Isolation and characterization of rice (Oryza sativa L.) β-Glucosidases

The objectives of this study) are: (1) partial purification and characterization of rice β-glucosidase, (2) determination of the physiological role of the enzyme during rice germination, and (3) histochemical localization of the enzyme. The method for partial purification of the enzyme was based on that of Schliemann (1984), which included differential solubility, cryoprecipitation, and cation exchange chromatography. The enzymes were characterized with respect to their molecular weights, pI value, pli and temperature profile of activity and stability, activity in the presence of selected denaturants and organic’ solvents, substrate specificity, and inhibition by several known β-glucosidase inhibitors.

To examine the physiological role of rice β-glucosidase, histochemical localization of the enzyme in dry seeds and application of inhibitors of the enzyme to the germinating seeds were carried out. The seeds were soaked in the presence or absence of β-glucosidase inhibitors, and the number of germinating seeds, growth and development of coleoptile and roots, and enzymatic activity of β-glucosidase and α-amylase were studied. To study histochemical localization of rice β-glucosidase, the chromogenic substrates were used. The substrates were incubated with cross and longitudinal sections of whole seeds and seedlings, tissue sections, protoplast and plastid preparations from 5-6-day-old coleoptiles. The development of the colors were observed under the light microscopes.

Among the cation exchange chromatography fractions, two distinct peaks of oNPGase and pNPgase activity were found: fraction-1 (Fr-1) and fraction-2 (Fr-2) forms. It was found that the two forms of rice β-glucosidase are different with respect to susceptibility to denaturation by SDS, substrate specificity and some physico-chemical properties. Fr-1 is susceptible to denaturation by SDS, and catalyzes specifically the hydrolysis of several β-galactosides (pNPGal, X-gal, and 6-BNGal) but not gentiobiose and cellobiose, and is stable over pH range (4 to 10). Fr-2, on the other hand, is more resistant to denaturing agents, catalyzes the hydrolysis of gentiobiose and cellobiose, but not any of the β-galactosides mentioned above; it is relatively stable at pH 9, and less stable at high temperatures than Fr-1. Both Fr-1 and Fr-2 are 120 kD native dimers, made up of 60 kD monomers.

In rice dry seeds, β-glucosidases were distributed in the aleurone layers and embryo parts. β-glucosidase inhibitors suppressed germination at the activation stage. The inhibitors Suppressed the expression of α-amylase and β-glucosidase during germination detected at the activity level. It is proposed, therefore, that the pre-existing f-glucosidase is involved in the regulation of availability and activity of a hormone (gibberellin) at the early step of germination that controls expression of hydrolytic enzymes such as α-amylase. In mature seeds, the Fr-1 is found mainly in the scutellum region and aleurone layers, while the Fr-2 form is in the axis of the embryo. In the seedling, the Fr-1 form is found in the scutellum, shoot and coleoptile, while the Fr-2 form is in the root. In young tissue of shoot and coleoptile, the enzyme is localized in the epidermis and vascular bundles. At the subcellular level it is localized to the plastids.