Ai Oikawa

An integrative approach to the identification of arabidopsis and rice genes involved in xylan and secondary wall development.

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.

Pectin Biosynthesis: GALS1 in Arabidopsis thaliana Is a β-1,4-Galactan β-1,4-Galactosyltransferase

GALS1, GALS2, and GALS3 are members of glycosyltransferase family GT92 in Arabidopsis thaliana. Loss-of-function mutants in the three corresponding genes are deficient in pectic β-1,4-galactan. GALS1 is shown to function as a β-1,4-galactan synthase in vitro, and GALS1 overexpressors have a 50% increased content of β-1,4-galactan in the cell walls. β-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 α-1,5-arabinans, β-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 β-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 β-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher β-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 β-1,4-galactopentaose. GALS1 specifically formed β-1,4-galactosyl linkages and could add successive β-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as β-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties.

The Cooperative Activities of CSLD2, CSLD3, and CSLD5 Are Required for Normal Arabidopsis Development

Glycosyltransferases of the Cellulose Synthase Like D (CSLD) subfamily have been reported to be involved in tip growth and stem development in Arabidopsis. The csld2 and csld3 mutants are root hair defective and the csld5 mutant has reduced stem growth. In this study, we produced double and triple knockout mutants of CSLD2, CSLD3, and CSLD5. Unlike the single mutants and the csld2/csld3 double mutant, the csld2/csld5, csld3/csld5, and csld2/ csld3/csld5 mutants were dwarfed and showed severely reduced viability. This demonstrates that the cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development, and that they are involved in important processes besides the specialized role in tip growth. The mutant phenotypes indicate that CSLD2 and CSLD3 have overlapping functions with CSLD5 in early plant development, whereas the CSLD2 and CSLD3 proteins are non-redundant. To determine the biochemical function of CSLD proteins, we used transient expression in tobacco leaves. Microsomes containing heterologously expressed CSLD5 transferred mannose from GDP-mannose onto endogenous acceptors. The same activity was detected when CSLD2 and CSLD3 were co-expressed but not when they were expressed separately. With monosaccharides as exogenous acceptors, microsomal preparations from CSLD5-expressing plants mediated the transfer of mannose from GDP-mannose onto mannose. These results were supported by immunodetection studies that showed reduced levels of a mannan epitope in the cell walls of stem interfascicular fibers and xylem vessels of the csld2/csld3/csld5 mutant.

Golgi-localized enzyme complexes for plant cell wall biosynthesis

The plant cell wall mostly comprises complex glycans, which are synthesized by numerous enzymes located in the Golgi apparatus and plasma membrane. Protein-protein interactions have been shown to constitute an important organizing principle for glycan biosynthetic enzymes in mammals and yeast. Recent genetic and biochemical data also indicate that such interactions could be common in plant cell wall biosynthesis. In this review, we examine the new findings in protein-protein interactions among plant cell wall biosynthetic enzymes and discuss the possibilities for enzyme complexes in the Golgi apparatus. These new insights in the field may contribute to novel strategies for molecular engineering of the cell wall.

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.

Inactivation of OsIRX10 leads to decreased xylan content in rice culm cell walls and improved biomass saccharification.

Dear Editor, Xylan polysaccharides constitute the major non-cellulosic components in secondary cell walls of dicots and in both primary and secondary cell walls of grasses (Scheller and Ulvskov,2010).Xylan is composed of a linear backbone of 13(1-4)-linked xylose (Xyl).In grasses,the xylan backbone is substituted with O3-1inked and O2-1inked arabinose residues and,to a lesser extent,α(1-3)-Iinked glucuronic acid residues.A unique feature of grass xylans is the presence of hydroxycinnamate esters (ferulic and p-coumaric acid residues)attached to C5 of some of the arabinosyl residues (Carpita,1996;Vogel,2008;Scheller and Ulvskov,2010).Xylan contributes to the recalcitrance of plant cell walls to enzymatic degradation probably through direct interaction with cellulose microfibrils and also through formation of ferulate cross-links (liyama et al.,1994;Yin et al.,2011).

Mannan synthase activity in the CSLD family

Cellulose Synthase Like (CSL) proteins are a group of plant glycosyltransferases that are predicted to synthesize β-1,4-linked polysaccharide backbones. CSLC, CSLF and CSLH families have been confirmed to synthesize xyloglucan and mixed linkage β-glucan, while CSLA family proteins have been shown to synthesize mannans. The polysaccharide products of the five remaining CSL families have not been determined. Five CSLD genes have been identified in Arabidopsis thaliana and a role in cell wall biosynthesis has been demonstrated by reverse genetics. We have extended past research by producing a series of double and triple Arabidopsis mutants and gathered evidence that CSLD2, CSLD3 and CSLD5 are involved in mannan synthesis and that their products are necessary for the transition between early developmental stages in Arabidopsis. Moreover, our data revealed a complex interaction between the three glycosyltransferases and brought new evidence regarding the formation of non-cellulosic polysaccharides through multimeric complexes.

The plant glycosyltransferase clone collection for functional genomics.

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate-Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell-wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full-length coding sequences without termination codons and are Gateway® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/.