title: ARAD proteins associated with pectic Arabinan biosynthesis form complexes when transiently overexpressed in planta.
abstract: Glycosyltransferase complexes are known to be involved in plant cell wall biosynthesis, as for example in cellulose. It is not known to what extent such complexes are involved in biosynthesis of pectin as well. To address this question, work was initiated on ARAD1 (ARABINAN DEFICIENT 1) and its close homolog ARAD2 of glycosyltransferase family GT47. Using bimolecular fluorescence complementation, Förster resonance energy transfer and non-reducing gel electrophoresis, we show that ARAD1 and ARAD2 are localized in the same Golgi compartment and form homo-and heterodimeric intermolecular dimers when expressed transiently in Nicotiana benthamiana. Biochemical analysis of arad2 cell wall or fractions hereof showed no difference in the monosaccharide composition, when compared with wild type. The double mutant arad1 arad2 had an arad1 cell wall phenotype and overexpression of ARAD2 did not complement the arad1 phenotype, indicating that ARAD1 and ARAD2 are not redundant enzymes. To investigate the cell wall structure of the mutants in detail, immunohistochemical analyses were carried out on arad1, arad2 and arad1 arad2 using the arabinan-specific monoclonal antibody LM13. In roots, the labeling pattern of arad2 was distinct from both that of wild type, arad1 and arad1 arad2. Likewise, in epidermal cell walls of inflorescence stems, LM13 binding differed between arad2 and WILD TYPE, arad1 or arad1 arad2. Altogether, these data show that ARAD2 is associated with arabinan biosynthesis, not redundant with ARAD1, and that the two glycosyltransferases may function in complexes held together by disulfide bridges.
authors.affiliation: Laboratory for Molecular Plant Biology, VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, University of Copenhagen, 1871, Frederiksberg C, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Harholt
authors.forename: Jacob Krüger
authors.initials: JK
authors.lastname: Jensen
authors.forename: Yves
authors.initials: Y
authors.lastname: Verhertbruggen
authors.forename: Casper
authors.initials: C
authors.lastname: Søgaard
authors.forename: Sophie
authors.initials: S
authors.lastname: Bernard
authors.forename: Majse
authors.initials: M
authors.lastname: Nafisi
authors.forename: Christian Peter
authors.initials: CP
authors.lastname: Poulsen
authors.forename: Naomi
authors.initials: N
authors.lastname: Geshi
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Sakuragi
authors.forename: Azeddine
authors.initials: A
authors.lastname: Driouich
authors.forename: J Paul
authors.initials: JP
authors.lastname: Knox
authors.forename: Henrik Vibe
authors.initials: HV
authors.lastname: Scheller
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002473:Cell Wall / Q000737:chemistry*; D004220:Disulfides / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D014644:Genetic Variation; D005838:Genotype; D016695:Glycosyltransferases / Q000378:metabolism; D009154:Mutation; D010368:Pectins / Q000096:biosynthesis*; D010430:Pentosyltransferases / Q000378:metabolism*; D010937:Plant Growth Regulators / Q000096:biosynthesis*; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000096:biosynthesis*; D016415:Sequence Alignment; D014026:Nicotiana / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D029681:Arabidopsis Proteins; D004220:Disulfides; D010937:Plant Growth Regulators; D011134:Polysaccharides; C030080:araban; D010368:Pectins; D016695:Glycosyltransferases; C576737:ARAD2 protein, Arabidopsis; D010430:Pentosyltransferases