Cell Wall Metabolism in Ripening Fruit

Abstract

Mature 'Bartlett' pear (Pyras communis) fruits were ripened at 20 C. Fruits at different stages of ripeness were homogenized, and extracts of the low speed pellet (crude cell wall) were prepared. These extracts contained polygalacturonase, pectin esterase, and activity against seven pnitrophenyl glycoside substrates. Polygalacturonase, a-galactosidase, and a-mannosidase increased in activity as the fruit ripened. CeOulase and activities against pear wall xylan and arabinan were absent from the extracts.The changes in cell wall composition which accompany the softening of ripening fruit apparently result from the action of enzymes produced by the fruit. Prominent among the enzymes implicated are PG3 and PE because striking changes in wall pectin content are observed in ripening fruits (1,13,14,21,22), and activities ofthese two enzymes often increase as ripening continues (21). In addition, primarily because of their presence in extracts of ripening fruits, a variety ofglycanases (10,21,26) and glycosidases (3,20,21) have been assigned an unspecified role in fruit cell wall metabolism.This paper describes measurements of the activity of a variety of carbohydrate-degrading enzymes in extracts of ripening 'Bartlett' pears. The potential for depolymerization of pear cell wall polysaccharides was assessed using model substrates containing the same types of glycosidic linkages that have been described for pear cell walls (1). Glycosidase activities were followed using pnitrophenyl substrates. The activities of several enzymes, notably PG, a-mannosidase, and a-galactosidase, increased substantially as the fruit approached the respiratory climacteric. The potential roles of the various enzymes in ripening-associated cell wall modification are discussed. Enzyme Assays. PE activity was determined as described by Nagel and Patterson (17) with the aid of an automatic titrating pH meter (Radiometer Titrometer II). MATERIALS AND METHODSPolysaccharide-degrading activities were followed by measuring the generation of reducing groups according to the method of Nelson (18), as modified by Somogyi (23). The standard reaction mixture contained enzyme and 0.1% (w/v) substrate in I ml of 100 mm Na-acetate (pH 5.0). Incubation was at 37 C. The substrates used were polygalacturonic acid (Sunkist Growers), carboxymethyl cellulose and larch xylan (Sigma), and araban (KochLight Laboratories, Ltd.).Prior to use, the xylan was purified according to the technique of McNeil and Albersheim (as described by Taiz and Honigman [24]) to remove abundant glucose-and mannose-containing contaminants. The araban also had to be treated prior to use in order to remove uronic acid-containing materials. Five g araban were refluxed for 16 h in 250 ml 5 N NaOH containing 4 g NaBH4. The preparation was cooled, neutralized with glacial acetic acid, and centrifuged for 5 min at 1,000g. Final clarification of the supernatant solution was accomplished by filtration through glass fiber (GF/C) paper. The filtrate was dialyzed against 10 mm K-phosphate (pH 7...

Activity and expression of polygalacturonase vary at different fruit ripening stages of sweet pepper cultivars

“…The results are similar to the findings of scientists who worked on the role of PG during fruit development in various kinds of fruits and found a correlation, as PG activity and softening occurs simultaneously in cherry (Barrett and Gonzalez, 1994), date (Hasegawa et al, 1969), durian (Ketsa and Daengkanit, 1999), kiwifruit (Wang et al, 2000), mango (Ketsa et al, 1998), papaya (Paull and Chen, 1983), pear (Ahmed and Labavitch, 1980), pepper (Rao and Paran, 2003;Arancibia and Motsenbocker, 2006), tobacco (Osteryoung et al, 1990), and tomato (Hobson, 1965;Crookes and Grierson, 1983;Huber, 1983;DellaPenna et al, 1987;Bird et al, 1988;Biggs and Handa, 1989;Smith et al, 1990;Fenwick et al, 1996).…”

“…The softening phenomenon influenced by PG in fruits like apples, cherries, dates, durians, grapes, kiwis, melons, peaches, pears, pineapples, tobacco, and tomatoes during ripening has been studied during the past two decades and substantial evidence has been collected, suggesting that the cell wall enzyme PG plays a major role in the entire ripening process (Hobson, 1965;Hasegawa et al, 1969;Ahmed and Labavitch, 1980;Paull and Chen, 1983;Osteryoung et al, 1990;Barrett and Gonzalez, 1994;Ketsa et al, 1998;Ketsa and Daengkanit, 1999).…”

Cell Wall Metabolism in Ripening Fruit

“…The increase in the low mol wt HGA as pears become overripe, apparently at the expense of higher mol wt HGA, indicates that the endogenous substrate is attacked by the endogenous PGs (18) but possibly is not degraded by these enzymes to the monomer or smaller oligomer (80% ethanolsoluble) form at a rapid rate. The level ofmethyl esterification (60%) may protect the HGA from extensive hydrolysis by the PGs (in spite of the presence of endogenous PE [2]) so that the 80% ethanol-insoluble forms accumulate in the ripening fruit.…”

“…Enzyme Preparations a-L-Arabinofuranosidase was prepared from ripe Bartlett pear fruit (2). Pear acetone powder, 0.4 g, prepared by the method of Dick et al (5) was suspended in 20 mL of cold 5 mM Na phosphate (pH 6.5) and the mixture centrifuged at l0,OOOg for 10 min.…”

“…The latter may be due to a lack of suitable hydrolytic enzymes in the ripening fruit or to a peculiar structure which protects the pectic polysaccharides from further degradation. Potent glycoside hydrolyzing activities of pear fruit, namely PG2 (16), and a-L-arabinofuranosidase (2) might be expected to rapidly degrade the watersoluble pectins which appear to accumulate in ripening pears (1).…”