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Lipid Peroxidation in Relation to Declining Vigour in Seeds of Soya (Glycine max L.) and Cabbage (Brassica oleracea L.)
J. PlantPhysiol. Vol. 133. pp. 452-456(1988)
Lipid Peroxidation in Relation to Declining Vigour in Seeds of Soya (Glycine max L.) and Cabbage (Brassica oleracea L.) MILSON
D.
HAILSTONES'~
and MICHAEL T.
SMITH*'~
* Department of Biology, University of Natal, Durban, 4001, Republic of South Africa ** UN/CSIR Research Unit for Plant Growth and Development, Department of Botany, University of Natal, Pietermaritzburg, 3200, Republic of South Africa Received March 23, 1988 . Accepted May 25, 1988
Summary The relationship between the decline of seed polyunsaturated fatty acids and seed vigour was investigated in seeds of cabbage and soya bean. A decline in the levels of polyunsaturated fatty acids of both seed types was clearly associated with a decline in germination percentage. Hydroperoxide levels were highly correlated with percentage germination in both soya (r = -0.88) and cabbage (r = -0.99). In cabbage, hydroperoxide levels were also significantly correlated with seed moisture contents (r = 0.83). These results suggest that a decrease in polyunsaturated fatty acids is associated with declining seed vigour in slowly aged seeds and adds support to a recent suggestion that the mechanisms of rapid and slow ageing may not be equivalent.
Key words: Lipid peroxidation, seed vigour, polyunsaturated fatty acids, soya bean seed, cabbage seed. Abbreviations: BHT, butylated hydroxytoluene; KSCN, potassium thiocyanate; TMAH, tetramethylammonium hydroxide.
Introduction It is well-established in the oil chemistry literature that the spontaneous oxidation of unsaturated fatty acids in lipids produces highly reactive free-radical intermediates, hydroperoxides and secondary products (Swern, 1964; Frankel, 1982). It has been proposed that the damage to membranes, en-
zymes and chromatin during seed ageing may be caused by similar free-radical peroxidation reactions (Koostra and Harrington, 1969; Villiers, 1973; Wilson and McDonald, 1986). Several experimental approaches have been made in an attempt to correlate lipid peroxidation with a decline in seed viability. In the first instance, peroxidation of seed lipids during storage could lead to changes in the relative percentages of constituent fatty acids due to the preferential breakdown of the polyunsaturated fatty acids. Results using this approach have, however, proved contradictory. Priestley and Leopold (1979) reported no changes in the levels of the fatty acids in © 1988 by Gustav Fischer Verlag, Stuttgart
soya beans subjected to accelerated ageing (40 °C, 100 % RH, 5 days), concluding that the oxidation of lipids was not involved in seed ageing. This contrasts with a later study by Stewart and Bewley (1980) where soya beans aged under almost similar conditions (45 °C, 100% RH, 4 days) did show a decline in the relative percentages of the polyunsaturated fatty acids. Apart from examining fatty acid levels, attempts have also been made to correlate declining seed vigour with a decline in antioxidants such as tocopherols. These are especially abundant in seed oils and have the ability to quench free radicals. During this process tocopherols are consumed, so that loss of tocopherols should be one of the manifestations of seed deterioration. However, no correlation between declining vigour and tocopherol levels has been observed. This has been taken as evidence for a lack of involvement of free radicals in seed deterioration (Priestley et al., 1980; Fielding and Goldsworthy, 1980). A third approach would be to measure lipid hydroperoxide levels, but this has yielded equivocal results. Pearce and
453
Lipid peroxidation and seed vigour
Abdel Samad (1980) could detect no hydroperoxides in rapidly aged peanut seeds, while Radrupal and Basu (1982) have reported a highly significant correlation between lipid peroxidation and percentage germination in seeds of wheat and mustard. In the light of the equivocal nature of the above studies, an investigation was undertaken on the relationship between vigour, and the hydroperoxide and fatty acid levels of different cultivars of soya bean and cabbage seeds.
Materials and Methods Seed material Cabbage seeds (Brassica oleracea L.) were obtained from the Mayford Seed Company and stored under ambient conditions. Soya bean seeds (Glycine max L.) were obtained from the Summer Grain Research Centre, Potchefstroom, and stored at 5°C.
Lipid esterification Methyl esters of the fatty acids were obtained using the organic base-catalysed technique of Metcalfe and Wang (1981). An aliquot of lipid was dissolved in 1 ml diethyl ether, and 0.5 ml of IN TMAH was added and the mixture shaken for 1 min at room temperature. One ml of water was then added. An aliquot of the upper ether phase was then analysed using a Pye Series 104 Gas Chromatograph fitted with a flame ionization detector. A 1.5 m x 4 mm I. D. glass column packed with 10 % Silar 5CP on Supelcoport 100 G was operated isothermally at 200°C with nitrogen (60 mi· min - 1) as carner.
Statistical analysis Statistical treatment of the data was carried out according to standard procedures (Campbell, 1974). Area percentages of fatty acids are given as the mean of two determinations.
Results Germination tests In the case of cabbage, 4 replicates of 100 seeds each were germinated in 9 cm Petri dishes on two sheets of Whatman No.1 filter paper and moistened initially with 5 ml of water. Percentage radicle emergence was recorded over 4 days. For soya, 5 replicates of 50 seeds were germinated in 14cm Petri dishes lined with paper towelling. Just sufficient water was added to dampen the paper (50 ml). Percentage radicle emergence was recorded over 4 days.
Moisture contents Two replicates of 1 g of intact seeds from each seed lot were dried in aluminium foil trays at 110°C overnight. Moisture contents were calculated as percentage of seed wet weight.
Hydroperoxide levels and seed vigour In the case of the soya beans, a significant correlation was observed (r = -0.88) between hydroperoxide levels and percentage germination (Table 1). In cabbage the relationship between these two parameters appeared to be non-linear. A regression of log y on log x yielded a highly significant correlation of r = -0.99 (Table 2). In soya, no relationship was observed between moisture content and either percentage germination or hydroperoxide levels (Table 1). In cabbage, the correlation of moisture content with percentage germination was also poor (Table 2). Hydroperoxide levels, however, showed a distinct relationship with moisture content. Again, the relationship appeared to be non-linear, a log y on x plot giving a significant positive correlation (r = 0.83, Table 2).
Lipid extraction Lipids were extracted using the solvent system of Khor and Chan (1985). Five grams of ground seed tissue was extracted with 20 ml of methylene chloride/methanol (2/1 v/v) containing BHT (0.0058 % w/v) for 30 min. Following centrifugation at 1500 xg in a benchtop centrifuge, the solvent was washed with a quarter volume of 1 % NaCl, recentrifuged, the lower phase aspirated and then dried down at 35°C under nitrogen.
Lipid fractionation Lipids were fractionated by silicic acid column chromatography (Beutelmann and Kende, 1977). A sample of lipid dissolved in 1 ml chloroform was placed onto the column and eluted with successive 20 ml volumes of chloroform, acetone and methanol, giving the neutral, glycolipid, and polar lipid fraction respectively. Eluates were dried down at 35°C under nitrogen.
Hydroperoxide determination Hydroperoxide levels were determined using a modification of the test of Stine et al. (1953). Twenty "I of 0.014M ferrous chloride were added to 5 ml of benzene/methanol and shaken. Five ILl of lipid followed by 20 "I of 30 % KSCN were then added with shaking. The absorbance was read at 505 nm against a blank of the reagents.
Changes in polyunsaturated fatty acid levels A decline in the levels of the polyunsaturated fatty acids with declining seed vigour was detected in both seed types. In the total lipid extract of cabbage, linolenic acid (18: 3) de-
Table 1: The percentage germination, hydroperoxide levels, and moisture contents of 6 cultivars of soya bean studied. Cultivar
Germination percentage
Ibis Impala A Impala B Pioneer Hartebeest Impala C
97± 2.1a 95± 8.6 95± 5.9 94± 2.5 89± 10.1 82±22.1
Hydroperoxide levels (absorbance) 0.026±0.02a 0.024±0.02 0.037±0.03 0.050±0.03 0.050±0.03 0.066±0.04
Correlation with: hydroperoxides -0.88* germination * Significant at the 5 % level. a standard error of mean, b mean and range of two determinations.
Moisture content (%w/w) 8.6±0.00b 8.3±0.05 8.9±0.00 9.8±0.05 5.1±0.00 7.9±0.00 -0.25 -0.43
454
MILSON D. HAILSTONES and MICHAEL T. SMITH
Table 2: The percentage germination, hydroperoxide levels, and moisture contents of 6 cultivars of cabbage studied. Cultivar
Germination percentage
Hydroperoxide levels (absorbance)
Moisture content (%w/w)
Golden Acre Glory of Enkhuizen Cape Spitz Savoy Perfection A
100±0.4"
0.072 ± 0.03"
3.59±0.3 b
98± 1.0 82± 1.0
0.104±0.01 0.169±0.02
6.47±0.1 6.77±0.2
76±5.0
0.097±0.01
5.78±0.0
60~--------------------------------~
W
~.
a:
Savoy Perfection B Savoy Perfection C
73±5.0
0.124±0.01
6.18±0.1
39±9.0
0.131±0.02
6.28±0.1
\ :-----------"'---0 3 11-.' . . , -'o~--:::::--:::: ==:::::; 18: 16: 0
,e ........ _
Correlation with: hydroperoxides -0.99*"1 germination
o~
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I
80
I
I
60
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40
20
% RADICLE EMERGENCE
regression log y on log x, excluding Cape Spitz, regression log y on x, * significant at the 5 % level, ** significant at the 0.1 % level, " standard error of mean, b mean and range of two determinations.
Fig. 2: Percentage fatty acid content of polar lipid fraction of cabbage seeds. Abbreviations as for Fig. 1.
1
2
clined by 4.9 % and linoleic acid (18: 2) by 8.4 %. The decline in the levels of both linolenic and linoleic acids was paralleled by a concomitant 9.3 % increase of oleic acid (18: 1, Fig. 1). Changes in the polar lipid fraction were more marked for linoleic acid, which decreased by 15.2 %, while linolenic acid decreased by only 3.7 %. The relative percentage of oleic acid almost doubled, increasing by 16.5 % (Fig. 2). In soya beans, linolenic acid levels of the total lipid fraction declined by 4.3 %, and linoleic acid by 6.8 %. Oleic acid
increased by 10.2 % (Fig. 3). In the polar lipid fraction differences in the levels of the fatty acids were small. Linolenic acid decreased by only 1.3 %. Linoleic acid showed no apparent change, and oleic acid increased by only 0.7 % (Fig. 4), in marked contrast to the changes observed in the total extract.
Discussion Before considering the results of the present study it is necessary to stress two important points. Firstly, many workers have employed accelerated ageing techniques in an attempt to find supportive evidence for
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% RADICLE EMERGENCE Fig. 1: Percentage fatty acid content of total lipid fraction of cabbage seeds. Cultivars: • Golden Acre; • Glory of Enkhuizen; ... Cape Spitz; 0 Savoy Perfection (Lot A); D Savoy Perfection (Lot B); ... Savoy Perfection (Lot C). Fatty acids: not; 16: 0, palmitic; 18: 0, stearic; 18: 1, oleic; 18: 2, linoleic; 18: 3, linolenic.
I
100
90
80
% RADICLE EMERGENCE Fig. 3: Percentage fatty acid content of total lipid fraction of soya bean seeds. Cultivars: • Ibis; • Impala (Lot A); ... Impala (Lot B); 0 Pioneer; 0 Hartebees; ... Impala (Lot C). Fatty acids: 16: 0, palmitic; 18: 0, stearic; 18: 1, oleic; 18: 2, linoleic; 18: 3, linolenic.
Lipid peroxidation and seed vigour 80
455
This contrasts with the results of soya beans where the total lipid extract showed the greatest change. This latter finding is supported by the work of Priestley and Leopold (1983), twho obtained a similar result from soya beans subjected to •__-._.l- -- - -- - - --0 __ - - - - - - - - - - - .. 18: 2 slow ageing. Overall, these results suggest that peroxidation O (3 60 t< of membranes, albeit slight, may be an important aspect of the ageing process, but storage lipids may be an equally >limportant target of peroxidative attacks (Wilson and McDoIt< L_ nald, 1986). u. The experiments reported here provide evidence of an asiI. 2;~ sociation between lipid peroxidation and declining vigour in • __ ... ~'"'o____ - 0 - ____ - - - - - - - - .. 16:0 < W seeds of soya and cabbage. a: < An increase in the level of oxygenated fatty acids has been -reported by Spencer et al. (1973) and Radrupal and Basu (1982) during seed storage. In the latter study, hydroperoxide 0levels were significantly correlated with germination perI 80 centage in seeds of wheat and mustard. These results showed 90 100 that an increase in hydro peroxides may be associated with % RADICLE EMERGENCE declining seed vigour, and implicated lipid peroxidation in Fig. 4: Percentage fatty acid content of polar lipid fraction of soya the ageing process. However, these results contrast with the bean seeds. Abbreviations as for Fig. 3. work of Pearce and Abdel Samad (1980) who could detect no hydroperoxides in aged peanut seeds. While we cannot comment fully on this latter observation it should be noted that lipid peroxidation in seed deterioration. This usually in- differences were found (Smith and West, unpublished revolves holding seed for several days under conditions of high sults) in the lipid hydroperoxides of two cultivars, and harrelative humidity (100 % RH) and temperature (40 cC). vests of peanut seeds using the Stine technique (see materials There is growing support for the opinion that the technique and methods). Studies to date in support of a decline in the levels of polyof rapid ageing may lead to changes within seeds that are not equivalent to those occurring in slow aged seeds (Priestley unsaturated fatty acids with ageing have proved equivocal. Priestley and Leopold (1979) reported no change in the relaand Leopold, 1983; Wilson and McDonald, 1986). In the context of the present argument we wish to define tive levels of polyunsaturated fatty acids in rapidly aged soya slow ageing as that taking place over months or years (rather beans. Stewart and Bewley (1980), also working on rapidly than days) at temperatures at or below 30 cC, and relative aged soya beans, demonstrated a decline in linoleic and lihumidities of below 60 %. nolenic acid, but pointed out that this change had occurred The results obtained from this study were obtained from after the seeds had lost viability. In a later study, Priestley different seed lots which had not been subjected to any ac- and Leopold (1983) examined changes taking place in soya celerated (unnatural) ageing and the vigour differences noted bean seeds stored at 4 cC and low relative humidity for 44 should be seen as the cumulative interaction of maturational, months. For seeds of cultivar Wayne, it was noted that a harvest and post-harvest storage effects. These can be ex12 % decline in viability was accompanied by a 2.5 % decline in the molar percentage of linolenic acid. pected to differ between seed lots. Secondly, due to the complex nature of the seed ageing In the results reported here a 15 % decline in soya bean process, it is difficult to assign causality to anyone pheno- seed viability was accompanied by a 4.3 % decline in the limenon. The relative importance of lipid peroxidation and its nolenic acid levels of the total lipids. While this, and other values, may appear small it should be exact causal relationship to seed deterioration remains to be noted that the area percentages are expressed as part of all the determined. While it has been noted that evidence for free-radical in- constituent fatty acids. If however the value is normalized, volvement in seed ageing is somewhat weak (Hal mer and relative to starting levels, a linolenic acid decline of 47 % is Bewley, 1984) it has been observed that if lipid oxidation is actually recorded. not the cause, evidence for membrane deterioration as an In conclusion, this study has shown that lipid peroxidation important component of seed deterioration continues to was significantly correlated with the decline in vigour in grow (Roberts, 1983). both cabbage and soya bean seeds. Furthermore, decrease in In an attempt to explain the increased leakage of solutes polyunsaturated fatty acids, was also seen to follow this deand electrolytes with seed age, Koostra and Harrington cline. Although these data do not prove causality, they do (1969) proposed that autoxidation of membrane lipids may suggest that lipid peroxidation, directly or indirectly, may lead to increased permeability. It was argued that if mem- playa causal role in seed deterioration. branes were the primary site of peroxidation, a greater deAcknowledgements gree of change would be expected in the polar lipid fraction. The present study indicated that a greater change occurred This work was carried out with the financial assistance of the in the polar lipid fraction of cabbage, but polyunsaturated Council for Scientific and Industrial Research. The help of Dr. G. K. Campbell and Mr. M. Dhlamini is also gratefully acknowledged. fatty acids of the total lipids also showed a marked decline.
456
MILSON D. HAILSTONES and MICHAEL T. SMITH
References BEUTELMANN, P. and H. KENDE: Membrane lipids in senescing flower tissue of Ipomea tricolor. Plant Physio!. 59, 888-893 (1977). CAMPBELL, R. c.: Statistics for Biologists. 2nd Ed. Cambridge University Press (1974). FIELDING, J. L. and A. GOLDSWORTHY: Tocopherol levels and ageing in wheat grains. Ann. Bot. 46, 453 - 456 (1980). FRANKEL, E. N.: Volatile lipid oxidation products. Prog. Lipid Res. 22, 1-33 (1982). HALMER, P. and J. D. BEWLEY: A physiological perspective on seed vigourtesting. Seed Sci. and Techno!. 12, 561-575 (1984). KHOR, H. T. and S. L. CHAN: Comparative studies of three solvent mixtures for the extraction of soybean lipids. J. Am. Oil Chern. Soc. 61, 98-99 (1985). KOOSTRA, P. T. and J. F. HARRINGTON: Biochemical effects of on membrane lipids of Cucumis sativus L. seed. Proc. into Seed Test. Ass. 34,329-340 (1969). METCALFE, L. D. and C. N. WANG: Rapid preparation of fatty acid methyl esters using organic base-catalysed transesterification. J. Chromatog. Sci. 19, 530-535 (1981). PEARCE, R. S. and 1. M. ABDEL SAMAD: Change in fatty acids content of polar lipids during ageing of seeds of peanut (Arachis hypogea L.)J. Exp. Bot. 31,1283-1290 (1980). PRIESTLEY, D. A. and A. C. LEOPOLD: Absence of lipid oxidation during accelerated ageing. Plant Physio!' 63, 726-729 (1979).
-
- Lipid changes during natural ageing of soybean seeds. Physio!. Plant. 59, 467 -470 (1983). PRIESTLEY, D. A., M. A. McBRIDE, and A. C. LEOPOLD: Tocopherol and organic free radical levels in soybean seeds during natural and accelerated ageing. Plant Physio!' 66, 715-719 (1980). RADRUPAL, A. B. and R. N. BASU: Lipid peroxidation and membrane damage in deteriorating wheat and mustard seeds. Indian J. Exp. Bot. 20, 465-470 (1982). ROBERTS, E. H.: Loss of viability during storage. In: THOMSON, J. R. (ed.): Advances in research and technology of seeds, part 8, 9-34, Pudoc, Wageningen (1983). SPENCER, G. F., F. R. EARLE, 1. A. WOLFF, and W. H. TALLENT: Oxygenation of unsaturated fatty acids in seeds during storage. Chern. and Phys. Lipids, 10,191-202 (1973). STEWART, R. R. C. and J. D. BEWLEY: Lipid peroxidation associated with accelerated ageing of soybean axes. Plant Physio!. 65, 245 - 248 (1980). STINE, M. c., H. A. HARLAND, S. T. COULTER, and R. JENESS: A modified peroxide test for detection of lipid oxidation in dairy products. J. Dairy Sci. 37,202-208 (1954). SWERN, D.: Bailey's industrial oil and fat products, 3rd edition. Interscience Publishers, New York (1964). VILLIERS, T. A.: Ageing and longevity of seeds in field conditions. In: HEYDECKER, W. (ed.): Seed ecology, 265-288. Butterworths, London (1973). WILSON, D. O. and M. B. McDONALD: The lipid peroxidation model of seed ageing. Seed Sci. and Techno!. 14,269-300 (1986).
Lipid Peroxidation in Relation to Declining Vigour in Seeds of Soya (Glycine max L.) and Cabbage (Brassica oleracea L.) MILSON
D.
HAILSTONES'~
and MICHAEL T.
SMITH*'~
* Department of Biology, University of Natal, Durban, 4001, Republic of South Africa ** UN/CSIR Research Unit for Plant Growth and Development, Department of Botany, University of Natal, Pietermaritzburg, 3200, Republic of South Africa Received March 23, 1988 . Accepted May 25, 1988
Summary The relationship between the decline of seed polyunsaturated fatty acids and seed vigour was investigated in seeds of cabbage and soya bean. A decline in the levels of polyunsaturated fatty acids of both seed types was clearly associated with a decline in germination percentage. Hydroperoxide levels were highly correlated with percentage germination in both soya (r = -0.88) and cabbage (r = -0.99). In cabbage, hydroperoxide levels were also significantly correlated with seed moisture contents (r = 0.83). These results suggest that a decrease in polyunsaturated fatty acids is associated with declining seed vigour in slowly aged seeds and adds support to a recent suggestion that the mechanisms of rapid and slow ageing may not be equivalent.
Key words: Lipid peroxidation, seed vigour, polyunsaturated fatty acids, soya bean seed, cabbage seed. Abbreviations: BHT, butylated hydroxytoluene; KSCN, potassium thiocyanate; TMAH, tetramethylammonium hydroxide.
Introduction It is well-established in the oil chemistry literature that the spontaneous oxidation of unsaturated fatty acids in lipids produces highly reactive free-radical intermediates, hydroperoxides and secondary products (Swern, 1964; Frankel, 1982). It has been proposed that the damage to membranes, en-
zymes and chromatin during seed ageing may be caused by similar free-radical peroxidation reactions (Koostra and Harrington, 1969; Villiers, 1973; Wilson and McDonald, 1986). Several experimental approaches have been made in an attempt to correlate lipid peroxidation with a decline in seed viability. In the first instance, peroxidation of seed lipids during storage could lead to changes in the relative percentages of constituent fatty acids due to the preferential breakdown of the polyunsaturated fatty acids. Results using this approach have, however, proved contradictory. Priestley and Leopold (1979) reported no changes in the levels of the fatty acids in © 1988 by Gustav Fischer Verlag, Stuttgart
soya beans subjected to accelerated ageing (40 °C, 100 % RH, 5 days), concluding that the oxidation of lipids was not involved in seed ageing. This contrasts with a later study by Stewart and Bewley (1980) where soya beans aged under almost similar conditions (45 °C, 100% RH, 4 days) did show a decline in the relative percentages of the polyunsaturated fatty acids. Apart from examining fatty acid levels, attempts have also been made to correlate declining seed vigour with a decline in antioxidants such as tocopherols. These are especially abundant in seed oils and have the ability to quench free radicals. During this process tocopherols are consumed, so that loss of tocopherols should be one of the manifestations of seed deterioration. However, no correlation between declining vigour and tocopherol levels has been observed. This has been taken as evidence for a lack of involvement of free radicals in seed deterioration (Priestley et al., 1980; Fielding and Goldsworthy, 1980). A third approach would be to measure lipid hydroperoxide levels, but this has yielded equivocal results. Pearce and
453
Lipid peroxidation and seed vigour
Abdel Samad (1980) could detect no hydroperoxides in rapidly aged peanut seeds, while Radrupal and Basu (1982) have reported a highly significant correlation between lipid peroxidation and percentage germination in seeds of wheat and mustard. In the light of the equivocal nature of the above studies, an investigation was undertaken on the relationship between vigour, and the hydroperoxide and fatty acid levels of different cultivars of soya bean and cabbage seeds.
Materials and Methods Seed material Cabbage seeds (Brassica oleracea L.) were obtained from the Mayford Seed Company and stored under ambient conditions. Soya bean seeds (Glycine max L.) were obtained from the Summer Grain Research Centre, Potchefstroom, and stored at 5°C.
Lipid esterification Methyl esters of the fatty acids were obtained using the organic base-catalysed technique of Metcalfe and Wang (1981). An aliquot of lipid was dissolved in 1 ml diethyl ether, and 0.5 ml of IN TMAH was added and the mixture shaken for 1 min at room temperature. One ml of water was then added. An aliquot of the upper ether phase was then analysed using a Pye Series 104 Gas Chromatograph fitted with a flame ionization detector. A 1.5 m x 4 mm I. D. glass column packed with 10 % Silar 5CP on Supelcoport 100 G was operated isothermally at 200°C with nitrogen (60 mi· min - 1) as carner.
Statistical analysis Statistical treatment of the data was carried out according to standard procedures (Campbell, 1974). Area percentages of fatty acids are given as the mean of two determinations.
Results Germination tests In the case of cabbage, 4 replicates of 100 seeds each were germinated in 9 cm Petri dishes on two sheets of Whatman No.1 filter paper and moistened initially with 5 ml of water. Percentage radicle emergence was recorded over 4 days. For soya, 5 replicates of 50 seeds were germinated in 14cm Petri dishes lined with paper towelling. Just sufficient water was added to dampen the paper (50 ml). Percentage radicle emergence was recorded over 4 days.
Moisture contents Two replicates of 1 g of intact seeds from each seed lot were dried in aluminium foil trays at 110°C overnight. Moisture contents were calculated as percentage of seed wet weight.
Hydroperoxide levels and seed vigour In the case of the soya beans, a significant correlation was observed (r = -0.88) between hydroperoxide levels and percentage germination (Table 1). In cabbage the relationship between these two parameters appeared to be non-linear. A regression of log y on log x yielded a highly significant correlation of r = -0.99 (Table 2). In soya, no relationship was observed between moisture content and either percentage germination or hydroperoxide levels (Table 1). In cabbage, the correlation of moisture content with percentage germination was also poor (Table 2). Hydroperoxide levels, however, showed a distinct relationship with moisture content. Again, the relationship appeared to be non-linear, a log y on x plot giving a significant positive correlation (r = 0.83, Table 2).
Lipid extraction Lipids were extracted using the solvent system of Khor and Chan (1985). Five grams of ground seed tissue was extracted with 20 ml of methylene chloride/methanol (2/1 v/v) containing BHT (0.0058 % w/v) for 30 min. Following centrifugation at 1500 xg in a benchtop centrifuge, the solvent was washed with a quarter volume of 1 % NaCl, recentrifuged, the lower phase aspirated and then dried down at 35°C under nitrogen.
Lipid fractionation Lipids were fractionated by silicic acid column chromatography (Beutelmann and Kende, 1977). A sample of lipid dissolved in 1 ml chloroform was placed onto the column and eluted with successive 20 ml volumes of chloroform, acetone and methanol, giving the neutral, glycolipid, and polar lipid fraction respectively. Eluates were dried down at 35°C under nitrogen.
Hydroperoxide determination Hydroperoxide levels were determined using a modification of the test of Stine et al. (1953). Twenty "I of 0.014M ferrous chloride were added to 5 ml of benzene/methanol and shaken. Five ILl of lipid followed by 20 "I of 30 % KSCN were then added with shaking. The absorbance was read at 505 nm against a blank of the reagents.
Changes in polyunsaturated fatty acid levels A decline in the levels of the polyunsaturated fatty acids with declining seed vigour was detected in both seed types. In the total lipid extract of cabbage, linolenic acid (18: 3) de-
Table 1: The percentage germination, hydroperoxide levels, and moisture contents of 6 cultivars of soya bean studied. Cultivar
Germination percentage
Ibis Impala A Impala B Pioneer Hartebeest Impala C
97± 2.1a 95± 8.6 95± 5.9 94± 2.5 89± 10.1 82±22.1
Hydroperoxide levels (absorbance) 0.026±0.02a 0.024±0.02 0.037±0.03 0.050±0.03 0.050±0.03 0.066±0.04
Correlation with: hydroperoxides -0.88* germination * Significant at the 5 % level. a standard error of mean, b mean and range of two determinations.
Moisture content (%w/w) 8.6±0.00b 8.3±0.05 8.9±0.00 9.8±0.05 5.1±0.00 7.9±0.00 -0.25 -0.43
454
MILSON D. HAILSTONES and MICHAEL T. SMITH
Table 2: The percentage germination, hydroperoxide levels, and moisture contents of 6 cultivars of cabbage studied. Cultivar
Germination percentage
Hydroperoxide levels (absorbance)
Moisture content (%w/w)
Golden Acre Glory of Enkhuizen Cape Spitz Savoy Perfection A
100±0.4"
0.072 ± 0.03"
3.59±0.3 b
98± 1.0 82± 1.0
0.104±0.01 0.169±0.02
6.47±0.1 6.77±0.2
76±5.0
0.097±0.01
5.78±0.0
60~--------------------------------~
W
~.
a:
Savoy Perfection B Savoy Perfection C
73±5.0
0.124±0.01
6.18±0.1
39±9.0
0.131±0.02
6.28±0.1
\ :-----------"'---0 3 11-.' . . , -'o~--:::::--:::: ==:::::; 18: 16: 0
,e ........ _
Correlation with: hydroperoxides -0.99*"1 germination
o~
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100
0.83*2 -0.32
I
I
80
I
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60
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40
20
% RADICLE EMERGENCE
regression log y on log x, excluding Cape Spitz, regression log y on x, * significant at the 5 % level, ** significant at the 0.1 % level, " standard error of mean, b mean and range of two determinations.
Fig. 2: Percentage fatty acid content of polar lipid fraction of cabbage seeds. Abbreviations as for Fig. 1.
1
2
clined by 4.9 % and linoleic acid (18: 2) by 8.4 %. The decline in the levels of both linolenic and linoleic acids was paralleled by a concomitant 9.3 % increase of oleic acid (18: 1, Fig. 1). Changes in the polar lipid fraction were more marked for linoleic acid, which decreased by 15.2 %, while linolenic acid decreased by only 3.7 %. The relative percentage of oleic acid almost doubled, increasing by 16.5 % (Fig. 2). In soya beans, linolenic acid levels of the total lipid fraction declined by 4.3 %, and linoleic acid by 6.8 %. Oleic acid
increased by 10.2 % (Fig. 3). In the polar lipid fraction differences in the levels of the fatty acids were small. Linolenic acid decreased by only 1.3 %. Linoleic acid showed no apparent change, and oleic acid increased by only 0.7 % (Fig. 4), in marked contrast to the changes observed in the total extract.
Discussion Before considering the results of the present study it is necessary to stress two important points. Firstly, many workers have employed accelerated ageing techniques in an attempt to find supportive evidence for
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% RADICLE EMERGENCE Fig. 1: Percentage fatty acid content of total lipid fraction of cabbage seeds. Cultivars: • Golden Acre; • Glory of Enkhuizen; ... Cape Spitz; 0 Savoy Perfection (Lot A); D Savoy Perfection (Lot B); ... Savoy Perfection (Lot C). Fatty acids: not; 16: 0, palmitic; 18: 0, stearic; 18: 1, oleic; 18: 2, linoleic; 18: 3, linolenic.
I
100
90
80
% RADICLE EMERGENCE Fig. 3: Percentage fatty acid content of total lipid fraction of soya bean seeds. Cultivars: • Ibis; • Impala (Lot A); ... Impala (Lot B); 0 Pioneer; 0 Hartebees; ... Impala (Lot C). Fatty acids: 16: 0, palmitic; 18: 0, stearic; 18: 1, oleic; 18: 2, linoleic; 18: 3, linolenic.
Lipid peroxidation and seed vigour 80
455
This contrasts with the results of soya beans where the total lipid extract showed the greatest change. This latter finding is supported by the work of Priestley and Leopold (1983), twho obtained a similar result from soya beans subjected to •__-._.l- -- - -- - - --0 __ - - - - - - - - - - - .. 18: 2 slow ageing. Overall, these results suggest that peroxidation O (3 60 t< of membranes, albeit slight, may be an important aspect of the ageing process, but storage lipids may be an equally >limportant target of peroxidative attacks (Wilson and McDoIt< L_ nald, 1986). u. The experiments reported here provide evidence of an asiI. 2;~ sociation between lipid peroxidation and declining vigour in • __ ... ~'"'o____ - 0 - ____ - - - - - - - - .. 16:0 < W seeds of soya and cabbage. a: < An increase in the level of oxygenated fatty acids has been -reported by Spencer et al. (1973) and Radrupal and Basu (1982) during seed storage. In the latter study, hydroperoxide 0levels were significantly correlated with germination perI 80 centage in seeds of wheat and mustard. These results showed 90 100 that an increase in hydro peroxides may be associated with % RADICLE EMERGENCE declining seed vigour, and implicated lipid peroxidation in Fig. 4: Percentage fatty acid content of polar lipid fraction of soya the ageing process. However, these results contrast with the bean seeds. Abbreviations as for Fig. 3. work of Pearce and Abdel Samad (1980) who could detect no hydroperoxides in aged peanut seeds. While we cannot comment fully on this latter observation it should be noted that lipid peroxidation in seed deterioration. This usually in- differences were found (Smith and West, unpublished revolves holding seed for several days under conditions of high sults) in the lipid hydroperoxides of two cultivars, and harrelative humidity (100 % RH) and temperature (40 cC). vests of peanut seeds using the Stine technique (see materials There is growing support for the opinion that the technique and methods). Studies to date in support of a decline in the levels of polyof rapid ageing may lead to changes within seeds that are not equivalent to those occurring in slow aged seeds (Priestley unsaturated fatty acids with ageing have proved equivocal. Priestley and Leopold (1979) reported no change in the relaand Leopold, 1983; Wilson and McDonald, 1986). In the context of the present argument we wish to define tive levels of polyunsaturated fatty acids in rapidly aged soya slow ageing as that taking place over months or years (rather beans. Stewart and Bewley (1980), also working on rapidly than days) at temperatures at or below 30 cC, and relative aged soya beans, demonstrated a decline in linoleic and lihumidities of below 60 %. nolenic acid, but pointed out that this change had occurred The results obtained from this study were obtained from after the seeds had lost viability. In a later study, Priestley different seed lots which had not been subjected to any ac- and Leopold (1983) examined changes taking place in soya celerated (unnatural) ageing and the vigour differences noted bean seeds stored at 4 cC and low relative humidity for 44 should be seen as the cumulative interaction of maturational, months. For seeds of cultivar Wayne, it was noted that a harvest and post-harvest storage effects. These can be ex12 % decline in viability was accompanied by a 2.5 % decline in the molar percentage of linolenic acid. pected to differ between seed lots. Secondly, due to the complex nature of the seed ageing In the results reported here a 15 % decline in soya bean process, it is difficult to assign causality to anyone pheno- seed viability was accompanied by a 4.3 % decline in the limenon. The relative importance of lipid peroxidation and its nolenic acid levels of the total lipids. While this, and other values, may appear small it should be exact causal relationship to seed deterioration remains to be noted that the area percentages are expressed as part of all the determined. While it has been noted that evidence for free-radical in- constituent fatty acids. If however the value is normalized, volvement in seed ageing is somewhat weak (Hal mer and relative to starting levels, a linolenic acid decline of 47 % is Bewley, 1984) it has been observed that if lipid oxidation is actually recorded. not the cause, evidence for membrane deterioration as an In conclusion, this study has shown that lipid peroxidation important component of seed deterioration continues to was significantly correlated with the decline in vigour in grow (Roberts, 1983). both cabbage and soya bean seeds. Furthermore, decrease in In an attempt to explain the increased leakage of solutes polyunsaturated fatty acids, was also seen to follow this deand electrolytes with seed age, Koostra and Harrington cline. Although these data do not prove causality, they do (1969) proposed that autoxidation of membrane lipids may suggest that lipid peroxidation, directly or indirectly, may lead to increased permeability. It was argued that if mem- playa causal role in seed deterioration. branes were the primary site of peroxidation, a greater deAcknowledgements gree of change would be expected in the polar lipid fraction. The present study indicated that a greater change occurred This work was carried out with the financial assistance of the in the polar lipid fraction of cabbage, but polyunsaturated Council for Scientific and Industrial Research. The help of Dr. G. K. Campbell and Mr. M. Dhlamini is also gratefully acknowledged. fatty acids of the total lipids also showed a marked decline.
456
MILSON D. HAILSTONES and MICHAEL T. SMITH
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- Lipid changes during natural ageing of soybean seeds. Physio!. Plant. 59, 467 -470 (1983). PRIESTLEY, D. A., M. A. McBRIDE, and A. C. LEOPOLD: Tocopherol and organic free radical levels in soybean seeds during natural and accelerated ageing. Plant Physio!' 66, 715-719 (1980). RADRUPAL, A. B. and R. N. BASU: Lipid peroxidation and membrane damage in deteriorating wheat and mustard seeds. Indian J. Exp. Bot. 20, 465-470 (1982). ROBERTS, E. H.: Loss of viability during storage. In: THOMSON, J. R. (ed.): Advances in research and technology of seeds, part 8, 9-34, Pudoc, Wageningen (1983). SPENCER, G. F., F. R. EARLE, 1. A. WOLFF, and W. H. TALLENT: Oxygenation of unsaturated fatty acids in seeds during storage. Chern. and Phys. Lipids, 10,191-202 (1973). STEWART, R. R. C. and J. D. BEWLEY: Lipid peroxidation associated with accelerated ageing of soybean axes. Plant Physio!. 65, 245 - 248 (1980). STINE, M. c., H. A. HARLAND, S. T. COULTER, and R. JENESS: A modified peroxide test for detection of lipid oxidation in dairy products. J. Dairy Sci. 37,202-208 (1954). SWERN, D.: Bailey's industrial oil and fat products, 3rd edition. Interscience Publishers, New York (1964). VILLIERS, T. A.: Ageing and longevity of seeds in field conditions. In: HEYDECKER, W. (ed.): Seed ecology, 265-288. Butterworths, London (1973). WILSON, D. O. and M. B. McDONALD: The lipid peroxidation model of seed ageing. Seed Sci. and Techno!. 14,269-300 (1986).