Purification, cloning and functional characterization of an endogenous beta-glucuronidase in Arabidopsis thaliana.
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 (Pro(35S):AtGUS2). Endogenous GUS activity assayed histochemically and biochemically was absent in atgus2-1 tissues and four times higher in Pro(35S):AtGUS2 lines. AGPs purified from atgus2-1 and Pro(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 Pro(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.
Eudes
Aymerick
A
INRA, Centre de Versailles, Institut Jean-Pierre Bourgin, Laboratoire de Biologie Cellulaire, 78026 Versailles cedex, France.
Mouille
Grégory
G
Thévenin
Johanne
J
Goyallon
Arnaud
A
Minic
Zoran
Z
Jouanin
Lise
L
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002845:Chromatography; D003001:Cloning, Molecular; D017343:Genes, Plant; D020723:Glucuronic Acid / Q000378:metabolism; D005966:Glucuronidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D018546:Hypocotyl / Q000254:growth & development; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000302:isolation & purification / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism; D018517:Plant Roots / Q000254:growth & development; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics; D013696:Temperature; D014170:Transformation, Genetic
D029681:Arabidopsis Proteins; D009088:Mucoproteins; D010940:Plant Proteins; D011134:Polysaccharides; D018749:RNA, Plant; C006619:arabinogalactan proteins; D020723:Glucuronic Acid; D005966:Glucuronidase