Partial Purification and Properties of Phleinase Induced in Stem Base of Orchardgrass after Defoliation

Abstract

Phleinase induced in stem base of orchardgrass (Dactylis glomerata L.) after defoliation was partially purified with ammonium sulfate precipitation, DEAE-Sephadex chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. The molecular weight of phleinase was 57,000 as determined by gel chromatography. The kg/ha) was applied after the second cutting. On July 25, the stubbles were dug out and the vegetative stems of 2.5 cm from the base were collected.Preparation of Crude Enzyme. Two hundred and fifty g of the stem bases were cut into slices (about 2 mm in thickness) and homogenized in 4 volumes of cold 0.1 M K-phosphate (pH 7.2) containing 5 mM ,3-mercaptoethanol with 10% insoluble PVP for 2 min using a Waring Blendor. The homogenate was squeezed through four layers of gauze and centrifuged at 14,000g at 4C for 20 min. Solid (NH4)2SO4 was added to the supernatant and the precipitate between 40 and 70% saturation was collected by centrifugation. The precipitate was dissolved in 20 ml of 20 mm K-phosphate (pH 7.2) and filtrated on a column of Sephadex G-25 (2 x 26 cm) with the same buffer as eluant. All subsequent procedures were carried out at about 4C. The protein fractions were combined and dialyzed against two changes of 20 volumes of 50 mM Na-acetate buffer (pH 5.4) for 4 h with stirring. The protein precipitated during dialysis was removed by centrifugation and the supernatant was used as the starting material for further purification.Preparation of Phlein and Triticin. Two hundred g ofthe stem bases of orchardgrass collected in late November were cut into slices and homogenized in 3 volumes of cold 10 mm ,B-mercaptoethanol solution. The homogenate was squeezed through gauze, and the filtrate was centrifuged at 14,000g at 4°C for 15 min. Each of 50 ml of 5% ZnSO4-7H20 and 0.3 N Ba(OH)2 was successively added to the supernatant, and the resulting precipitate was removed by centrifugation. Four volumes of ethanol were added to the supernatant to collect the precipitate by centrifugation. The precipitate was dissolved in 200 ml of distilled H20 in a boiling water bath for 3 min and cooled in running tap water, followed by centrifugation. The supernatant was treated with 20 g of the mixture of Amberlite and IRA45(OH-), and shaken occasionally for 30 min at room temperature, followed by filtration through a filter paper in vacuo. Four volumes of ethanol were added to the filtrate and the resulting precipitate was collected by centrifugation. The precipitate was suspended in 150 ml of 80% ethanol (v/v)

Fructan biosynthesis in excised leaves of Lolium temulentum VII. Sucrose and fructan hydrolysis by a fructan‐polymerizing enzyme preparation

“…This release of reducing sugar shows that the FSA fraction is both distinct from invertase and capable of the net hydrolysis of Neosugar fructans under these conditions of assay. The M r of 57 kDa is identical to that of the FEH of Dactylis glomerata (Yamomoto & Mino, 1985) and the results closely parallel those reported for L. rigidum where an invertase of M r 132 kDa was separated from an FEH of M r 68 kDa using SEC . The values for L. temulentum fructan hydrolase and invertase fall within the range of 57-68 kDa and 40-155 kDa reported for FEH and acid invertases, respectively, from grasses Obenland et al, 1993 ;Henson & Livingston, 1996).…”

Hydrolysis of fructan in grasses: a β‐(2‐6)‐linkage specific fructan‐β‐fructosidase from stubble of Lolium perenne

“…Up to now, however, only FEH from roots of Chicorium intybus (Claessens, Van Laere & De Proft, 1990) and Helianthus tuberosus (Marx, Nosberger & Frehner, 1997) has been purified to apparent homogeneity as revealed by silver-stained gels from lD-SDS-PAGE. In monocots, FEH has been partly purified from the stem bases of Dactylis glomerata (Yamamoto & Mino, 1985) and Hordeum vulgare (Henson, 1989), from the shoots of Lolium temulentum (Simpson, Walker & Pollock, 1991) and several fractions from Lolium rigidum (Bonnett & Simpson, 1993. The FEH from D. glomerata and an FEH fraction from L. rigidum showed pronounced /?-(2-6)-cleaving activity (Yamamoto & Mino, 1985;Bonnett & Simpson, 1995); the other mentioned enzyme preparations illustrated predominantly /?-(2-l)-cleaving activity.…”

“…In monocots, FEH has been partly purified from the stem bases of Dactylis glomerata (Yamamoto & Mino, 1985) and Hordeum vulgare (Henson, 1989), from the shoots of Lolium temulentum (Simpson, Walker & Pollock, 1991) and several fractions from Lolium rigidum (Bonnett & Simpson, 1993. The FEH from D. glomerata and an FEH fraction from L. rigidum showed pronounced /?-(2-6)-cleaving activity (Yamamoto & Mino, 1985;Bonnett & Simpson, 1995); the other mentioned enzyme preparations illustrated predominantly /?-(2-l)-cleaving activity. Recently, an FEH with pronounced y^-(2-6)-linkage specificity has been purified from Avena sativa (Henson & Livingston, 1996).…”

Rise of fructan exohydrolase activity in stubble of Lolium perenne after defoliation is decreased by uniconazole, an inhibitor of the biosynthesis of gibberellins

“…Defoliation by grazing or cutting reduces the amount of the leaf surface and thereby the supply of photosynthate. Fructans stored in the stem base (or stubble defined as the remaining shoot tissues including both sheaths and bases of expanding leaves) are mobilized and used for the regeneration of new shoots (Yamamoto & Mino, 1985;Danckwerts & Gordon, 1987;Gonzalez et al, 1989). Examining changes in concentration of non-structural carbohydrates in individual leaf sheaths and bases of elongating leaves during regrowth following defoliation, Volenec (1986) showed that fructan concentrations decline in all sheath tissue as well as in elongating leaf bases.…”

“…Examining changes in concentration of non-structural carbohydrates in individual leaf sheaths and bases of elongating leaves during regrowth following defoliation, Volenec (1986) showed that fructan concentrations decline in all sheath tissue as well as in elongating leaf bases. The enzyme responsible for hydrolysis of fructan in plants is fructan exohydrolase (FEH, EC 3 .2.1.80), the activity of which increased in Dactylis, Phleum, Festuca and Lolium after defoliation (Yamamoto & Mino, 1985;Prud'homme ct al., 1992). EEH is located in vacuoles (Frehner, Keller & Wiemken, 1984;Wagner, Wiemken & Matile, 1986;Darwen & John, 1989), and has been partially purified from stem bases of Dactylis glomerata (Yamamoto & Mino, 1987), stems and leaf sheaths of Hordeum vulgare (Henson, 1989) and shoot tissues of L. temulentum and L. rigidum (Simpson, Walker & Pollock, 1991;Bonnett & Simpson, 1995).…”