Comparison of Enzymatic Properties and Gene Expression Profiles of Two Tuberonic Acid Glucoside .BETA.-Glucosidases from Oryza sativa L.

Abstract

Jasmonic acid (JA) is a plant hormone that regulates stress responses such as insect herbivory, pathogen attack, 1, 2) plant growth and development. 36) The existence of various JA analogs, for example, a methylated form at carbon 1, 7) a hy-droxylated form at carbon 11 or 12, 8, 9) and a conjugated form at carbon 1 with an amino acid and its analog, 10) makes it difficult to understand the role of each compound in plant immunity and growth. Tuberonic acid (TA, 12-hydroxyjas-monic acid) was isolated from potato (Solanum tuberosum L.) as a tuber-inducing compound, and its glucoside was also found to be a tuberization compound. 11, 12) A previous study using labeled [2-14 C] JA showed that JA is metabolized to TA and TA glucoside (TAG), and the resulting TA or TAG is transported to other parts of the plant body including the underground part. 12) These data suggest that the metabolites from JA, TA and TAG, also have a crucial role in stress responses as well as JA. However, very little is known about the physiological function of TA and TAG other than tuber-ization. To better understand their function, analysis of in vivo mutual conversion between TA and TAG is required. A recent work showed the universal existence of TA and TAG in higher plants and the purification and some properties of a rice glucosyltransferase that is active toward TA. 13) In our previous study, the occurrence of β-glucosidase activity converting TAG to TA in rice was demonstrated, and a rice TAG glucosidase 1 (OsTAGG1) was purified. 14) Here we describe the purification of another OsTAGG isozyme (designated OsTAGG2) and the comparison of some enzymatic properties and gene expression profiles between OsTAGG1 and OsTAGG2. The plant (O. sativa L. cv. Kitaake) materials for use in enzyme purification and gene expression analysis were grown in an experimental field at Hokkaido University, Sap-poro, Japan, and a growth chamber, respectively. The chamber conditions for temperature, moisture, and illumination were set at 30C, 60% and 530 μmol/m 2 /s under a 10.5-h light photoperiod, respectively. OsTAGG activity was assayed following the previous report. 14) Briefly, the reaction mixture (0.1 mL) contained 10 μM TAG, 20 mM sodium acetate buffer (pH 4.5) and the Abstract: Rice tuberonic acid glucoside-hydrolyzing β-glucosidase (OsTAGG) produces physiologically active tuberonic acid (TA) from its glucoside (TAG). We have previously reported the identification and some properties of OsTAGG1. Here, we describe the isolation and enzymatic properties of another OsTAGG isozyme (OsTAGG2). OsTAGG2 was purified from rice by seven purification procedures with a 2,800-fold purification. Like OsTAGG1, the purified OsTAGG2 migrated as two bands with molecular masses of 40 and 26 kDa on SDS-PAGE. Results from N-terminal sequencing and peptide mass fingerprinting of both polypeptides suggested that both bands were derived from a single polypeptide. The Km and Vmax values of OsTAGG2 toward TAG were 146 μM and 38.0 μmol/min/mg, and were 4.6-fold and 2.6-fold higher than those of OsTAGG1, respectively. OsTAGG2 as well as OsTAGG1 preferentially hy-drolyzed TAG among several natural glucosides used in this study. Quantitative real-time reverse tran-scriptase-mediated PCR analysis revealed that OsTAGG1 and OsTAGG2 are differentially expressed in response to wounding; the expression of OsTAGG1 is down-regulated, whereas that of OsTAGG2 is up-regulated by wound treatment.