Purification, cloning and functional characterization of an endogenous beta-glucuronidase in Arabidopsis thaliana

• Published in: Plant and cell physiology 2008-09, Vol.49 (9), p.1331-1341
• Main Authors: Eudes, A.(Laboratoire de Biologie Cellulaire, Versailles (France)), Mouille, G, Thevenin, J, Goyallon, A, Minic, Z, Jouanin, L
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Arabidopsis; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis - growth & development; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; ARABIDOPSIS THALIANA; Arabinogalactan protein; Beta-glucuronidase; Chromatography; CLONACION MOLECULAR; CLONAGE MOLECULAIRE; Cloning, Molecular; Escherichia coli; Family 79 glycoside hydrolase; GENE; GENES; Genes, Plant; GENETIC MARKERS; Glucuronic Acid - metabolism; Glucuronidase - genetics; Glucuronidase - isolation & purification; Glucuronidase - metabolism; HIPOCOTILOS; Hydrogen-Ion Concentration; Hypocotyl - growth & development; Hypocotyl and root growth; HYPOCOTYLE; HYPOCOTYLS; Life Sciences; MARCADORES GENETICOS; MARQUEUR GENETIQUE; MOLECULAR CLONING; Molecular Sequence Data; Mucoproteins - isolation & purification; Mucoproteins - metabolism; Plant Proteins - isolation & purification; Plant Proteins - metabolism; Plant Roots - growth & development; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; Plants, Genetically Modified - enzymology; Plants, Genetically Modified - genetics; Polysaccharides - metabolism; PROTEINAS; PROTEINE; PROTEINS; RACINE; RAICES; RNA, Plant - genetics; ROOTS; Temperature; Transformation, Genetic; TRANSGENIC PLANTS
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/pcp/pcn108

Beta-glucuronidase (GUS) activities have been extensively characterized in bacteria, fungi, and animals, and the bacterial enzyme GUSA from Escherichia coli is commonly used as a reporter for gene expression studies in plants. Although endogenous GUS activity has been observed in plants, the nature and function of the enzymes involved remain elusive. Here we report on tissue-specific localization, partial purification and identification of AtGUS2, a GUS active under acidic conditions from Arabidopsis thaliana. This enzyme belongs to the GH79 family in the Carbohydrate-Active Enzymes database, which also includes mammalian heparanases that degrade the carbohydrate moieties of cell surface proteoglycans, and fungal enzymes active on arabinogalactan proteins (AGPs). We characterized a knockout insertion line (atgus2-1) and transgenic lines overexpressing AtGUS2 (Prosub(35S):AtGUS2). Endogenous GUS activity assayed histochemically and biochemically was absent in atgus2-1 tissues and four times higher in Prosub(35S):AtGUS2 lines. AGPs purified from atgus2-1 and Prosub(35S):AtGUS2 seedlings showed higher and markedly lower glucuronic acid content, respectively. Our results suggest that endogenous GUS activity influences the sugar composition of the complex polysaccharide chains of AGPs. We also show that transgenics display hypocotyl and root growth defects compared to wild-type plants. Hypocotyl and root lengths are increased in Prosub(35S):AtGUS2 seedlings, whereas hypocoyl length is reduced in atgus2-1 seedlings. These data are consistent with a role for the carbohydrate moieties of AGPs in cell growth.