Influence of Calcium on Fruit Firmness and Cell Wall Degrading Enzyme Activity in Elstar Apples during Storage D. Kittemann, D.A. Neuwald and J. Streif Kompetenzzentrum Obstbau odensee avendorf-ravensburg Germany Keywords: 1-MCP, endo(1-4)-β-d-glucanase, polygalacturonase, pectate lyase, pectinmethylesterase, respiration Abstract Softening processes in apples during storage and shelf-life are related to cell wall degrading enzymes involved in the hydrolysis of cell wall components. Calcium also has a role in cell wall stability as calcium treatments can extend storage life, decrease fruit softening rate, and reduce both respiration and ethylene production. Our experiment investigates the influence of calcium infiltration in combination with different storage treatments, including the use of 1-MCP, on the softening behaviour of Elstar apples. We measured the activity of the cell wall degrading enzymes (endo-(1-4)-β-d-glucanase; polygalacturonase; pectate lyase; pectin methylesterase) to explain differences in fruit softening rates. Calcium treatments decreased respiration, ethylene production and fruit softening, but increased cell wall degrading enzyme activity. In general, the effect of calcium infiltration treatments on the measured parameters was comparable to the effect of 1-MCP. INTRODUCTION Fruit storage aims to maintain fruit quality, especially fruit flesh firmness, and extensive research has been conducted to better understand the physical and physiological changes that are occurring during ripening. Fruit softening is mainly related to dissolution of the middle lamella, that results in a reduction in intercellular adhesion when hemicellulosic and pectic cell wall polysaccharides (rummel and Harpster, 21), are degraded by enzymes, such as polygalacturonase (PG), pectate lyase (PL), endoglucanase (EGase) or pectinmethylesterase (PME). Calcium helps stabilise cell walls and as a result, it can affect softening behaviour of apples (Siddiqui et al., 1995). Infiltration with CaCl 2 can reduce apple respiration rates (angerth et al., 1972), ethylene production (Sams et al., 1984) and can delay fruit senescence during ripening and storage (Wills, 1982). Application of calcium sprays is a standard practice in German apple orchards to increase fruit storability and to control physiological disorders, such as bitter pit (Link et al., 22). Controlled atmosphere storage (CA) allows improved storability and quality preservation of climacteric fruits, such as apple. Recently the ethylene-inhibitor 1-MCP has become available as a practical tool for use in apple CA storage because it helps maintain fruit quality both during storage and shelf-life. However, one possible disadvantage of these technologies is that after 1-MCP application and CA storage, depending on the ripening stage and storage duration, fruit sometimes do not soften at all. Some consumer tests have shown that apples may be too firm and therefore negatively evaluated by the consumer (McCormick et al., 29). This work investigates the influence of calcium infiltration in combination with different storage treatments, including the use of 1-MCP, on the ripening and softening behaviour of Elstar apples and the activity of cell wall degrading enzymes. MATERIALS AND METHODS Elstar apples were obtained from the Kompetenzzentrum Obstbau odensee research orchard situated near Lake Constance, Southern Germany. After hand sorting to remove damaged and undersized fruit and to get homogenous fruit material apples were Proc. 6 th International Postharvest Symposium Eds.: M. Erkan and U. Aksoy Acta Hort. 877, ISHS 21 137
treated with 1-MCP after 1 day at 1 C or not treated and kept at 1 C in air for 24h. Afterwards fruit were vacuum infiltrated (1 mbar) at room temperature with 3 mm mannitol only or in combination with 15 mm CaCl 2 for 1 min and stored at 1 C in either air or CA (1% O 2 ; 3% CO 2 ) for 4 months followed by 1 days shelf-life at 2 C. Directly after infiltration samples of flesh tissue (an equatorial slice including the skin) were taken for mineral analysis. Calcium content was measured by standard atomic absorption spectroscopy (AAS) method. Fruit firmness and enzyme activity were measured at harvest, after storage and after shelf-life. Firmness Fruit flesh firmness was measured using a semi automatic penetrometer (Güss/ Fruit Texture Analyser, SA) between the blushed and unblushed side of the apple (8 fruit 3 reps) after removing the skin. Results were expressed as kg/cm 2. CO 2 and Ethylene Production CO 2 and ethylene production were measured on 12 fruits (3 4) after harvest and after storage during a 1 day shelf-life at 2 C according to Xuan and Streif (25). Results were expressed as ml CO 2 *(kg*h) -1 and ml H 2 O 4 *(kg*h) -1. Enzyme Extraction and Assay Material for enzyme analysis was taken from an equatorial slice of flesh tissue using 24 fruit (8 3 reps.) per treatment, frozen with liquid nitrogen, freeze dried, powdered and kept at -3 C until use. The methods for extraction and measurement of enzyme activity were those as described by Ortiz and Lara (28). Extraction was carried out using 1 mg of freeze dried fruit powder according to Lohani et al. (24). A 1% (w/v) pulp suspension was prepared by homogenizing and centrifuging freeze dried tissue in an adequate buffer (2 mm Tris-HCl, ph 7.; 2 mm Cys-HCl; 2 mm EDTA;,5% (v/v) Triton X-1). PG was determined according to Pathak and Sanwall (1998). Results are given as specific activity, where one unit is defined as the liberation of 1 µmol of galacturonic acid per minute. PL activity was measured as described by Lohani et al. (24). One unit of PL activity is defined as A 235 per minute. EGase activity was determined according to Miller (1959), defining one Unit of EGase activity as the release of 1 µmol of glucose per minute. PME activity was measured as described by Hagermann and Austin (1986). One unit of PME activity is described as A 62 per minute. The total protein content was determined according to radford et al. (1976) using the IORAD enzyme essay kit with bovine serum albumin as a standard. Statistical Analysis Statistical analyses were carried out with MINITA Release 14 software, to calculate a one-factorial analysis of variance and onferroni-test for comparison of the means (p<,5). Standard deviations were calculated using Microsoft Office-Excel 27 software. RESULTS AND DISCUSSION Mineral Content Infiltration treatments are a rapid and effective postharvest method to increase the calcium content of apples (Scott and Wills, 1977). The calcium content in apples directly after CaCl 2 infiltration was about 1% higher compared with fruit infiltrated with mannitol only (9.42 mg and 4.48 mg calcium/1 g FW). CO 2 and Ethylene Production Calcium treatment reduced the respiration rate (Fig. 1) and ethylene production (data not shown) of apples after storage, especially when air-stored. In calcium infiltrated 138
apples, but without 1-MCP application, respiration rates were comparable with fruit treated with 1-MCP, with and without calcium. Apples without calcium and without 1-MCP were characterized by a higher respiration rate and higher ethylene production during shelf-life. For CA storage this effect was much lower than for air. This influence of calcium on the respiration rate and ethylene production in apples is also described by Song and angerth (1993), where CaCl 2 infiltration decreased CO 2 and ethylene production. Firmness Calcium treatment affected flesh firmness (Fig. 2). Apples held in air, with Cainfiltration, but without 1-MCP treatment were firmer and softened slower than non calcium treated. 1-MCP treatment gave no additional increase in firmness. Firmness of calcium treated fruit in air but without 1-MCP was comparable to both 1-MCP treatments, with or without calcium. This calcium-infiltration effect is also described by Glenn et al. (199), who found higher firmness and tensile strength in apples after calcium infiltration (4% w/v) compared with an untreated control. In CA, there was no impact of Ca-infiltration on fruit firmness. 1-MCP treated apples were firmer than those without 1-MCP, both under air and CA and especially during shelf-life. This positive effect of 1-MCP on fruit firmness is widely known and described in the literature (Fan et al., 1999; Zanella, 24; Streif et al., 26). Activity of Cell Wall Degrading Enzymes The cell wall degrading enzymes (EGase, PG, PL, PME) show a clear influence of calcium-treatment, 1-MCP, and storage condition (Fig. 3). In air the treatments without 1-MCP and calcium and in CA the treatments without 1-MCP and with or without calcium, respectively show the lowest firmness and also the lowest enzymatic activity, both after storage and shelf-life. In air, calcium treated fruit consistently had higher enzyme activity in comparison with untreated fruit. As also observed for firmness there was no influence of calcium treatments on enzyme activity under CA, either after storage or after shelf-life. Fruit treated with 1-MCP show a higher enzyme activity than fruit without 1-MCP, both in RA and CA. These results are partially comparable with Ortiz and Lara (28) for peach, where PG activity in 1-MCP treated fruit after 7 days shelf-life was higher than in untreated fruit. In contrast, Jeong et al. (22) suppressed increases in PG activity in 1-MCP treated avocado. However, our results are surprising, as most of the enzymes analysed here are supposed to be ethylene dependent and negatively correlated to firmness (Huber et al., 23). There can be different reasons for this result. One possibility is, that at the time of sampling, apples treated with 1-MCP or with calcium were less developed in ripeness than untreated fruit, so that they still show a higher enzyme activity while the untreated apples are already past their maximum enzyme activity. A second and maybe more suitable explanation is that in fruit treated with either calcium or 1-MCP, the substrate for the enzyme is not directly available or the enzymes are not activated, so that the measured activity in vitro does not effectively represent in vivo depolymerisation of cell wall components. According to Huber et al. (21) endogenous levels of PG and PME do not always correlate well with trends in pectin depolymerisation, indicating that the activity of these and likely other enzymes is restricted in vivo. Possible factors controlling in vivo hydrolysis are changes in apoplastic ph, cell wall inorganic ion levels, non enzymatic proteins including the noncatalytic β-subunit and expansions, wall porosity, and steric hindrances. ased on our results there must be an effect of calcium and/or1-mcp treatment to impede cell wall degrading enzymes in vivo. The use of calcium chelators may negate the prior influence of enzymes or other factors on pectin solubility (Huber et al., 21). The mechanisms by which calcium and 1-MCP affect cell wall enzyme activity might be different from each other. Work currently in progress including analysis of the cell wall 139
components and pectin fractions will help to further explain our results. CONCLUSIONS Our results show a clear influence of calcium on softening behaviour and the activity of cell wall degrading enzymes in apples. Although calcium infiltration is not a practical tool for commercial use, this work supports the importance of calcium to help stabilise the cell wall and maintain fruit firmness during fruit ripening, as flesh firmness in calcium treated apples was comparable with that of 1-MCP treated fruit after 4 months air storage. As the in vitro activity of EGase, PG, PL, and PME was higher in both calcium and 1-MCP treated apples, both experimental treatments may restrict these enzymes in vivo. ACKNOWLEDGEMENTS We thank Isabel Lara and Abel Ortiz from the University of Lleida in Spain for help with the cell wall analysis methods and for technical advice, and Roy McCormick for improvements to the manuscript. Literature Cited angerth, F., Dilley, D.R. and Dewey, D.H. 1972. Effect of postharvest calcium treatments on internal breakdown and respiration of apple fruits. J. Amer. Soc. Hort. Sci. 97:679-682. radford, M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. iochem. 72:248-254. rummel, D.A. and Harpster, M.H. 21. Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Mol. iol. 47:311-34. Fan, X., lankenship, S.M. and Mattheis, J.P. 1999. 1-methylcyclopropene inhibits apple ripening. Journal of the American Society for Horticultural Science 124:69-695. Glenn, G.M. and Poovaiah,.W. 199. Calcium-mediated postharvest changes in texture and cell wall structure and composition in Golden Delicious apples. J. Amer. Soc. Hort. Sci. 115:962-968. Hagermann, A.E. and Austin, P.J. 1986. Continuous spectophotometric assay for plant pectin methyl-esterase. J. Agri. Food Chem. 34:44-444. Huber, D.J., Jeong, J. and Mao, L.-C. 23. Softening during ripening of ethylene-treated fruits in response to 1-methylcyclopropene application. Acta Hort. 628:193-22. Huber, D.J., Karakurt, Y. and Jeong, J. 21. Pectin degradation in ripening and wounded fruits. Rev. ras. Fisiol. Veg. 13:224-241. Jeong, J., Huber, D.J. and Sargent, S.A. 22. Influence of 1-methylcyclopropene (1- MCP) on ripening and cell wall matrix polysaccharides of avocado (Persea americana) fruit. Postharvest iol. Technol. 25:241-256. Link et al. 22. Lucas Anleitung zum Obstbau. Verlag Eugen Ulmer. Stuttgart. 32:234. Johnson, D.S. 1979. New techniques in the post-harvest treatment of apple fruits with calcium salts. Commun. Soil Sci. Plant Anal. 1:373-382. Lohani, S., Trivedi, P.K. and Nath, P. 24. Changes in activities of cell wall hydrolases during ethylene-induced ripening in banana: effect of 1-MCP, AA and IAA. Postharvest iol. Technol. 31:426-428. McCormick, R., Kittemann, D. and Streif, J. 29. Consumer preferences of Elstar apples at differing ripeness treated with 1-MCP. Acta Hort. 585-99-13. Miller, G.L. 1959. Use of dinitrosalycilic acid reagent for determination of reducing sugar. Anal. Chem. 31:426-428. Moran, F., Nasuno, S. and Starr, M.P. 1968. Extracellular and intracellular polygalacturonic acid trans eliminase of Erwinia carotovora. Arch. iochem. iophys. 123:298-36. Ortiz, A. and Lara, I. 28. Cell wall modifying enzyme activities after storage of 1-MCP treated peach fruit. Acta Hort. 796:137-142. 14
Pathak, N. and Sanwall, G.G. 1998. Multiple forms of polygalacturonase from banana fruits. Phytochem. 48:249-255. Sams, C.E. and Conway, W.S. 1984. Effect of calcium infiltration on ethylene production, respiration rate, soluble polyuronide content and quality of Golden Delicious apple fruit. J. Amer. Soc. Hort. Sci. 19:53-57. Scott, K.J. and Wills, R..H. 1977. Vacuum infiltration of calcium chloride: a method for reducing bitter pit and senescence of apples during storage at ambient temperatures. HortScience 12:71-72. Siddiqui, S. and bangerth, F. 1995. Effect of pre-harvest application of calcium on flesh firmness and cell-wall composition of apples- influence of fruit size. Journal of Horticultural Science7:263-269. Song, J. and angerth, F. 1993. The effect of calcium infiltration on respiration, ethylene and aroma production of Golden Delicious apple fruits. Acta Hort. 326:131-137. Streif, J. and Gasser, F. 26. MCP und DCA heißen die neuen Zauberformeln. W agrar. 41/26:2-21. Wills, R..H. 1982. Inhibiton of ripening of avocados with calcium. Sci. Hort. 16:323-33. Xuan, H. and Streif, J. 25. Effect of 1-MCP on the respiration and ethylene production as well as on the formation of aroma volatiles in Jonagold apple during storage. Acta Hort. 682:123-121. Zanella, A. 24. Dynamische CA-Lagerung und Anwendung von 1-MCP. esseres Obst. 9/24:11-13. Figures CO2- production [ml* (kg*h) -1 ] 1 9 8 7 6 5 4 3 2 1 1 2 3 4 5 6 7 8 9 1 mannitol without MCP mannitol with MCP calcium + mannit. without MCP calcium + mannit. with MCP days of shelf-life Fig. 1. Influence of calcium on the CO 2 -production of Elstar apples with and without 1- MCP during 1 d at 2 C after 4 months air storage (error bars = SD). 141
firmness [kg*(cm²) -1 ] 1 9 8 7 6 5 4 3 2 1 a/a RA CA a a a a a a A A A A A A b b - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP mannitol calcium+mannit. mannitol calcium+mannit. mannitol calcium+mannit. mannitol calcium+mannit. harvest after 4 month storage 4 month storage + shelf-life after 4 month storage 4 month storage + shelf-life Fig. 2. Influence of calcium on firmness of Elstar apples with and without 1-MCP at harvest, after 4 months air or CA storage at 1 C plus shelf-life (1 d at 2 C); the statistical analysis was carried out separately for RA and for CA storage; p<,5. 142
Endoglucanase (EGase) EGase specific activity [µmol Glc*(mg*protein*t) -1 ] 3 25 2 15 1 5 b c/cd d RA d a CD C C CA CD C D A - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP mannitol calcium+mannit. mannitol calcium+mannit. mannitol calcium+mannit. mannitol calcium+mannit. harvest after 4 month storage 4 month storage + shelf-life after 4 month storage 4 month storage + shelf-life Polygalacturonase (PG) PG specific activity [µmol GalUa*(mg*protein*min) -1 ] 7 6 5 4 3 2 1 a/a cd RA CA b d b d C C C AC d C C C A - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP mannitol calcium+mannit. mannitol calcium+mannit. mannitol calcium+mannit. mannitol calcium+mannit. harvest after 4 month storage 4 month storage + shelf-life after 4 month storage 4 month storage + shelf-life Pectatlyase (PL) PL specific activity [ Abs.*(mg*protein*min) -1 ] 2 1,6 1,2,8,4 a/a c b RA c b CA - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP - MCP + MCP mannitol calcium+mannit. mannitol calcium+mannit. mannitol calcium+mannit. mannitol calcium+mannit. harvest after 4 month storage 4 month storage + shelf-life after 4 month storage 4 month storage + shelf-life Fig. 3. Influence of calcium on the EGase, PG and PL activity in Elstar apples at harvest, after 4 months air or CA storage plus shelf-life (1 d at 2 C), with and without 1-MCP; the statistical analysis was carried out separately for RA and for CA storage; p<,5. 143
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