Akira Tateishi

Isolation, characterization, and cloning of α -L -arabinofuranosidase expressed during fruit ripening of Japanese pear

α-l-Arabinofuranosidase (α-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 α-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 α-l-arafase belonged to glycoside hydrolase family 3, which includes some β-xylosidases. The purified enzyme hydrolyzed mainly p-nitrophenyl α-l-arabinofuranoside and also reacted bifunctionally with p-nitrophenyl β-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 α-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.

α-l-Arabinofuranosidase from cell walls of Japanese pear fruits

Abstract Cell wall-bound glycosidase activities were measured in pre-ripe and ripe fruits of Japanese pears (Pyrus serotina Rehd. var. culta. cv. Hosui). α- l -Arabinofuranosidase (EC. 3.2.1.55) activity increased dramatically with fruit ripening and its activity was assayed during fruit development and ripening. After the fruit enlargement stage, cell wall-bound α- l -arabinofuranosidase activity increased 15-fold with fruit ripening. The enzyme was solubilized from cell walls using the chelator trans-1,2-cyclohexanediamine-N,N,N′,N′-tetraacetic acid and the solubilized enzyme purified using DEAE-cellulose, hydroxyapatite, Mono Q and Sephadex G-100 chromatography. The purified enzyme was a Mr 42 000 monomer on SDS-PAGE. Optimum pH activity was 5.0 and the Km value was 34 mM for p- nitrophenyl-α- l -arabinofuranoside .

Does farm fungicide use induce azole resistance in Aspergillus fumigatus

Azole resistance of Aspergillus fumigatus isolates has been reported worldwide and it would appear to be mainly due to a point mutation in the 14α-sterol demethylase (CYP51A) gene, which is the target enzyme for azoles. The mutation has been confirmed in isolates from patients who received long-term itraconazole (ITZ) therapy and from agricultural fields where high levels of azole fungicides were employed. However, the relationship between farm environments and azole-resistant A. fumigatus has not been fully studied. In this investigation, 50 isolates of A. fumigatus were obtained from a farm where tetraconazole has been sprayed twice a year for more than 15 years. The mean minimum inhibitory concentration (MIC) of isolates was 0.74 (0.19-1.5) mg/L against ITZ, which was below the medical resistance level of ITZ. The sequence of CYP51A from isolates indicated no gene mutations in isolates from the farm. Antifungal susceptibility of isolates to tetraconazole showed that spraying with tetraconazole did not induce resistance to tetraconazole or ITZ in A. fumigatus.

Flavonoids in the extract and exudate of the roots of leguminous crops

The colonization level of arbuscular mycorrhizal fungi varies with the crop species (I so be et al., 1998), owing to differences in root structure and in the composition of root exudates (Baon et al., 1994; Mandelbaum et al., 2000). Root exudates affect the spore germination, hyphal growth and colonization of arbuscular mycorrhizal fungi (Gianinazzi-Pearson et al., 1989; Vierheilig et al., 1990). Root exudates contain many types of compound, such as amino acids, reducing sugars, organic acids, flavonoids and plant hormones. Moreover, many reseachers have reported that flavonoids stimulate the spore germination, hyphal growth and colonization of arbuscular mycorrhizal fungi (Baptista et al., 1994; Becard et al., 1992; Siqueira et al., 1991; Tsai et al., 1991; Vierheilig et al., 1998). However, Becard et al. (1992) reported that the effect of the flavonoid on the hyphal growth of arbuscular mycorrhizal fungi varies with the kind of flavonoid. To date, however, there have been few reports on the flavonoid composition in root extracts and exudates. In this study we examined the flavonoid content in the root extracts of various crops and root exudates of kidney bean.

Heterologous expression of tomato glycoside hydrolase family 3 α-L-arabinofuranosidase/β-xylosidases in tobacco suspension cultured cells and synergic action of a family 51 isozyme under antisense suppression of the enzyme

Four cDNA clones (SlArf/Xyl1-4) encoding α-l-arabinofuranosidase/β-xylosidase belonging to glycoside hydrolase family 3 were obtained from tomato (Solanum lycopersicum) fruit. SlArf/Xyl1 was expressed in various organs. Its level was particularly high in flower and leaves but low in fruit. SlArf/Xyl3 was highly expressed in flower. On the contrary, SlArf/Xyl2 and 4 were expressed in early developmental stage in various organs. Comparison with SlArf/Xyl4, SlArf/Xyl2 expression was observed in earlier stages. The active recombinant proteins were obtained by using BY-2 tobacco (Nicotiana tabacum) suspension cultured cells. The SlArf/Xyl1 and 2 recombinant proteins showed a bi-functional activity of α-l-arabinofuranosidase/β-xylosidase while the SlArf/Xyl4 protein possessed a β-xylosidase activity predominantly. Neither enzyme activities were detected for the SlArf/Xyl3 protein under the same conditions. Although SlArf/Xyl2 possessed a bi-functional activity, it preferentially hydrolyzed arabinosyl residues from tomato hemicellulosic polysaccharides. Antisense suppression of SlArf/Xyl2 resulted in no apparent changes in the enzyme activities, monosaccharide composition or fruit phenotype. Increment of a family 51 α-l-arabinofuranosidase expression rather than that of family 3 resulted in a restoring the activity in SlArf/Xyl2-suppressed fruit. The ability of recombinant SlArf/Xyl2 to hydrolyze both arabinan and arabinoxylan is nearly identical to that of α-l-arabinofuranosidases belonging to family 51. Our results suggested that BY-2 cells are a useful expression system for obtaining active cell wall hydrolyzing enzymes. In addition, an α-l-arabinofuranosidase activity derived from SlArf/Xyl2 would be essential in young organ development and the action of the enzyme could be restored by the other enzyme belonging to a different family under a defective condition.