Jack Egelund

Arabinogalactan-Proteins: Key Regulators at the Cell Surface?

Arabinogalactan-proteins (AGPs) are undoubtedly one of the most complex families of macromolecules found in plants, perhaps matched only by the polyphenolics (lignins/cutins/suberins) and pectins. Their complexity arises from the incredible diversity of the glycans decorating the protein backbone,

O-Glycosylated Cell Wall Proteins Are Essential in Root Hair Growth

Sequential protein posttranslational modifications facilitate cell wall self-assembly and root hair elongation in Arabidopsis. Root hairs are single cells that develop by tip growth and are specialized in the absorption of nutrients. Their cell walls are composed of polysaccharides and hydroxyproline-rich glycoproteins (HRGPs) that include extensins (EXTs) and arabinogalactan-proteins (AGPs). Proline hydroxylation, an early posttranslational modification of HRGPs that is catalyzed by prolyl 4-hydroxylases (P4Hs), defines the subsequent O-glycosylation sites in EXTs (which are mainly arabinosylated) and AGPs (which are mainly arabinogalactosylated). We explored the biological function of P4Hs, arabinosyltransferases, and EXTs in root hair cell growth. Biochemical inhibition or genetic disruption resulted in the blockage of polarized growth in root hairs and reduced arabinosylation of EXTs. Our results demonstrate that correct O-glycosylation on EXTs is essential for cell-wall self-assembly and, hence, root hair elongation in Arabidopsis thaliana.

Arabidopsis thaliana RGXT1 and RGXT2 Encode Golgi-Localized (1,3)-α-d-Xylosyltransferases Involved in the Synthesis of Pectic Rhamnogalacturonan-II

Two homologous plant-specific Arabidopsis thaliana genes, RGXT1 and RGXT2, belong to a new family of glycosyltransferases (CAZy GT-family-77) and encode cell wall (1,3)-α-d-xylosyltransferases. The deduced amino acid sequences contain single transmembrane domains near the N terminus, indicative of a type II membrane protein structure. Soluble secreted forms of the corresponding proteins expressed in insect cells showed xylosyltransferase activity, transferring d-xylose from UDP-α-d-xylose to l-fucose. The disaccharide product was hydrolyzed by α-xylosidase, whereas no reaction was catalyzed by β-xylosidase. Furthermore, the regio- and stereochemistry of the methyl xylosyl-fucoside was determined by nuclear magnetic resonance to be an α-(1,3) linkage, demonstrating the isolated glycosyltransferases to be (1,3)-α-d-xylosyltransferases. This particular linkage is only known in rhamnogalacturonan-II, a complex polysaccharide essential to vascular plants, and is conserved across higher plant families. Rhamnogalacturonan-II isolated from both RGXT1 and RGXT2 T-DNA insertional mutants functioned as specific acceptor molecules in the xylosyltransferase assay. Expression of RGXT1- and RGXT2-enhanced green fluorescent protein constructs in Arabidopsis revealed that both fusion proteins were targeted to a Brefeldin A–sensitive compartment and also colocalized with the Golgi marker dye BODIPY TR ceramide, consistent with targeting to the Golgi apparatus. Taken together, these results suggest that RGXT1 and RGXT2 encode Golgi-localized (1,3)-α-d-xylosyltransferases involved in the biosynthesis of pectic rhamnogalacturonan-II.

Arabinogalactan-proteins and the research challenges for these enigmatic plant cell surface proteoglycans.

Arabinogalactan-proteins (AGPs) are complex glycoconjugates that are commonly found at the cell surface and in secretions of plants. Their location and diversity of structures have made them attractive targets as modulators of plant development but definitive proof of their direct role(s) in biological processes remains elusive. Here we overview the current state of knowledge on AGPs, identify key challenges impeding progress in the field and propose approaches using modern bioinformatic, (bio)chemical, cell biological, molecular and genetic techniques that could be applied to redress these gaps in our knowledge.

Molecular characterization of two Arabidopsis thaliana glycosyltransferase mutants, rra1 and rra2 , which have a reduced residual arabinose content in a polymer tightly associated with the cellulosic wall residue

Two putative glycosyltransferases in Arabidopsis thaliana, designated reduced residual arabinose-1 and -2 (RRA1 and RRA2), are characterized at the molecular level. Both genes are classified in CAZy GT-family-77 and are phylogenetically related to putative glycosyltranferases of Chlamydomonas reinhardtii. The expression pattern of the two genes was analyzed by semi-quantitative RT-PCR using mRNA extracted from various organs of bolting Arabidopsis thaliana plants. In addition, promoter::gusA analysis of transgenic Arabidopsis thaliana containing a fusion between either the RRA-1 or -2 promoter fragment and the gusA reporter gene showed that whereas the RRA1 promoter was primarily active in the apical meristem, the expression pattern of the RRA2 promoter was more diverse but also highly active in the meristematic region. In addition, T-DNA mutant insertion lines of both RRA-1 and -2, were identified and characterized at the molecular and biochemical level. Monosaccharide compositional analyses of cell wall material isolated from the meristematic region showed a ca. 20% reduction in the arabinose content in the insoluble/undigested cell wall residue after enzymatic removal of xyloglucan and pectic polysaccharides. These data indicate that both RRA-1 and -2 play a role in the arabinosylation of cell wall component(s).

A Complementary Bioinformatics Approach to Identify Potential Plant Cell Wall Glycosyltransferase-Encoding Genes

Plant cell wall (CW) synthesizing enzymes can be divided into the glycan (i.e. cellulose and callose) synthases, which are multimembrane spanning proteins located at the plasma membrane, and the glycosyltransferases (GTs), which are Golgi localized single membrane spanning proteins, believed to participate in the synthesis of hemicellulose, pectin, mannans, and various glycoproteins. At the Carbohydrate-Active enZYmes (CAZy) database where e.g. glucoside hydrolases and GTs are classified into gene families primarily based on amino acid sequence similarities, 415 Arabidopsis GTs have been classified. Although much is known with regard to composition and fine structures of the plant CW, only a handful of CW biosynthetic GT genes—all classified in the CAZy system—have been characterized. In an effort to identify CW GTs that have not yet been classified in the CAZy database, a simple bioinformatics approach was adopted. First, the entire Arabidopsis proteome was run through the Transmembrane Hidden Markov Model 2.0 server and proteins containing one or, more rarely, two transmembrane domains within the N-terminal 150 amino acids were collected. Second, these sequences were submitted to the SUPERFAMILY prediction server, and sequences that were predicted to belong to the superfamilies NDP-sugartransferase, UDP-glycosyltransferase/glucogen-phosphorylase, carbohydrate-binding domain, Gal-binding domain, or Rossman fold were collected, yielding a total of 191 sequences. Fifty-two accessions already classified in CAZy were discarded. The resulting 139 sequences were then analyzed using the Three-Dimensional-Position-Specific Scoring Matrix and mGenTHREADER servers, and 27 sequences with similarity to either the GT-A or the GT-B fold were obtained. Proof of concept of the present approach has to some extent been provided by our recent demonstration that two members of this pool of 27 non-CAZy-classified putative GTs are xylosyltransferases involved in synthesis of pectin rhamnogalacturonan II (J. Egelund, B.L. Petersen, A. Faik, M.S. Motawia, C.E. Olsen, T. Ishii, H. Clausen, P. Ulvskov, and N. Geshi, unpublished data).

A putative Arabidopsis thaliana glycosyltransferase, At4g01220, which is closely related to three plant cell wall-specific xylosyltransferases, is differentially expressed spatially and temporally.

Plant cell wall polysaccharides are amongst the most complex, heterogeneous and abundant bio-molecules on earth. This makes the biosynthetic enzymes, namely the glycosyltransferases and polysaccharide synthases, important research targets in plant science and biotechnology. As an initial step to characterize At4g01220, a putative Arabidopsis thaliana encoding glycosyltransferases in CAZy GT-family-77 that is similar to three known xylosyltransferases involved in the biosynthesis of the pectic polysaccharide, rhamnogalacturonan II, we conducted an expression analysis. In transgenic Arabidopsis thaliana plants containing a fusion between the At4g01220 promoter and the gusA reporter gene we found the expression to be spatially and developmentally regulated. Analysis of Nicotiana benthamiana transfected with the At2g01220::YFP fusion protein revealed that the fusion protein resided in a Brefeldin A-sensitive compartment consistent with a sub-cellular location in the Golgi apparatus. In addition, in silico expression analysis from the Genevestigator database revealed that At4g01220 was up-regulated upon treatment with isoxaben, an inhibitor of cellulose synthesis, which, together with a co-expression analysis that identified a number of plant cell wall co-related biosynthetic genes, suggests involvement in cell wall biosynthesis with pectin being a prime candidate. The data presented provide insights into the expression, sub-cellular location and regulation of At4g01220 under various conditions and may help elucidate its specific function.

Carbohydrate Microarrays in Plant Science

Almost all plant cells are surrounded by glycan-rich cell walls, which form much of the plant body and collectively are the largest source of biomass on earth. Plants use polysaccharides for support, defense, signaling, cell adhesion, and as energy storage, and many plant glycans are also important industrially and nutritionally. Understanding the biological roles of plant glycans and the effective exploitation of their useful properties requires a detailed understanding of their structures, occurrence, and molecular interactions. Microarray technology has revolutionized the massively high-throughput analysis of nucleotides, proteins, and increasingly carbohydrates. Using microarrays, the abundance of and interactions between hundreds and thousands of molecules can be assessed simultaneously using very small amounts of analytes. Here we show that carbohydrate microarrays are multifunctional tools for plant research and can be used to map glycan populations across large numbers of samples to screen antibodies, carbohydrate binding proteins, and carbohydrate binding modules and to investigate enzyme activities.