enzymes. The central process of polysaccharide biosynthesis is the action of glycosyltransferases (GTs), the enzymes responsible for the formation of glycosidic bonds. The overall objective of the project is to identify and assign a function to genes possibly involved in the biogenesis of plant cell wall polysaccharides and to determine how they interact in the coordinatOO biosynthesis of highly complex biopolymers. With the objective to identify new Arabidopsis GT genes, a bioinformatic strategy was designed that 100 to the identification of more than 150 candidate protein sequences. Among them, 20 are considerOO as strong candidates since known GT signatures were clearly evidenced. To improve the functional annotation of plant GT genes, we took advantage of structural data available for sorne GT families to further explore the sequence determinants that confer substrate specificities. This approach which relies on the use of molecular modeling methods was applied to the large GT8 family that comprises approximatel~ 40 Arabidopsis GT genes of unknown functioIl. ln addition, we contributed to the functional characterization of selected candidate genes. Our effort concemed a small number of genes that were characterized in the lab through plant mutant analysis and heterologous expression in insect cells.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Phylogenetic tree of GT43 proteins from selected species.

<p>A multiple alignment of protein sequences from Arabidopsis (At), rice (<i>Oryza sativa</i>, Os), poplar (<i>Populus trichocarpa</i>, Pt), spikemoss (<i>Selaginella moellendorffii</i>, Sm) and human (<i>Homo sapiens</i>, Sp) was used to generate a neighbor-joining tree using MEGA5. Bootstrap values are indicated and the scale bar shows evolutionary distances in units of the number of amino acid substitutions per site. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105014#s2" target="_blank">Materials and Methods</a> section for sequence IDs.</p

Xylose content in cell wall polysaccharides isolated from stems of 6-week-old mutant plants transformed with the different constructs.

<p>Cell walls were prepared from stems, hydrolyzed in TFA, and the xylose content determined by HPAEC. The nomenclature for the constructs used to transform the <i>irx9</i> and <i>irx14</i> plants is explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105014#pone-0105014-t001" target="_blank">Table 1</a>. The bars show average ± SD (n = 4). Averages that are not significantly different (ANOVA, Tukey's test, p>0.05) are indicated with the same letter.</p

Microcopy analysis of stems from 6-week-old plants.

<p>The LM10 anti-xylan monoclonal antibody was used for immunodetection of xylan in transverse stem sections. The nomenclature for the constructs used to transform the <i>irx9</i> and <i>irx14</i> plants is explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105014#pone-0105014-t001" target="_blank">Table 1</a>. Irregular xylem phenotype (indicated with arrows) was observed in the <i>irx9</i> and <i>irx14</i> mutants, as well as in the <i>irx14</i> plants transformed with the 35S:IRX14-1 construct. All the other transformants showed normal vessel phenotype.</p

Representative photos of wild type, mutants and transformed plants.

<p>The nomenclature for the constructs used to transform the <i>irx9</i> and <i>irx14</i> plants is explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105014#pone-0105014-t001" target="_blank">Table 1</a>. Rosettes were from 4-week-old plants and mature plants were 6-week-old.</p