β-Galactosidases in Ripening Tomatoes

Abstract

Tomatoes (Lycopersicon escadentum L.) contained a high level of aigalactosidase activity which was due to three forms of the enzyme. During tomato ripening, the sum of their activities remained relatively constant, but the levels of the individual forms of 8-galactosidase changed markedly. The three enzymes were separated by a combination of chromatography of DEAE-Sephadex A-50 and Sephadex G-100. During ripening of tomatoes, /8-galactosidases I and III levels decreased but the P-galactosidase II level increased more than 3-fold. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. However, the enzymes differed in molecular weight, Km value with p-nitrophenyl-flgalactoside, and stability with respect to pH and temperature. /3-Galactosidase II was the only enzyme capable of hydrolyzing a polysaccharide that was isolated from tomatoes and that consisted primarily of a-1, 4-linked galactose. The ability of tgalactosidase II to degrade the galactan and the increase in its activity during tomato ripening suggest a possible role for this enzyme in tomato softening.During fruit ripening, the most apparent cell-wall change is an increase in water-soluble polyuronide (6)(7)(8)15). The solubilization of polyuronide is generally attributed to the action of polygalacturonase, which appears in many fruits near the onset of ripening (6,15,19). Another change that occurs, at least in apples, strawberries, and tomatoes, is a pronounced loss of galactose from the cell walls. This process does not appear to be related to cell-wall degradation by polygalacturonase (9,17), but possibly to a galactanase. Bartley (2) found a f8-galactosidase in apples that degraded galactan and increased in activity during apple ripening. Tomatoes also contain ,B-galactosidase, but it has been concluded that this enzyme is not responsible for the hydrolysis of cell wall galactans (5). Furthermore, Gross and Wallner (5) rate at which it hydrolyzes p-nitrophenyl-,B-galactoside (Sigma). The reaction mixture consisted of 0.5 ml of 0.1 M citrate (pH 4.0), 0.4 ml of 0.1% BSA, 0.1 ml of diluted enzyme, and 0.4 ml of 13 mm substrate. After 15 min at 37°C, the reactions were terminated by the addition of 2 ml of 0.2 M sodium carbonate, and the liberated p-nitrophenol was measured at 415 nm. One unit of ,8-galactosidase was defined as the amount that hydrolyzed 1 ,umol ofp-nitrophenyl-,f-galactoside/ 15 mi.Galactanase was assayed by measuring the release of reducing sugars from a galactose-rich polysaccharide isolated from tomatoes (see below). The reaction mixture consisted of 0.4 ml of 0.1 M sodium acetate (pH 4.0), 0.4 ml of enzyme solution diluted with 0.2% BSA, and 0.2 ml of 1% polysaccharide. After 1 h at 37°C, the reaction was terminated by heating in boiling water, and the solution was analyzed for reducing groups by the arsenomolybdate method (13). The reaction rates in the ,B-galactosidase and galactanase assays were linear with respect to both enzyme concentration and incubation time.T...

Multiple forms of cotyledonary b-galactosidases from Vigna unguiculata quiescent seeds

“…These data indicate that the partially purified cotyledonary β-gal I, β-gal II and β-gal III extracted from Pitiúba cowpea were heat stable up to 50°C, and that their thermostability is similar to the β-galactosidases isolated from cotyledons of Vigna sinensis (Biswas 1987) and Vigna radiata (Kundu et al 1990), as well as from β-galactosidases isolated from other plants sources (Konno & Katoh 1986, Simos & Georgatsos 1988. However, they differ from the ones obtained with β-galactosidases isolated from radish (Sekimata et al 1989) and tomato seeds (Pressey 1983), which were inactive at 55°C or had only 50% of their initial activities at temperature ranging from 48 to 52°C, respectively.…”

“…These results are similar to those found for cotyledonary β-galactosidases from Vigna sinensis (Biswas 1987) and Vigna radiata (Kundu et al 1990), as well as for seeds (Agrawal & Bahl 1968, Li et al 1975, Matheson & Saini 1977 al. 1988, Sekimata et al 1989, Giannakouros et al 1991, Buckeridge & Reid 1994, shoots (Konno & Tsumuki 1993), leaves (Sawicka & Kacperska 1995) and fruits (Pressey 1983, Ogawa et al 1990, Ranwala et al 1992) of different species.…”

“…2000Sekimata et al 1989, Simos et al 1989, Kundu et al 1990, Giannakouros et al 1991, Enéas-Filho et al 1995. It has been suggested that these enzymes are associated to the depletion of oligo-and polysaccharides during the initial phases of seed germination (Matheson & Saini 1977, Corchete & Guerra 1986, Biswas 1987, Enéas-Filho et al 1995, involved in the metabolism of cell wall constituints (Corchete & Guerra 1987a, Edwards et al 1988, Dopico et al 1989, Konno & Katoh 1992, Konno & Tsumuki 1993, Gómez et al 1995, Kitagawa et al 1995, and in the process of fruit ripening (Pressey 1983, Ogawa et al 1990, Veau et al 1993, Ali et al 1995. β-galactosidase activity has been detected in quiescent seeds (Dey 1984) as well as during germination and seedling establishment (Agrawal & Bahl 1968, Corchete & Guerra 1987b, Kundu et al 1990, Buckeridge & Reid 1994, Enéas-Filho et al 1995.…”

Temporal Sequence of Cell Wall Disassembly in Rapidly Ripening Melon Fruit1

“…The loss of Gal can occur independently of PG activity (Carrington et al, 1993), suggesting the involvement of other classes of enzymes such as ␤-galactosidases/␤-galactanases, which have been associated with many ripening fruits (Ross et al, 1993(Ross et al, , 1994Carey et al, 1995;Lashbrook et al, 1997), and which may act on several classes of polysaccharides, including the galactan side chains of rhamnoglacturonans (Pressey, 1983). Loss of galactans has been demonstrated to accompany or even precede increased solublization of polyuronides (Gross and Wallner, 1979;Kim et al, 1991), and purified or partially purified ␤-galactosidase has been shown to catalyze an apparent decrease in the molecular size of pectins in vitro in tomato (De Veau, 1993) and melon fruits (Ranwala et al, 1992).…”

Cell Wall Metabolism in Ripening Fruit (IX. Synthesis of Pectic and Hemicellulosic Cell Wall Polymers in the Outer Pericarp of Mature Green Tomatoes (cv XMT-22)

“…In the combined pectic fraction (Table 111), linkages identified as typical for tomato pectic polysaccharides (based on the report of Pressey [1983] and the thorough analysis of Seymour et al 119901) comprised more than 90% of the total identified linkages. The most abundant linkages were 1,5-arabinosyl (furanosyl) residues (23% of the total), 1,4-galactosyl residues (18% of the total), and terminal galactosyl, and 1,2-and/or 1,4-xylosyl residues (not separable under the chromatographic conditions employed [15% of the total]).…”