Functional identification of an Arabidopsis pectin biosynthetic homogalacturonan galacturonosyltransferase.

Galacturonosyltransferases (GalATs) are required for the synthesis of pectin, a family of complex polysaccharides present in the cell walls of all land plants. We report the identification of a pectin GalAT (GAUT1) using peptide sequences obtained from Arabidopsis thaliana proteins partially purified for homogalacturonan (HG) alpha-1,4-GalAT activity. Transient expression of GAUT1 cDNA in the human embryonic kidney cell line HEK293 yielded uridine diphosphogalacturonic acid:GalAT activity. Polyclonal antibodies generated against GAUT1 immunoabsorbed HG alpha-1,4-GalAT activity from Arabidopsis solubilized membrane proteins. blast analysis of the Arabidopsis genome identified a family of 25 genes with high sequence similarity to GAUT1 and homologous genes in other dicots, in rice, and in Physcomitrella. Sequence alignment and phylogenetic Bayesian analysis of the Arabidopsis GAUT1-related gene family separates them into four related clades of GAUT and GAUT-like genes that are distinct from the other Arabidopsis members of glycosyltransferase family 8. The identification of GAUT1 as a HG GalAT and of the GAUT1-related gene family provides the genetic and biochemical tools required to study the function of these genes in pectin synthesis.

[1]  R. Carlson,et al.  Rhizobium fredii and Rhizobium meliloti produce 3-deoxy-D-manno-2-octulosonic acid-containing polysaccharides that are structurally analogous to group II K antigens (capsular polysaccharides) found in Escherichia coli , 1993, Journal of bacteriology.

[2]  T. Takao,et al.  Structural Studies by Stepwise Enzymatic Degradation of the Main Backbone of Soybean Soluble Polysaccharides Consisting of Galacturonan and Rhamnogalacturonan , 2002, Bioscience, biotechnology, and biochemistry.

[3]  S. Aubourg,et al.  Organization and structural evolution of four multigene families in Arabidopsis thaliana: AtLCAD, AtLGT, AtMYST and AtHD-GL2 , 2000, Plant Molecular Biology.

[4]  Hong Ma,et al.  The GAOLAOZHUANGREN1 gene encodes a putative glycosyltransferase that is critical for normal development and carbohydrate metabolism. , 2004, Plant & cell physiology.

[5]  O. Thomas,et al.  Solid-supported enzymatic synthesis of pectic oligogalacturonides and their analysis by MALDI-TOF mass spectrometry. , 2003, Carbohydrate research.

[6]  M. Bar-Peled,et al.  The Biosynthesis of UDP-Galacturonic Acid in Plants. Functional Cloning and Characterization of Arabidopsis UDP-d-Glucuronic Acid 4-Epimerase1 , 2004, Plant Physiology.

[7]  C. Raetz,et al.  A facile enzymatic synthesis of uridine diphospho-[14C]galacturonic acid. , 2000, Analytical biochemistry.

[8]  H. Scheller,et al.  QUASIMODO1 is expressed in vascular tissue of Arabidopsis thaliana inflorescence stems, and affects homogalacturonan and xylan biosynthesis , 2005, Planta.

[9]  G. Coupland,et al.  Mutation of a family 8 glycosyltransferase gene alters cell wall carbohydrate composition and causes a humidity-sensitive semi-sterile dwarf phenotype in Arabidopsis , 2003, Plant Molecular Biology.

[10]  Christopher P. Bonin,et al.  The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4,6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-L-fucose. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[11]  F. Wurm,et al.  Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. , 1996, Nucleic acids research.

[12]  R. Visser,et al.  If Homogalacturonan Were a Side Chain of Rhamnogalacturonan I. Implications for Cell Wall Architecture1 , 2003, Plant Physiology.

[13]  T. Tsukamoto,et al.  Successive Glycosyltransfer Activity and Enzymatic Characterization of Pectic Polygalacturonate 4-α-Galacturonosyltransferase Solubilized from Pollen Tubes ofPetunia axillaris Using Pyridylaminated Oligogalacturonates as Substrates , 2002, Plant Physiology.

[14]  D. Northcote,et al.  A function of the Golgi apparatus in polysaccharide synthesis and transport in the root-cap cells of wheat. , 1966, The Biochemical journal.

[15]  B. Henrissat,et al.  A census of carbohydrate-active enzymes in the genome of Arabidopsis thaliana. , 2001 .

[16]  Philip Yeagle,et al.  The Structure of Biological Membranes , 2004 .

[17]  Y. Meyer,et al.  Three Glycosylated Polypeptides Secreted by Several Embryogenic Cell Cultures of Pine Show Highly Specific Serological Affinity to Antibodies Directed against the Wheat Germin Apoprotein Monomer , 1995, Plant physiology.

[18]  D. Mohnen,et al.  Development of a filter assay for measuring homogalacturonan: alpha-(1,4)-Galacturonosyltransferase activity. , 2005, Analytical biochemistry.

[19]  John P. Huelsenbeck,et al.  MrBayes 3: Bayesian phylogenetic inference under mixed models , 2003, Bioinform..

[20]  H. Scheller,et al.  Pectin biosynthesis: a solubilized α1,4-galacturonosyltransferase from tobacco catalyzes the transfer of galacturonic acid from UDP-galacturonic acid onto the non-reducing end of homogalacturonan , 1999, Planta.

[21]  Derrick J. Zwickl.  :Phylogenetic Trees Made Easy: A How-To Manual , 2008 .

[22]  Tadashi Ishii,et al.  Rhamnogalacturonan II: structure and function of a borate cross-linked cell wall pectic polysaccharide. , 2004, Annual review of plant biology.

[23]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[24]  M. León-Barrios,et al.  Alcian blue fixation allows silver staining of the isolated polysaccharide component of bacterial lipopolysaccharides in polyacrylamide gels , 1991, Electrophoresis.

[25]  R. Verma,et al.  The biosynthesis of the branched-chain sugar d-apiose in plants: functional cloning and characterization of a UDP-d-apiose/UDP-d-xylose synthase from Arabidopsis. , 2003, The Plant journal : for cell and molecular biology.

[26]  S. Munro,et al.  Activity of the yeast MNN1 alpha-1,3-mannosyltransferase requires a motif conserved in many other families of glycosyltransferases. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[27]  S. Satoh,et al.  A pectin glucuronyltransferase gene is essential for intercellular attachment in the plant meristem , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[28]  D. Mohnen,et al.  Solubilization and characterization of a galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan , 1998 .

[29]  William Mackie,et al.  Pectin: cell biology and prospects for functional analysis , 2001, Plant Molecular Biology.

[30]  R. Verma,et al.  The biosynthesis of L-arabinose in plants: molecular cloning and characterization of a Golgi-localized UDP-D-xylose 4-epimerase encoded by the MUR4 gene of Arabidopsis. , 2003, The Plant cell.

[31]  A. Orellana,et al.  The catalytic site of the pectin biosynthetic enzyme alpha-1,4-galacturonosyltransferase is located in the lumen of the Golgi. , 2001, Plant physiology.

[32]  Staffan Persson,et al.  Identification of genes required for cellulose synthesis by regression analysis of public microarray data sets. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Y. Takeuchi,et al.  In Vitro Biosynthesis of Homogalacturonan by a Membrane-bound Galacturonosyltransferase from Epicotyls of Azuki Bean , 2001, Bioscience, biotechnology, and biochemistry.

[34]  John P. Huelsenbeck,et al.  MRBAYES: Bayesian inference of phylogenetic trees , 2001, Bioinform..

[35]  A. Kumar,et al.  Cell-Free Synthesis of Pectin (Identification and Partial Characterization of Polygalacturonate 4-[alpha]-Galacturonosyltransferase and Its Products from Membrane Preparations of Tobacco Cell-Suspension Cultures) , 1995, Plant physiology.

[36]  M. Bar-Peled,et al.  A Bifunctional 3,5-Epimerase/4-Keto Reductase for Nucleotide-Rhamnose Synthesis in Arabidopsis1 , 2004, Plant Physiology.

[37]  P. Zimmermann,et al.  GENEVESTIGATOR. Arabidopsis Microarray Database and Analysis Toolbox1[w] , 2004, Plant Physiology.

[38]  Barry G. Hall,et al.  Phylogenetic Trees Made Easy: A How-To Manual , 2001 .

[39]  Electrical properties of intact rabbit aortic endothelium , 2001 .

[40]  Jeffrey B. Harborne,et al.  Methods in plant biochemistry , 1989 .

[41]  L. Staehelin,et al.  Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants , 1991, The Journal of cell biology.

[42]  K. Moremen,et al.  Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi. , 1998, Glycobiology.

[43]  G. Seymour,et al.  Pectins and their manipulation , 2002 .

[44]  Sophie Bouton,et al.  QUASIMODO1 Encodes a Putative Membrane-Bound Glycosyltransferase Required for Normal Pectin Synthesis and Cell Adhesion in Arabidopsis Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.004259. , 2002, The Plant Cell Online.

[45]  Joseph M. Dale,et al.  Empirical Analysis of Transcriptional Activity in the Arabidopsis Genome , 2003, Science.

[46]  Alan G. Darvill,et al.  The Pectic Polysaccharides of Primary Cell Walls , 1990 .

[47]  A. Imberty,et al.  Structure/function studies of glycosyltransferases. , 1999, Current opinion in structural biology.

[48]  Rod B. Watson,et al.  Localization of Organelle Proteins by Isotope Tagging (LOPIT)*S , 2004, Molecular & Cellular Proteomics.

[49]  K. Liljebjelke,et al.  Enzymatic synthesis and purification of uridine diphosphate [14C]galacturonic acid: a substrate for pectin biosynthesis. , 1995, Analytical biochemistry.

[50]  B. Ridley,et al.  Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. , 2001, Phytochemistry.