An Isoflavone Conjugate-hydrolyzing β-Glucosidase from the Roots of Soybean (Glycine max) Seedlings

Abstract

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 ␤-glucosidase. Here we report on the purification and cDNA cloning of this important ␤-glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing ␤-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-␤-D-glucoside) to produce free genistein (k cat , 98 s ؊1 ; K m , 25 M at 30°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 ␤-glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.

Defense enzyme activities and biochemical variations of Pelargonium zonale in response to nanosilver application and dark storage

“…In the current work, the β-GLU enzyme response to NS application in 2 Pelargonium cultivars was completely different (Figure 6). In plants, β-GLU could deliver many important functions, including bioactivation of defense compounds, cell wall degradation, activation of phytohormones, and lignification and abscisic acid liberation (Suzuki et al, 2006). These defense compounds are regarded as protective mechanisms against the toxicity of the plant's own chemical defense system, to add to solubility and to facilitate storage (Lee et al, 2006).…”

Different mechanisms for phytoalexin induction by pathogen and wound signals in Medicago truncatula

“…G2 preferred isoflavonoid over flavonoid glucosides, and G4, with a broad substrate preference, was down-regulated by YE. Other ␤-glucosidases have been shown to exhibit preference for specific (iso)flavonoid compounds, such as a set of chickpea isoenzymes possessing K m values for isoflavones of at least 100-fold lower than for other phenolic glycosides (34), enzymes from white lupin roots that, like the Medicago enzymes, can cleave isoflavone glucosides but not kaempferol 3-O-␤-glucoside (35), and an enzyme from soybean roots (GmICHG) that has very high specificity and turnover for gensitein and daidzein malonyl glucosides (36). This enzyme is found in the cell wall fraction in soybean roots, and, of the Medicago enzymes reported here, is most closely related to G2 (70% at the amino acid level) (SI Fig.…”

β-Glucosidases

“…Cyanogenic b-glucosides, including linamarin from clover, cassava and various other plants, dhurrin from sorghum, and prunasin from cherry and other stone fruits, are hydrolyzed to release an a-hydroxynitrile, which then breaks down either enzymatically or spontaneously to release cyanide and an aldehyde [41,42]. Noncyanogenic defense compounds, such as c-and b-hydroxynitriles and isoflavones in legumes, other flavonoids, coumarins, hydroxaminic acids, such as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) in maize and wheat, and saponins are also stored as b-D-glucosides, which are hydrolyzed by specific b-glucosidases [44][45][46][47][48][49][50][51][52][53][54].…”