Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.     

Kiwifruit β-galactosidase: Isolation and activity against specific fruit cell-wall polysaccharides

A -galactosidase (EC 3.2.1.23) capable of degrading a number of fruit cell-wall polysaccharides in vitro, was isolated from ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson cv. Hayward). The enzyme has a molecular weight of approximately 60 kDa by gel permeation and consists of several basic isoforms. Several polypeptides were enriched during purification, with 33-, 46- and 67-kDa bands being predominant after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The optimum activity of the enzyme against p-nitrophenyl--d-galactopyranoside was at pH 3.2, but against a galactan purified from kiwifruit cell walls, it was at pH 4.9. The enzyme was specific for galactosyl residues in the -configuration, releasing galactose from a variety of kiwifruit cell-wall polysaccharide fractions including cell wall material, Na₂CO₃-soluble pectin, high-molecular-weight galactan, xyloglucan, and galactoglucomannan. A galactosylated glucuronomannan found throughout the kiwifruit plant was also a substrate for the enzyme. The results indicate that the enzyme attacks the non-reducing end of galactose side chains, cleaving single galactose residues which may be attached to the 2, 3, 4, or 6 position of the aglycone. Activity of the enzyme in-vitro was too low to account for the total loss of galactose from the cell walls during ripening. If the -galactosidase of this study is solely responsible for the removal of galactose from the cell wall during ripening then its in-vivo activity must be much greater than that observed in-vitro.Abbreviations CWM cell wall material - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We thank Bronwyn Culling and Teresa Wegrzyn for assistance and acknowledge a contribution towards the cost of the research from the New Zealand Kiwifruit Marketing Board.     

Activity of cell wall-associated enzymes in ripening olive fruit

Enzymatically active cell wall isolaled from olive (Olea europaea) fruit was employed Hi investigate some hydrolytic enzymes bound to the cell wall and the changes in these during ripening. Seven glycosidases. β-glucosidase (EC 3.2.1.21) α-galactosidase (EC 3.2.1.22). β-galactosidase (EC 3.2.1.23). α-arabinosidase (EC 3.2.1.55), α-mannosidase (EC 3.2.1,24). β-xylosidase (EC 3.2.1.37) and β-N-acetylglucosamidase (EC 3.2.1.30). as well as Cx-cellulase (EC 3.2.1.4) and endo-polygalacturonase (EC 3.2.1.15). were identified in the cell wall preparation, at four stages of ripeness (mature green. changing colour, black and black-ripe). Activities of all these cell wall-associated enzymes fionicallv and covalently linked) were determined either by cell wall incubation with artificial substrate or after extraction from the cell wall with buffers of high salt concentration (Cx-cellulase). and were compared to those of forms solubilized from acetone powders with 500 nM citrate buffer (cytoplasmic and/or apoplastic plus ionically hound to cell wall) In general, the activities of low ionic strength buffer-soluble enzymes were found to be much higher than those of the bound enzymes. The bound enzymes are present in the fruit at the green colour stage, whereas the activities of the soluble enzymes only increased from the changing colour stage onwards. The tenacity of binding of enzymes to the wall was investigated by treating the walls with high salt and measuring residual activity. The nature of the ionic and covalent binding and the changes during ripening were also established for wall-hound glycosidase During ripening there was a marked change in the percentages of covalently- and tonically linked activities of β-glucosidase and β-galaclosidase: al the changing colour stages about 75–80% of the bound active in was present in high ionic strength buffer while al the black-ripe stage it was only 15–20. A possible role for these cell wall degradative enzymes in olive softening is discussed.     

Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.     

Endopolygalacturonase: a Candidate Gene for <Emphasis Type="Italic">Freestone</Emphasis> and <Emphasis Type="Italic">Melting Flesh</Emphasis>in Peach

Peach fruit are handled, processed, and marketed according to their stone adhesion and fruit softening type. Uncertainty exists over whether these simply inherited traits are controlled by two linked loci, Freestone (F) and Melting flesh (M) or one multi-allelic locus, and whether M is controlled by the cell wall degrading enzyme, endopolygalacturonase. From morphological and molecular analysis of two related segregating populations of peach, we conclude that a single locus containing at least one gene for endopolygalacturonase, controls both F and M with at least three effective alleles. A simple diagnostic PCR test is now available for the three major phenotypes of freestone melting flesh (FMF), clingstone melting flesh (CMF), and clingstone non-melting flesh (CNMF).     

丙烯对柿果实采后细胞壁物质代谢和几种生理指标的影响

丙烯处理的柿果实,其软化进程显著加快,呼吸速率和乙烯释放高峰提前,且峰值升高,细胞壁各组分的代谢速度加快,柿果实中多聚半乳糖醛酸酶（PG）和纤维素酶（Cx）活性提高,且高峰提前。     

柿果实采后软化过程中细胞壁组分代谢和超微结构的变化

柿果实采后果胶酯酶活性迅速上升,其活性与果实硬度的下降呈明显的负相关。多聚半乳糖醛酸酶活性增加缓慢,但其活性与果实硬度的下降无明显相关性。β-半乳糖苷酶活性迅速增加,其活性与果实硬度的下降呈明显的负相关。纤维素酶活性呈逐渐上升趋势,与果实硬度的下降也呈明显的负相关。伴随着细胞壁水解酶活性的增加,果实原果胶和纤维素含量迅速下降,而水溶性果胶含量则迅速上升。柿果刚采收时细胞壁结构完整,3d后细胞壁中胶层基本被溶解,甚至初生壁也局部发生降解。     

Note Floristiche Valdarnesi

Abstract

The involvement of cellulase (endo-b-l,4-glucanase, EC 3.2.1.4) in a number of different cell separation events which occur in higher plants has been well established. Besides their significance for the plant growth and differentiation, these events can be economically important since they also comprise softening of fleshy fruits and abscission of fruits, flowers and leaves. In higher plants cellulase is present in a number of different biochemical isoforms which are encoded by different genes. This finding is in accordance with the wide range of physiological events which require the intervention of cellulase activity, and whose peculiarities and amplitudes can be quite various.     

Responses of banana fruit to treatment with 1-methylcyclopropene

Experiments were conducted to determine levels of 1-methylcyclopropene (1-MCP) exposure needed to prevent ethylene-stimulated banana fruit ripening, characterise responses of ethylene-treated fruit to subsequent treatment with 1-MCP, and to test effects of subsequent ethylene treatment on 1-MCP-treated fruit softening. Fruit softening was measured at 20°C and 90% relative humidity. One hour exposure at 20°C to 1000 nl 1-MCP/l essentially eliminated ethylene-stimulated ripening effects. Exposure for 12 h at 20°C to just 50 nl 1-MCP/l was similarly effective. Fruit ripening initiated by ethylene treatment could also be delayed with subsequent 1-MCP treatment. However, 1-MCP treatment only slowed down ripening of ethylene-treated fruit when applied at 1 day after ethylene and was ineffective when applied 3 or 5 days after ethylene treatment. The ripening response of fruit treated with 1-MCP and subsequently treated with ethylene varied with interval time between 1-MCP and ethylene treatments. As time increased, the response of 1-MCP-treated fruit to ethylene was enhanced. Responses to 0.1, 1, 10 or 100 µl ethylene/l concentrations were similar. Enzyme kinetic analysis applied to 1-MCP effects on ethylene-induced softening of banana fruit suggested that 1-MCP inhibition is by noncompetitive antagonism of ethylene binding.