Isolation, Characterization, and Cloning of α-l-Arabinofuranosidase Expressed during Fruit Ripening of Japanese Pear

Abstract

a-L-Arabinofuranosidase (a-L-arafase) was purified from fruit of Japanese pear (Pyrus pyrifolia). The enzyme solubilized from the cell wall by NaCl and Triton X-100 had the homogeneity of a single 62-kD polypeptide on SDS-PAGE after purification through the steps of hydroxyapatite, anion-exchange chromatography, and size-exclusion chromatography. A related cDNA clone was isolated (PpARF2). The transcript and related protein were detected solely in the ripening fruit corresponding to the increase of a-L-arafase activity. Transcripts of PpARF2 were not detected in buds, leaves, roots, or shoots of the Japanese pear. The deduced amino acid sequences of PpARF2 had low identity with those of other plants or bacteria. This a-L-arafase belonged to glycoside hydrolase family 3, which includes some b-xylosidases. The purified enzyme hydrolyzed mainly p-nitrophenyl a-L-arabinofuranoside and also reacted bifunctionally with p-nitrophenyl b-D-xylopyranoside. However, it released only arabinose from native cell wall polysaccharides prepared from Japanese pear and from sugar beet arabinan. The enzyme did not release xylose from arabinoxylan and xylan. The only activity of the a-L-arafase presented here was hydrolyzing the arabinosyl residue from native polysaccharides, whereas it showed bifunctional activity against artificial substrates. According to the expression pattern and properties of the enzyme, it is a new member of the glycoside hydrolase family 3 isolated from fruit, and it may be responsible for modification of the cell wall architecture during fruit softening.The modification of cell wall architecture is involved in plant growth, development, and formation of shape. The cooperative biosynthesis and degradation of several cell wall components are necessary, and numerous cell wall-related enzymes are implicated in these processes. Fruit softening or textural changes are important factors that decide fruit quality, and they are caused by modification of cell wall polysaccharide architecture during fruit ripening. Several cell wallmetabolizing enzymes contribute to the changes in cell wall architecture (Fischer and Bennett, 1991). During fruit ripening, pectic and some hemicellulosic polysaccharides become increasingly soluble and depolymerize with the release of neutral sugar residues from side chains of matrix polysaccharides (Huber and O'

β-Galactosidase and α-L-Arabinofuranosidase Activities and Gene Expression in European and Chinese Pear Fruit During Ripening

“…This concurs with previous findings that showed the expression of PpARF2 to be ripeningrelated in the Japanese pear (Tateishi et al, 2005a). The observed lag between enzymatic activity and transcript accumulation of PcARF1 suggests a possibility that its activity could not be detected by the method of using the artificial substrate p-nitrophenyl-α-D-arabinofuranoside, and the measurable α-Af activity may have been a result of PpARF2.…”

“…Indeed, GH family 51 has most of the α-Af genes such as from the fruit of Japanese pear, apple, and tomato, and Arabidopsis. Based on substrate specificity, PpARF2 was shown to possess α-Af as well as β-xylosidase activity (Tateishi et al, 2005a).…”

“…The probes for β-Gal and α-Af were from cDNAs corresponding to full length sequences of Japanese pear β-Gal and α-Af genes designated PpGAL1, PpGAL2, PpGAL3, PpGAL4, PpGAL5, PpGAL6, and PpARF2, whose accession nos. are AB046543, AB190363, AB190364, AB190365, AB190366, AB190367, and AB195230, respectively (Tateishi et al, 2005a(Tateishi et al, , 2005b. In addition, a PcARF1 probe obtained from full length cDNA corresponding to the European pear α-Af gene which was cloned as mentioned above was also used.…”

“…More recently, α-Af enzyme PpARF2 was purified from Japanese pear fruit and shown to possess bifunctional activity of α-Af/β-xylosidase as an enzymatic property (Tateishi et al, 2005a). In our previous study, we characterized the expression of different members of the β-Gal gene family in 'La France' pear fruit during growth and the possible role of ethylene regulation during postharvest ripening (Mwaniki et al, 2005).…”

“…A loss of neutral sugars of the pectic side chains has also been associated with pectin 86 solubilization, and is thought to possibly contribute to pectin mobilization during ripening (Dawson et al, 1992;Nara et al, 2001;Redgwell et al, 1992). These neutral sugar components usually exist as side chains attached to rhamnosyl residues of the rhamnogalacturonan backbone, and their release during fruit ripening is assumed to increase the sensitivity of enzymatic degradation or accessibility of other glycan hydrolases (Tateishi et al, 2005a).…”