Purification, functional characterization, cloning, and identification of mutants of a seed-specific arabinan hydrolase in Arabidopsis.
This work describes the purification and characterization of an enzyme that exhibits arabinan hydrolase activity in seeds of Arabidopsis thaliana. The enzyme, designated XYL3, had an apparent molecular mass of 80 kDa when purified to homogeneity, and was identified using MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) as a putative beta-D-xylosidase that belongs to family 3 of glycoside hydrolases encoded by gene At5g09730. XYL3 hydrolysed synthetic substrates such as p-nitrophenyl-alpha-L-arabinofuranoside and p-nitrophenyl-beta-D-xyloside with similar catalytic efficiency. XYL3 released L-arabinose from (1-->5)-alpha-L-arabinofuranobiose, arabinoxylan, sugar beet arabinan, and debranched arabinan. The enzyme hydrolysed both arabinosyl-substituted side group residues and terminal arabinofuranosyl residues (1-->5)-alpha-linked to the arabinan backbone. This indicates that XYL3 is able to degrade all terminal arabinosyl residues and suggests that it participates in the in-vivo hydrolysis of arabinan. Analysis of gene expression patterns by semi-quantitative RT-PCR, in-situ hybridization and a promoter-GUS fusion demonstrated that AtBX3 was specifically expressed in the seed endosperm at the globular stage of the embryo. Immunolocalization using LM6 anti-arabinan antisera found that arabinan, the XYL3 substrate, was also present in this seed tissue. T-DNA null mutants for AtBX3 were identified. The mutant plants lacked the alpha-L-arabinofuranosidase and beta-D-xylosidase activities corresponding to XYL3. Mutants showed reduced seed size and are delayed in seedling germination compared with the wild type.
Minic
Zoran
Z
Laboratoire de Biologie Cellulaire - Institut National de la Recherche Agronomique, Route de St-Cyr, F-78026 Versailles Cedex, France.
Do
Cao-Trung
CT
Rihouey
Christophe
C
Morin
Halima
H
Lerouge
Patrice
P
Jouanin
Lise
L
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D002473:Cell Wall / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004252:DNA Mutational Analysis; D004187:Disaccharides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D035264:Flowers / Q000201:enzymology; D005638:Fruit / Q000201:enzymology; D015870:Gene Expression; D005960:Glucosides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D014995:Xylosidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004187:Disaccharides; D005960:Glucosides; D006027:Glycosides; D011134:Polysaccharides; C434937:arabinobiose; C030080:araban; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase