Calcium alters senescence rate of postharvest muskmelon fruit disks

Postharvest Biologyand Techndogy ELSEVIER

Postharvest Biology and Technology

7 (1996) 91-96

Calcium alters senescence rate of postharvest muskmelon fruit disks Gene Lester * US. Department

of Agriculture, Agricultural Research Service, Subtropical Agricultural Research Laboraroq 2301 South International Boulevard, Weslaco, TX 78596, USA

Accepted 15 March 199.5

Abstract

Percent total chlorophyll loss in whole muskmelon (Cucumis melo L. var. reticulatus Naud.) fruit disks incubated in control (no salt), 0.02, 0.04, 0.08, 0.16 or 0.32 M CaC12, MgCl2 or KC1 plus 0.35 M mannitol for ten days at 22°C in the dark was delayed only in calcium solutions, with the greatest retention occurring in 0.04 M content. Percent total chlorophyll loss was hastened in 0.16 and 0.32 M concentrations regardless of the cation. Differences in the chemical, senescence-associated, properties of the plasma membrane (PM) from hypodermal mesocarp tissue of postharvest muskmelon disks were compared before and after floating disks in control, 0.04 or 0.16 M CaC12, MgCl2 or KC1 plus 0.35 M mannitol for ten days at 22°C in the dark. Total phospholipid, protein, and H+-ATPase (EC 3.6.1.35) activity loss, in the PM were delayed only with 0.04 M CaC12 and hastened with most all other chloride solutions vs. no salt. The free sterol : phospholipid ratio and percent unsaturation of phospholipid fatty acids was the highest in the PM from fruit disks incubated in 0.04 M CaC12 and were the highest and lowest, respectively, in 0.16 M CaC12 vs. no salt. This study suggests that muskmelon fruit senescence is affected by calcium treatment, by affecting the PM, possibly involving the regulation of phospholipid composition and content. Keywords:

Cucumis

melo

var. reticulatus; Melons; H+-ATPase; Lipids; Plasma membrane

1. Introduction Calcium-mediated cross linking of pectic polymers has been cited as a possible mechanism controlling softening in fruits (Bangerth, 1979; Fry, 1986; Stow, 1993). However, there is growing evidence that the molecular mechanism of calcium’s action in fruit softening is in part due to its effect on membrane integrity (Ferguson, 1984; Paliyath and Droillard, 1992). Additionally, calcium has been shown to * Fax: 210 565-6652. 0925-5214/96/$15.00 0 1996 Elsevier SSDI 0925-5214(95)00020-8

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G. Lester I Postharvest Biology and Technology 7 (1996) 91-96

be effective in reducing chlorophyll and protein loss and inhibiting plant tissue senescence (Poovaiah et al., 1988). Muskmelon fruit softening may (McCollum et al., 1989; Ranwala et al., 1992) or may not (Lester and Dunlap, 1985) be associated with content changes of the major cell wall fractions. But melon softening and senescence does appear to be associated with changes in membrane integrity (Lester and Bruton, 1986) specifically the plasma membrane (PM) from hypodermal mesocarp tissue (Lester, 1988; Lester and Stein, 1993). Because there is evidence that calcium regulates fruit softening and senescence at the membrane level, the objective of this study was to determine the effect of calcium on cellular senescence of postharvest muskmelon hypodermal mesocarp disks. 2. Materials and methods Plant material

Fruits for percent total chlorophyll retention were ‘retail’ muskmelons with a mean weight of 1.5 kg, free of defects, and purchased from a local supermarket in Weslaco, Texas. Fruits for the physicochemical analyses were ‘Explorer’ muskmelons with a mean weight of 1.5 kg, free of defects, and were harvested just after sunrise from a commercial field located near Rio Grande City, Texas. All fruits were classified as mature green, fully abscised. Fruits were washed in 20% (v/v) sodium hypochlorite 5.25% solution, and sterilized, distilled water containing 50 ,~l 1-l polyoxyethylene-sorbitan monolaurate (Tween 20). Hypodermal mesocarp disks (10 x 1 mm) were taken from a plug of tissue 5 mm under the epidermis. All tissue plugs were removed under aseptic conditions from within the equatorial region of the fruit. Ground-spot areas were not sampled. Chloride salt treatments

Calcium, magnesium and potassium chloride salt treatments of muskmelon fruit disks were done using a modification of the procedure of Cheour et al. (1992). Five disks were floated in sterile solutions of 20 ml control (no salt), 0.02, 0.04, 0.08, 0.16 or 0.32 M CaC12, MgC12 or KC1 plus 0.35 M mannitol in 125 ml Erlenmeyer flasks for ten days in the dark at 22 f 2°C with three replications per chloride salt treatment. The levels of chloride salt treatment of fruit disks for PM tissue analyses were chosen after preliminary tests showed the greatest delay of chlorophyll content occurred at 0.04 M and accelerated above 0.08 M CaC12. Fifteen fruit disks were used per replication/treatment,

it = 9.

Chlorophyll analyses

Chlorophyll was analyzed on five disks per replication/treatment. was determined by the aqueous acetone procedure of Arnon (1949).

Chlorophyll

Isolation of PA4 and ATPase, lipid andprotein analyses

PM isolation was conducted on a combination of three replications/treatment, to achieve a combined final fresh weight of 35 f 3 g, resulting in n = 3. Vanadate-

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93

sensitive, magnesium-dependent, and KN03-insensitive H+-ATPase was determined as previously described (Lester and Stein, 1993). Total free sterol, total phospholipids and protein were determined on the PM as previously described (Lester and Stein, 1993). Protein content was determined by the method of Bradford (1976). Statistical analyses

Analysis of variance was used to evaluate control (no salt) and chloride salt concentration differences for whole fruit disk chlorophyll content and for PM physicochemical data (SAS, 1988). Duncan’s multiple range test (P 5 0.05) was used to discern between molar concentrations when F values were significant. 3. Results and discussion

Various concentrations of calcium, magnesium and potassium chloride solutions affected the percent total chlorophyll retention of muskmelon hypodermal mesocarp disks (Table 1). Percent total chlorophyll retention from whole fruit disks incubated in 0.02, 0.04, 0.08 or 0.16 M MgC12 or KC1 were not significantly different from control (no salt). Whereas with CaC12, percent chlorophyll retention at 0.02, 0.04 and 0.08 was significantly greater than no salt, with 0.04 M CaC12 having the highest percent retention. These data demonstrate that moderate calcium vs. magnesium or potassium chloride solutions delays senescence of muskmelon fruit disks as measured by percent total chlorophyll retention. However, percent chlorophyll retention from disks incubated in 0.32 M solutions, regardless of chloride salt, was significantly less than no salt signifying that calcium, magnesium or potassium chloride solutions greater than 0.16 M are probably highly toxic. These data on whole fruit disks of muskmelon do concur with a similar study performed on cabbage (Brassica oleracea L., var. capitata) leaf disks (Cheour et al., 1992), in which relatively moderate CaC12 concentrations delayed, while high CaC12 concentrations accelerated loss of chlorophyll on a whole tissue basis.

Table 1 Percent chlorophyll retention of muskmelon hypodermal mesocarp disks incubated in control (no chloride salt), 0.02, 0.04, 0.08, 0.16 or 0.32 M CaC12, MgClz or KCI plus 0.35 M mannitol for ten days at 22 It 2°C in the dark. Values are means where n = 3. Salt concentration

Percent

(M)

CaClz

MgClz

KC1

Control 0.02 0.04 0.08 0.16 0.32

68 87 96 82 63 59

6.5 63 63 62 62 57

67 66 65 62 58 55

5

4

6

Duncan’s

P ‘: 0.05

chlorophyll

retention

from whole disks

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Biology and Technology 7 (1996) 91-96

Table 2 Percent total phosphohpids, protein and H+-ATPase activity retention in the plasma membrane of muskmelon hypodermal mesocarp disks incubated in control (no chloride salt), 0.04 and 0.16 M CaC12, MgC12 or KC1 plus 0.35 M mannitol for ten days at 22 f 2°C in the dark. Values are means where n = 3. Chloride salt concentration

Percent

retention

in plasma

membrane Protein

Phospholipids

H+-ATPase

(M) Control 0.04 0.16 Duncan’s

P 5 0.05

Ca

Mg

K

Ca

Mg

K

Ca

Mg

K

79 96 72

80 74 72

81 76 75

76 88 69

16 70 14

77 73 71

86 96 65

78 13 69

18 76 71

3

3

3

3

2

2

3

4

3

Isolation of PM from hypodermal mesocarp tissue disks incubated in control (no salt), 0.04 or 0.16 M CaC12, MgC12 or KC1 showed that with 0.04 M CaCl2 PM senescence was slowed, whereas with all other salt solutions senescence was hastened, compared to no salt (Table 2). Percent retention of PM total phospholipids was significantly greater with 0.04 M CaClz and significantly less with all other salt solutions vs. no salt. Also, percent retention of protein and H+-ATPase activity in PM followed that of the phospholipids where 0.04 M CaCl2 slowed, while higher CaClz and most all other salt solutions hastened the loss, compared to no salt. These data demonstrate that 0.04 M exogenous calcium chloride, and not potassium or magnesium, appears to be beneficial to the structural, functional or physiological properties of melon hypodermal PM tissue. Physicochemical changes in PM structure and properties occur during maturation and senescence of muskmelon hypodermal mesocarp tissue and these changes are characterized, as previously described, by a decline in membrane phospholipids, proteins, H+-ATPase activity and increased efflux of electrolytes (Lester and Stein, 1993). These physicochemical changes are typical for all fruits (Paliyath and Droillard, 1992). The present findings indicate that application of calcium can delay these physicochemical senescence related changes by reducing the loss of PM phospholipids, protein and Hf-ATPase activity. The decrease in PM fluidity which is characterized by increased efflux of electrolytes is regulated by, in general, the degree of unsaturated fatty acids and the ratio of sterols : phospholipids (Quinn, 1981). Calcium in this study may have a direct effect on PM leakiness (microviscosity) possibly by influencing free sterol increase and phospholipid loss and by reducing percent saturated fatty acid content (Table 3). Percent unsaturated fatty acids were greatest with 0.04 M CaClz and least with 0.16 M compared with no salt. A loss in membrane unsaturated fatty acids is highly negatively correlated with increased membrane leakiness (loss of microviscosity) in potato tubers (Solunum tuberosum L.) (Knowles and Knowles, 1989) and in muskmelon fruit (Cucumis melo L.) (Lester and Stein, 1993). A loss of membrane phospholipids and an increase in sterol content resulting in an increase in the sterol to phospholipid ratio causing

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Table 3 Total free sterols, total phospholipids and percent phospholipid fatty acid unsaturation in the plasma membrane of muskmelon hypodermal mesocarp disks incubated in control (no salt), 0.04 or 0.16 M CaCl2 plus 0.35 M mannitol for ten days at 22 f 2°C in the dark. Values are means where n = 3. Free sterols (nmol)

Phospholipids (nmol)

Unsaturation

(M) Control 0.04 0.16

2.1 f 0.3a 2.2 f 0.2 3.4 f 0.3

6.8 f 0.2 8.5 f 0.3 6.1 f 0.3

51.1 55.8 46.8

Calcium

Duncan’s

chloride

P 5 0.05

(%)

2.1

a Values are &SD.

a destabilization of the membrane bilayer and nonbilayer structures is a direct measure of increased membrane senescence (Paliyath and Thompson, 1990). The sterol : phospholipid ratio of the PM in this study was lowest with 0.04 M CaC12 and highest with 0.16 M CaClz. However, this sterol : phospholipid ratio increase in PM from fruit disks incubated in 0.16 M CaCl2 was not due only to the apparent loss in membrane phospholipids, as in the case with no salt, but due to an additional increase in sterol content. The results described in this report indicate that at the membrane level a delay in senescence, as evidenced by slowing the decline in physicochemical changes, occurs in muskmelon fruit disks incubated in 0.04 M CaC12 compared to no salt. However, an increase in senescence, described by the same physicochemical changes, occurs when disks are incubated in 0.16 M CaC12. Muskmelon fruit senescence as described herein is altered by calcium affecting the physicochemical function of the hypodermal PM tissue. Calcium chloride at 0.04 M delays and at 0.16 M hastens senescence of muskmelon fruit, possibly by affecting the apparent rate of PM lipid degradation and possibly the osmotic tonicity of the cell. The mechanism by which the lipid degradation is influenced is beyond the scope of this present study, but it may involve binding to negatively charged head-groups of phospholipids. Alternatively it may be influenced by controlling the critical calcium concentration of the cytosol through Ca’-ATPase (E.C. 3.6.1.38) in turn controlling lipolytic enzymes (Cheour et al., 1992) or calcium-dependent protein kinase phosphorylation of the PM H+-ATPase (Roberts and Harmon, 1992). The precise way by which calcium controls muskmelon hypodermal mesocarp senescence is thus still obscure and requires additional investigation. References Amon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol., 24: I-15. Bangerth, F., 1979. Calcium related physiological disorders of plants. Annu. Rev. Phytopathol., 17: 97-122. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 72: 248-254.

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Cheour, F., Arul, J., Makhlouf, J. and Willemont, C., 1992. Delay of membrane lipid degradation by calcium treatment during cabbage leaf senescence. Plant Physiol., 100: 1656-1660. Ferguson, I.B., 1984. Calcium in plant senescence and fruit ripening. Plant Cell Environ., 7: 477-489. Fry, SC., 1986. Cross-linking of matrix polymers in the growing cell walls of angiosperms. Armu. Rev. Plant Physiol., 37: 165-186. Knowles, N.R. and Knowles, L.O., 1989. Correlations between electrolyte leakage and degree of saturation of polar lipids from aged potato (Solanum ruberosum L.) tuber tissue. Ann. Bot., 63: 331-338. Lester, G., 1988. Comparisons of ‘Honey Dew’ and netted muskmelon fruit tissues in relation to storage life. HortScience, 23: 180-182. Lester, G.E. and Bruton, B.D., 1986. Relationship of netted muskmelon fruit water loss to postharvest storage life. J. Am. Sot. Hortic. Sci., 111: 727-731. Lester, G.E. and Dunlap, J.R., 1985. Physiological changes during development and ripening of ‘Perlita’ muskmelon fruits. Sci. Hortic., 26: 323-331. Lester, G.E. and Stein, E., 1993. Plasma membrane physicochemical changes during maturation and postharvest storage of muskmelon fruit. J. Am. Sot. Hortic. Sci., 118: 223-227. McCollum, TG., Huber, D.J. and Cantliffe, D.J., 1989. Modification of polyuronides and hemicelluloses during muskmelon fruit softening. Physiol. Plant., 76: 303-308. Paliyath, G. and Droillard, M.J., 1992. The mechanisms of membrane deterioration and disassembly during senescence. Plant Physiol. Biochem., 30: 789-812. Paliyath, G. and Thompson, J.E., 1990. Evidence for early changes in membrane structure during postharvest development of cut carnation flowers. New Phytol., 1114: 555-562. Poovaiah, B.W., Glenn, G.M. and Reddy, A.S.N., 1988. Calcium and fruit softening: Physiology and biochemistry. Hortic. Rev., 10: 107-153. Quinn, P.J., 1981. The fluidity of cell membranes and its regulation. In: D. Noble and TL. Blindell (Editors), Prog. in Biophys. Mol. Biol., Vol. 38. Pergamon Press, New York, NY, pp. l-104. Ranwala, A.P., Suematsu, C. and Masuda, H., 1992. The role of beta-galactosidases in the modification of cell wall components during muskmelon fruit ripening. Plant Physiol., 100: 1318-1325. Roberts, D.M. and Harmon, A.C., 1992. Calcium-modulated proteins: Targets of intracellular calcium signals in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol., 43: 375-414. SAS, 1988. SAS User’s Guide: Statistics. SAS Institute, Cary, N.C. Stow, J., 1993. Effect of calcium ions on apple fruit softening during storage and ripening. Postharvest Biol. Technol., 3: 1-9.