An isoflavone conjugate-hydrolyzing beta-glucosidase from the roots of soybean (Glycine max) seedlings: purification, gene cloning, phylogenetics, and cellular localization.
Soybeans (Glycine max (L.) Merr.) and certain other legumes excrete isoflavones from their roots, which participate in plantmicrobe interactions such as symbiosis and as a defense against infections by pathogens. In G. max, the release of free isoflavones from their conjugates, the latent forms, is mediated by an isoflavone conjugate-hydrolyzing beta-glucosidase. Here we report on the purification and cDNA cloning of this important beta-glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing beta-glucosidase from roots of G. max seedling (GmICHG), is a homodimeric glycoprotein with a subunit molecular mass of 58 kDa and is capable of directly hydrolyzing genistein 7-O-(6 ''-O-malonyl-beta-d-glucoside) to produce free genistein (k(cat), 98 s(-1); K(m), 25 microM at 30 degrees C, pH 7.0). GmICHG cDNA was isolated based on the amino acid sequence of the purified enzyme. GmICHG cDNA was abundantly expressed in the roots of G. max seedlings but only negligibly in the hypocotyl and cotyledon. An immunocytochemical analysis using anti-GmICHG antibodies, along with green fluorescent protein imaging analyses of Arabidopsis cultured cells transformed by the GmICHG:GFP fusion gene, revealed that the enzyme is exclusively localized in the cell wall and intercellular space of seedling roots, particularly in the cell wall of root hairs. A phylogenetic analysis revealed that GmICHG is a member of glycoside hydrolase family 1 and can be co-clustered with many other leguminous beta-glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.
Suzuki
Hirokazu
H
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 6-6-11, Sendai 980-8579, Japan.
Takahashi
Seiji
S
Watanabe
Ryoko
R
Fukushima
Yusuke
Y
Fujita
Naoki
N
Noguchi
Akio
A
Yokoyama
Ryusuke
R
Nishitani
Kazuhiko
K
Nishino
Tokuzo
T
Nakayama
Toru
T
D002478:Cells, Cultured; D003001:Cloning, Molecular; D018548:Cotyledon / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D018546:Hypocotyl / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D007529:Isoflavones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D018517:Plant Roots / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D036226:Seedlings / Q000166:cytology / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D013025:Glycine max / Q000166:cytology / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D007529:Isoflavones; D001617:beta-Glucosidase