Microsomal Membrane Changes during the Ripening of Apple. Fruit'. Received for publication November 1, 1982 and in revised form June 3, 1983. SUSAN ...
Plant Physiol. (1983) 73, 636-638 0032-0889/83/73/0636/03/$00.50/0
Microsomal Membrane Changes during the Ripening of Apple
Fruit' Received for publication November 1, 1982 and in revised form June 3, 1983
SUSAN LURIE AND RUTH BEN-ARIE Division of Fruit and Vegetable Storage, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel ABSTRACT The changes in leakage and viscosity of microsomal membranes from apples (Malus sylvestris cv Calville de San Sauveur) at different stages of ripening were examined. These changes were correlated with those in the lipid composition of the membranes, sterols, phospholipids, and fatty acids of the phospholipids. The greatest changes in membrane properties occurred as the fruit reached its cfimacteric and this corresponded with a change in the sterol:phospholipid ratio in the membranes. Changes were also found in fatty acid unsaturation level, but primarily in the postclimacteric stage of ripening.
During fruit ripening, progressive loss of membrane function occurs. It has been suggested that the changes in membrane structures at the onset of ripening lead to increased permeability of membranes resulting in a decompartmentalization of cellular components and increased catabolic processes causing eventual senescence (17). In climacteric fruits, this becomes pronounced in the climacteric and postclimacteric stages. The passive permeability characteristics of lipoprotein membranes are mainly influenced by the lipid component (7) and a change in permeability should be reflected by a change in either the composition or arrangement of membrane lipids. This, in turn, should be seen as a change in the membrane fluidity. There are several reports indicating a correspondence between changes in membrane function and changes in membrane fluidity (8, 9, 18). There is also evidence of changes in membrane viscositythe converse of fluidity-as plant tissues senesce (3, 13), but no work has been done on changes in ripening fruit. This paper describes the lipid composition of the microsomal fraction of apple fruit as it ripens, and compares it with changes in the membrane viscosity and permeability.
periments were performed (A and B) at different harvest dates and for each stage of ripeness two apples were sampled from each experiment. The apples were peeled, the core removed, and 50 g of flesh was ground in a blender for 1 min in 100 ml of 250 mm sucrose, 25 mm Tris, 3 mM EDTA, and 1 mm DTT. The slurry was filtered through six layers of cheesecloth, brought to pH 7.2 with NaOH, and centrifuged for 15 min at 13,000g. The supernatant was centrifuged for 30 min at 80,000g and the pellet resuspended in 1 ml of 10 mm Tris-HCI (pH 7.2), 1 mm DTT (11). Protein was determined by the method of Bradford (5). Microviscosity was measured in an Elscint polarized fluorometer using the fluorescent membrane probe 1,6-diphenyl- 1,3,5hexatriene (DPH) after the method of Shinitzky and Barenholz (19). A sample containing 10 to 20 ,ug protein/ml was incubated with 1 ,uM DPH in 0.1 M phosphate buffer (pH 7.2) for 20 min in a shaker at room temperature. The polarized fluorescence was then read at 25°C. The formula for calculating the apparent microviscosity (X7) measured by the fluorescent probe DPH was:
2p_ _ 0.46 - p
111/12-I 0.73 - 0.27 111/12
where p is the fluorescence polarization and I 1/12 is the ratio of the light emission which is oriented parallel or perpendicular to the direction of polarization of the excitation light. A full derivation of this formula can be found in Shintzky and Barenholz
(19).
After measuring membrane microviscosity, the microsomal fraction was extracted for lipids using a chloroform:methanol (8:5, v/v) mixture according to the method of Renkonen et al. (16). The total membrane lipid fraction obtained was dried in a rotavapor (Buchi) redissolved in 1 ml hexane, and stored under N2 at -200C. Aliquots were taken for total phospholipid and sterol contents. The mole content of phospholipids was determined by phosphorus analysis after conversion to phosphoric acid with 70% HC104 at 1900C for 60 min by the method of Bottcher et al. (4). Sterol MATERIALS AND METHODS in the lipid extracts was determined colorimetrically by the Apples, Malus sylvestris cv Calville de San Sauveur, were used method of Chiamori and Henri (6), using cholesterol as a stanfor the experiments. Individual apples were placed in I-L con- dard. tainers at 20°C with an air flow of 60 ml/min; respiration and A final aliquot of the total lipid fraction was used for fatty acid ethylene were measured daily. Ethylene was measured on a FID analysis. It was run on a precoated silica gel G TLC plate (Merck), gas chromatograph (Packard) with an alumina column. At the 250 ,m in thickness. The plates were run in hexane:ether:acetic appropriate stage of ripeness, as shown by the fruit's ethylene acid (80:20:1) for 2 h and then air-dried. The origin containing production pattern, apples were removed for analysis. Two ex- the phospholipids was removed, washed with 5 ml chloroform:methanol (1:1), and then dried under N2. The phospholipid 'Supported by a grant from the United States-Israel Binational Agri- fraction was prepared for fatty acid analysis by methylation in 4 cultural Research and Development Fund (BARD). Contribution from ml of 5% H2SO4 in methanol (v/v) held at 800C for 1 h. The the Agricultural Research Organization, The Volcani Center, Bet Dagan, fatty acid methyl esters were partitioned into hexane and anaIsrael. No. 533-E, 1982 series. lyzed using a Packard FID gas chromatograph equipped for 636
637
MEMBRANES AND APPLE FRUIT RIPENING Table 1. Ethylene Production by Apples at Different Stages ofRipening and the Microviscosity of the Microsomal Membranes
Exp. Stage Stage
C2H4 Microviscosity Leakage 2h ~~~~~~~~~~~~i
Early climacteric
Midclimacteric Peak climacteric
Postclimacteric
A B A B A B A B
gl/kg
poise
%
apple- h 54 51 61 67 100 102 55 27
3.17 3.75 3.11 3.67 4.05 4.30 4.45 4.67
20.5 20.6
23.2 24.2 23.9 25.7
Table II. Effect of Stage of Ripeness ofApple on the Sterol and Phospholipid Contents of Microsomal Membranes Data are from the apples in experiment A. Sterol Stage of Phospholipid St/PL Ripeness (St) (PL) ratio mol/50 gfresh wt 6.77 1.08 0.16 Early climacteric 0.16 1.39 8.91 Midclimacteric 0.23 1.48 6.43 Peak climacteric 2.87 8.75 0.33 Postclimacteric temperature programming. Separations were done on a 1.82-m-
long 2-mm i.d. glass column (Supelco) packed with GP 10% SP2330 on 100/ 120 Chromosorb W. The heating program was 160 to 200°C, 5°C/min. Reference methylated fatty acids were run for identification purposes. Leakage of potassium was measured in apple discs (1 cm diameter) with 8 discs weighing about 2 g. These were incubated in 25 ml of 0.6 M mannitol for 2 h and the leakage into the medium was measured with a potassium ion electrode (Orion). The discs were frozen, thawed, and boiled for 20 min to extract total potassium, which was measured in the same manner. RESULTS The course of apple fruit ripening was followed by monitoring the ethylene production of the fruit. The pattern was that of a typical climacteric fruit: ethylene increased to a peak production and then declined (Table I). At the different stages ofthis ripening
the viscosity and semipermeable properties of microsomal membranes were measured (Table 1). There are no viscosity changes in the membranes until the fruit reaches its peak climacteric stage. Then there is a sudden increase in membrane viscosity and this increases further as the fruit enters the postclimacteric phase. In parallel, the membranes become more permeable to potassium ions as the climacteric is reached. The changes in lipid components of the membranes show a similar picture. The ratio of sterol to phospholipid (Table II) does not alter in the preclimacteric and midclimacteric stages, but increases in the climacteric and continues to rise in the postclimacteric. This change in ratio is caused by an increase in the sterol content of the membranes rather than a decrease in the phospholipid fraction. There are also changes occurring in the fatty acids of the membrane phospholipids, as shown in Table III. The two major changes are in palmytic (16:0) and linoleic (18:2) acids, the former increasing as the fruit ripens and the latter decreasing. In experiment A, the major change in these two fatty acids occurs between the climacteric and postclimacteric stages. In experiment B, the change begins before the climacteric peak and continues into the postclimacteric. By expressing the fatty acid data as various ratios found in the literature (Table III), a clearer pattern emerges. The ratio of linolenic (18:3) to linoleic (18:2) acid does not change consistently as the fruit ripens. The ratio of saturated to unsaturated fatty acids remains relatively constant until the postclimacteric stage, when there is a large increase. This is a reflection not just of an increase in palmytic acid as a percent of the total, but also of decreases in all three of the unsaturated 18 carbon chain fatty acids. Perhaps the best reflection of the changes in the components of the phospholipid fatty acids is the double bond index. This shows a steady decrease from stage to stage of fruit ripening, which means a steady decrease in the degree of unsaturation of the fatty acids. process,
DISCUSSION In apple fruits, there are changes in the lipid components of the microsomal membranes as the fruit ripens. The most dramatic change takes place between the midclimacteric and climacteric peaks. This is when there is a sharp increase in membrane viscosity, and the membrane sterol to phospholipid ratio mirrors this change. The fact that there are no large changes at earlier stages of ripening suggests that these membrane changes are a consequence, and not a cause, of the ripening process, as had been suggested previously (17). However, we were working with a crude microsomal membrane fraction and cannot rule
Table III. Effect of Stage of Ripeness ofApples on the Fatty Acid Content of the Phospholipids in the
Microsomal Membranes Stage of Ripeness
16:0
18:0
Fatty Acids 18:1 18:2
18:3
18.3 18.2
% total area
Saturated Unsaturated
Double Bond
Index'
ratio
A
Preclimacteric Midclimacteric Peak climacteric Postclimacteric
19 17 17
29
6 6 3 4
0.338 0.255 0.413 0.321
0.45 0.41 0.41 0.70
1.42 1.33 1.23 1.16
50 15 0.309 17 46 0.374 44 15 0.325 41 0.299 13 no. double bonds) + 100.
0.38 0.40 0.46 0.72
1.48 1.45 1.32 1.22
2 3 1 1
47 47 47 39
16 12 20 13
B 17 6 Preclimacteric 4 Midclimacteric 16 2 Peak climacteric 20 2 28 Postclimacteric a Double bond index = 2: (peak area % x
3 2 1 1
638
LURIE AND BEN-ARIE
out the possibility that ripening might be brought about by changes in a specific membrane fraction. Ripening of fruits has many processes in common with senescence of other plant organs. From the literature, it appears that there are similarities as well as differences in the behavior of the membrane lipids. In work done on senescing flower tissue, it was found that phospholipid content began to decline even before signs of senescence were evident (1, 2). In flower petals, this decrease in phospholipid content was accompanied by an increase in membrane permeability (20). Borochov et al. (3) found that as flowers aged the membrane viscosity increased and this was correlated with an increase in the free sterol:phospholipid ratio. This was due to a decrease in the phospholipid content, while the sterol fraction remained essentially constant. It is of interest to note that their ratios are higher than those found in apple fruit, varying from 0.28 in young petals to 0.44 in aged petals. It has been well documented that as seeds age there is an increase in solute leakage and a decrease in phospholipid content (10, 14, 15). Thompson's group, using X-ray diffraction, demonstrated in senescing bean cotyledons, as well as in algae, that senescence of membranes involves a phase change whereby the proportion of gel to liquid-gel lipid increases progressively (12, 13, 21). This altered physical state of lipid represents a substantial increase in membrane viscosity and contributes to a loss of membrane function in senescing tissue. In algal cultures, the unsaturated to saturated fatty acid ratio of the membrane lipid, while fluctuating with age, did not show any consistent trend that could be related to the change in lipid membrane state. On the other hand, in bean cotyledons the neutral lipids, including sterol esters, were deemed to be the cause of the membrane phase changes. The general conclusion appears to be that the major changes in the lipid components of the membranes during senescence are in the sterol:phospholipid ratio. This may be due to loss of phospholipids-as in flowers and seeds-or, as we found in ripening apple fruit, to increase in sterol content. There have been thus far only negative reports on the involvement of the phospholipid fatty acids in the senescence-related changes in membrane function or structure. We have found, however, that in the transition from climacteric to postclimacteric, the increased saturation of fatty acids also contributes to increased membrane viscosity. In terms of the double bond index, there are changes throughout the ripening process and, in terms of
Plant Physiol. Vol. 73, 1983
saturated-unsaturated acids ratio, there is a large increase in ratio at later stages of ripening. Therefore, we conclude that both the sterols and the phospholipids play an important role in the membrane changes that accompany ripening. 1.
2.
3. 4.
5. 6. 7.
8. 9. 10. 11.
12.
13. 14. 15.
16. 17.
18. 19. 20. 21.
LITERATURE CITED BEUTELMANN P, H KENDE 1977 Membrane lipids in senescing flower tissue of Ipomoea tricolor. Plant Physiol 59: 888-893 BOROCHoV A, A HALEVY, H BOROCHOV, M SHINITZKY 1978 Microviscosity of plasmalemmas in rose petals as affected by age and environmental factors. Plant Physiol 61: 812-815 BOROCHoV A, A HALEVY, M SHINITZKY 1976 Increase in microviscosity with aging in protoplast plasmalemma of rose petals. Nature 263: 158-159 BOTTCHER G, C VAN GENT, C PRIES 1961 A rapid and sensitive sub micro phosphorus determination. Anal Chim Acta 24: 203-204 BRADFORD M 1976 A new method of protein determination. Anal Biochem 72: 248-251 CHIAMORI I, R HENRI 1959 Study of method of determination of total cholesterol and cholesterol esters. Am J Clin Pathol 31: 305-309 COLLANDER R 1959 In FC Steward, ed, Plant Physiology, A Treatise, Vol 2. Academic Press, New York, pp 3-162 ELETR S, D ZAKIM, D VESSEY 1973 A spin label study of the role of the phospholipids in the regulation of membrane bound microsomal enzymes. J Mol Biol 78: 351-362 ESFAHANI M, A LIMBRICK, S KNUTrON, T OKA, S WAKIL 1971 The molecular organization of lipids in the membrane of E. coli: phase transitions. Proc Natl Acad Sci USA 68: 3181-3184 HODGES TK, RT LEONARD 1973 Purification of a plasma membrane bound adenosine triphosphate from plant roots. Methods Enzymol 4: 392-406 KOOSTRA P, J HARRINGTON 1969 Biochemical effects of age on membranal lipids of Cucumis sativus L. seed. Proc Int Seed Test Assoc 34: 329 McKERSIE B, J THOMPSON 1977 Lipid crystallization in senescent membranes from cotyledons. Plant Physiol 59: 803-807 McKERSIE B, J THOMPSON 1979 Phase properties of senescing plant membranes: role of the neutral lipids. Biochim Biophys Acta 550: 48-58 POWELL A, S MATTHEWS 1977 Deteriorative changes in pea seeds (Pisum sativum L.) stored in humid or dry conditions. J Exp Bot 28: 227-236 POWELL A, S MATTHEWS 1981 Association of phospholipid changes with early stages in seed aging. Ann Bot 47: 709-712 RENKONEN 0, T KOSUNEN, 0 RENKONEN 1963 Extractions of serum inositides and other phosphatides. Ann Med Exp Biol Fenniae (Helsinki) 41: 375-381 SACHER IA 1967 Senescence. Symp Soc Exp Biol 21: 269-273 SHECHTER E, L LETELLIER, T GULIK-KRzYwIcKI 1974 Relations between structure and function in cytoplasmic membrane vesicles isolated from an E. coli fatty acid auxotroph. Eur J Biochem 49: 61-76 SHINITZKY M, Y BARENHOLZ 1978 Fluidity parameters of lipid regions determined by fluorescence polarization. Biochim Biophys Acta 515: 367-394 SUTTLE J, H KENDE 1980 Ethylene action and loss of membrane integrity during petal senescence in Tradescantia. Plant Physiol 65: 1067-1072 THOMPSON J, C MAYFIELD, W INNiss, D BUTLER, J KRUUW 1978 Senescencerelated changes in the lipid transition temperature of microsomal membranes from algae. Physiol Plant 43: 114-120
