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Effect of the Embryonic Axis on the Development of Glyoxysomes, Plastids and Mitochondria in Cotyledons of Germinated Cucumber Seeds
J.PlantPhysiol. Vol. 132.pp. 279-283 {1988}
Effect of the Embryonic Axis on the Development of Glyoxysomes, Plastids and Mitochondria in Cotyledons of Germinated Cucumber Seeds 1 YUKIO MOROHASHI )
and HISASHI MATSUSHIMA2)
I) Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183 2) Department of Regulation Biology, Faculty of Science, Saitama University, Urawa 337 Oapan) Received March 31 , 1987 . Accepted August 19, 1987
Summary The effect of the presence of the embryonic axis was examined on the following parameters of activities of cellular organelles in dark-grown cucumber cotyledons: activities of malate synthase, isocitrate lyase and 3-hydroxy-acyl-CoA dehydrogenase with glyoxysomes, ribulose bis-phosphate carboxylase activity and rate of chlorophyll formation with plastids, and activities of some respiratory enzymes (succinate and malate dehydrogenase and cytochrome oxidase) and respiratory activities (0 2 uptake and its cyanide-sensitivity, and respiratory control) with mitochondria. The development of these parameters depends upon the presence of the axis, and benzyladenine can bring about a full replacement of the action of the axis on the development of all the parameters except the two, respiratory control and cyanide-sensitivity of mitochondrial respiration. As to these two parameters, benzyladenine showed little effect on their development. On the basis of these results, the role of the axis in the development of the organelles in cotyledons is discussed.
Key words: CucumlS sativus, axis-effect, henzyladenine, glyoxysome, mitochondrion, plastid. Abbreviations: BA, benzyladenine; CN, cyanide; Rubisco, ribulose bisphosphate carboxylase_ Introduction The cotyledons of oil-storing seeds of some plant species, such as cucumber, watermelon and sunflower, undergo drastic changes in function and in cell structure after germination in the light; the storage function is gradually replaced by the photosynthetic one (Kagawa et al. 1973). The development or transformation of plastids and glyoxysomes is deeply involved in this process. But the understanding of the mechanism controlling these changes is still rather incomplete. It is known that the phytochrome system is involved (Schopfer and Apel 1983). In sunflower or watermelon cotyledons, however, asubstantial increase in activity of enzymes of plastids and microbodies has been observed even in the absence of light, if cytokinins are supplied to the cotyledons (Theimer et al. 1976, Longo et al. 1979 a, b, Lampugnani et al. 1980). In addition to plastids and microbodies, r'§"1
1988 by Gust av F Ischer Verlag, Stuttgart
activities of mitochondria in excised watermelon cotyledons also increase when the cotyledons are treated with cytokinins (Longo et al. 1979 a, b). On the other hand, it has been suggested that the greening of dark-grown cucumber cotyledons after the transfer to light is stimulated by cytokinins which are transported from the axis (presumably from the radicle) (Dei 1978). Taking these observations into consideration, it is possible that the embryonic axis has critical effects on the development of the organelles in cotyledons through supplying cytokinins. However, most of the studies on the role of the axis in the development of the organelles, though not many in number, have dealt mainly with glyoxysomes in realtion to mobilization of reserve lipids (Davies and Slack 1981) and have paid little attention to the other organelles. In the present study, we examined the effects of the axis on the development of glyoxysomes, plastids and mitochondria in cucumber cotyledons.
280
YUKIO MOROHASHI and HISASHI MArSUSHIMA
Materials and Methods
bisphosphate and [14C]NaHC0 3 (Amersham) at 30°C according to Peterson and Huffaker (1975). Chlorophyll was determined by the method of Arnon (1949). Protein was estimated according to Breidenbach et al. (1966).
Plant materials Cucumber seeds (Cucumis sativus L., cv. Shinfushinarijibae) were soaked in water for 15 h at 25°C, then put on filter paper in Petri dishes wetted with water, and incubated at 28 °C in the dark. The cotyledons were excised from the embryonic axis on the 2nd day after imbibition and further incubated on filter paper wetted with water, or BA-solution (0.3 mM). In some experiments, a part (1110 or less) of the seed coat was stripped off from the cotyledons when the axis was removed, and incubated on wet filter paper. The BAtreated, or husked, cotyledons were subjected to analysis one day after the treatments. When activities of chlorophyll formation and of Rubisco were examined, cotyledons were transferred to light (white fluorescent lamp, 2000 lux) at indicated times, kept for 4 h at 25 oC, and then subjected to analyses.
Preparation ofenzymes and mitochondnal fractions Washed mitochondrial fractions were prepared from cotyledons as described previously (Morohashi 1980), except that the bovine serum albumin concentration was raised to 0.3 % (w/v). The glyoxysomal enzymes were extracted as mentioned before (Morohashi 1986). For the extraction of Rubisco, cotyledons were disrupted in a Polytron homogenizer in Tris-S0 4 buffer (0.2M, pH 8.0). The homogenate was centrifuged at 35,000 g for 15 min and the resulting supernatant was used as a source of the enzyme.
Results Effect ofaxis-removal Glyoxysomal enzymes: Malate synthase, 3-hydroxyacylCoA dehydrogenase and isocitrate lyase activities in darkgrown, attached cucumber cotyledons greatly increased during the experimental period (Fig. 1). When the axis was removed, the development of malate synthase and isocitrate lyase activities was retarded. These results are in good agreement with the findings of Davies and Chapman (1979). On the other hand, as to 3-hydroxyacyl-CoA dehydrogenase, excision of the axis showed little effects on the development of its activity. Thus, enzymes housed in the same organelle respond differently to the removal of the axis. Similar situations have been reported with castor bean endosperm; the development of isocitrate lyase was slower in embryo-less endosperm, while the development of 3-hydroxyacyl-CoA dehydrogenase was unaffected by the removal of the embryo (Marriott and Northcote 1975). At present there is no definite explanation for such different responses to the axis-removal.
Assays The rate of respiration of mitochondria was assayed polarographically with succinate as substrate (10 mM) as previously mentioned (Morohashi 1980). The activities of enzymes, except for Rubisco, were determined spectrophotometrically at 30°C. The methods employed were essentially the same as described in the literature: succinate dehydrogenase (Pierpoint 1963); malate dehydrogenase and cytochrome oxidase (Nawa and Asahi 1971); 3-hydroxyacyl-CoA dehydrogenase (Overath et al. 1967); malate synthase (Cooper and Beevers 1969); isocitrate lyase (Dixon and Kornberg 1959). Rubisco activity was determined by measuring the acid-stable radioactivity produced in the reaction between ribulose
Rubisco activity and chlorophyll formation Cotyledons from dark-grown, 2-day-old seedlings could produce little chlorophyll when transferred to light, but in older seedlings the ability to form chlorophyll markedly increased (Fig. 2). In detached cotyledons, the development of the ability of chlorophyll synthesis was severely retarded. The increase in Rubisco activity, which was observed in attached cotyledons, also was delayed when the axis was excised (Fig. 3).
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Fig. 1: Changes in activities of malate synthase (A), 3-hydroxyacyl-CoA dehydrogenase (B) and isocitrate lyase (C) in darkgrown cucumber cotyledons. The mean of, and the difference between (vertical bar), two replicates are shown .• , attached cotyledons; 0, detached cotyledons; A, detached and BA-treated cotyledons; 6, detached and husked cotyledons.
Organelle development in cucumber cotyledons
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Fig. 2: Chlorophyll content after 4 h treatment with white light in cucumber cotyledons grown in the dark for 2, 3 and 4 days. Symbols are the same as in Fig. 1.
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Fig. 4: Changes in state 3 respiration rate (A) and respiratory control (RC) ratio (B) of washed mitochondria isolated from dark-grown cucumber cotyledons. Shown are the mean and the SE of the mean (vertical bar) of 3 replicates. Substrate, 10 mM succinate. Symbols are the same as in Fig. 1. The specific rates of respiration for washed mitochondria from 3-day-old attached, detached, detached and BAtreated, and detached and partially husked cotyledons were 121,69, 97 and 92 nmol O 2 min -\ (mg proteint \, respectively.
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Fig. 3: Changes in Rubisco activity in dark-grown cucumber cotyledons. The mean of, and the difference between (vertical bar), of two replicates are shown. Symbols are the same as in Fig. 1.
Mitochondrial respiration and enzymes Mitochondrial activities (state 3 respiration, respiratory control) markedly increased during the period from 2 to 4
days after imbibition in attached cotyledons, but in detached ones the increases were severely inhibited (Fig. 4). In Fig. 4 the respiration activity was expressed on a per cotyledon basis, because it is possible that washed mitochondria isolated from cotyledons treated in different ways are contaminated with other organelles and organelle fragments to various degrees and that this may have some effect on the specific activities of mitochondrial respiration. But, even if the activities were expressed on a per protein weight basis, situations were not much altered (see the legend for Fig. 4). Activities of mitochondrial enzymes examined (succinate and malate dehydrogenase, and cytochrome oxidase) also increased in attached cotyledons during the experimental period (Fig. 5). In detached cotyledons, the increase was more or less delayed (Fig. 5). The sensitivity of state 3 respiration to eN (1 mM) was lower in mitochondria isolated from detached cotyledons than those isolated from attached ones (Table 1). In the presence of eN plus salicylhydroxamate, an inhibitor of the alternative respiration, oxygen uptake was severely (93 -100 %) inhibited in all samples, indicating the eN-insensitive respiration is mainly due to the alternative pathway.
282
YUKIO MOROHASHI
and
HISASHI MATSUSHIMA
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Fig. 5: Changes in activities of succinate (A) and malate (B) dehydrogenase, and cytochrome oxidase (C) in dark-grown cucumber cotyledons. The mean of, and the difference between (vertical bar), of two replicates are shown. Symbols are the same as in Fig. 1.
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Table 1: Cyanide-sensitivity of washed mitochondria isolated from dark-grown cucumber cotyledons. The mean of, and the difference between (in parenthesis), two replicates are shown. Cotyledons 2-day-old, attached 3-day-old, attached detached detached, husked detached, BA-treated
Inhibition by CN (%) 81 (2) 89 (1) 76 (1)
72 (5)
Table 2: Fresh weight of dark-grown cucumber cotyledons. Cotyledons
Fresh weight (g/IOO cotyledons)
2-day-old, attached 3-day-old, attached detached detached, husked detached, BA-treated
1.65 1.85 1.63 1.83
2.01
73 (3)
Effect of BA-treatment and husking In BA-treated, or partially husked, detached cotyledons, the values of all parameters examined, except for respiratory control ratio and CN-sensitivity of mitochondrial respiration, were increased, although the degrees of increase were variable depending on the parameters and on the treatments (Figs. 1-5)_On the other hand, respiratory control ratio and CN-sensitivity were little changed irrespective of the treatments (Fig. 4 and Table I}. Discussion Attached cotyledons of cucumber grow well and the testa ruptures during the period between the 2nd and the 3rd day after imbibition, while detached cotyledons expand little and the testa remains unruptured. This is reflected in the changes in fresh weight of cotyledons (Table2). When a part of the testa is removed from detached cotyledons, the cotyledons can expand as well as attached ones (Table 2). It seems as if the testa is a mechanical barrier to the expansion growth of cotyledons. BA-treated cotyledons are endowed with the ability to expand (Hutton et al. 1982, Longo et al. 1982, Table 2), and the testa is actually ruptured in BA-treated axis-
removed seeds. It is thought that, in germinated seeds, cytokinins are transported to the cotyledons from the embryonic radicle and that they induce the growth of the cotyledons (van Staden 1983). This is supposed to be one of the reasons why attached cotyledons can grow well and why detached ones cannot. It has also been suggested that the testa of cucumber seeds is a barrier for oxygen permeability (Davies and Slack 1981). Therefore, the presence of the unruptured testa may result in inadequate supply of oxygen to cotyledons, in addition to the mechanical hindrance of expansiongrowth of cotyledons. One of, or both of, these factors might be responsible for the delay of growth of cotyledons and for the retardation of the development of physiological and biochemical activities in detached cotyledons. The development of glyoxysomal enzymes is accelerated in husked detached cotyledons (Fig. I). Davies and Chapman (1979) have also reported similar results. It seems, therefore, that under the conditions where the cotyledons can grow well the development of glyoxysomal enzymes occurs in cotyledons without the axis attached; in other words, if the cotyledons are allowed to expand and! or if they are adequately supplied with oxygen, cytokinins from the radicle are not necessarily required for the development of glyoxysomal enzymes. On the other hand, the removal of the testa could only partially restore the retardation of the development of activ-
Organelle development in cucumber cotyledons ities of chlorophyll synthesis and Rubisco (Figs. 2 and 3). But BA-treatment could almost fully restore these activities (Figs. 2 and 3). Therefore, it is supposed that the hindrance of the expansion-growth of cotyledons and/or the poor supply of oxygen are not the only cause for the retardation of the plastid development; the supply of cytokinins to cotyledons from the axis seems to be indispensable for the plastids development in cotyledons. Respiratory and enzymatic activities of mitochondria in husked, or BA-treated, detached cotyledons were increased to the levels of those in attached ones (Figs. 4 and 5). However, respiratory control ratio and eN-sensitivity of mitochondrial respiration in detached cotyledons remained unchanged after these treatments (Fig.4 and Table 1). Therefore, the retardation of the development of some qualitative properties of mitochondria in detached cotyledons cannot be explained only in terms of the absence of cytokinins. Other factor(s) controlling mitochondrial development in cotyledons must be taken into consideration. Some metabolites accumulate in detached cotyledons (Davies and Slack 1981). It is possible that some accumulated metabolites, such as free fatty acids (Baddeley and Hanson 1967), may have some inhibitory effect on mitochondrial activities when detached cotyledons are macerated. However, even though the concentration of bovine serum albumin was raised to 2 % in preparing mitochondria, it has no effect on the respiratory control ratio and on eN-sensitivity (data not shown). Furthermore, when mitochondria are prepared from a mixture consisting of the same number of attached and detached cotyledons, their respiratory control ratio lays between respective values for mitochondria prepared from the two cotyledons (data not shown). Therefore, the possibility is unlikely that the difference in mitochondrial properties between attached and detached cotyledons is due to any accumulated cytoplasmic factor(s). It is also possible that hormonal substances other than cytokinins are transported from the axis to the cotyledons and that these factors might be involved in the development of mitochondria in cotyledons. However, treatments of detached cotyledons with IAA, GA 3, or the combinations ofIAA, GA 3 , and BA, did not show any effect on respiratory control ratio and eN-sensitivity (data not shown). Further investigations are necessary to elucidate what factor(s) causes the difference in mitochondrial properties between attached and detached cotyledons. In conclusion, the axis-effects on the development of plastids and glyoxysomes in cotyledons of germinated cucumber seeds seem to be explained in terms of cytokininsupply by the axis to the cotyledons, although in the case of glyoxysomal development the involvement of cytokinins is supposed to be of an indirect nature. On the other hand, concerning mitochondrial development, factor(s) other than cytokinin-supply must be taken into consideration.
References ARNON, D. I.: Copper enzymes in isolated chloroplasts. Plant Physiol. 24, 1-15 (1949). BADDELEY, M. S. and J. B. HANSON: Uncoupling of energy-linked functions of corn mitochondna by linoleic acid and mono methyldecenylsuccinic acid. Plant Physiol. 42, 1702 -1710 (1967).
283
BREIDENBACH, R. W., P. CASTELFRANCO, and C. PETERSON: Biogenesis of mitochondria in germinating peanut cotyledons. Plant Physiol. 41, 803-809 (1966). COOPER, T. G. and H. BEEVERS: Mitochondria and glyoxysomes from castor bean endosperm. Enzyme constituents and catalytic capacity. J. BioI. Chern. 244, 3507 -3513 (1969). DAVIES, H. V. andJ. M. CHAPMAN: The control offood mobilisation in seeds of Cucumis sativus L. 1. The influence of embryonic axis and testa on protein and lipid degradation. Planta 146, 579 - 584 (1979). DAVIES, H. V. and P. T. SLACK: The control of food mobilization in seeds of dicotyledonous plants. New Phytol. 88, 41-51 (1981). DEI, M.: Inter-organ control of greening in etiolated cucumber cotyledons. Physiol. Plantarum 43, 94-98 (1978). DIXON, G. H. and H. L. KORNBERG: Assay methods for key enzymes of the glyoxylate cycle. Biochem. J. 72, 3p (1959). HUTTON, M. J., J. VAN STADEN, and J. E. DAVEY: Cytokinins in germinating seeds of Phaseolus vulgaris L. Ann. Bot. 49, 685-691 (1982). KAGAWA, T., D. 1. MCGREGOR, and H. BEEVERS: Development of enzymes in the cotyledons of watermelon seedlings. Plant Physiol. 51,66-71 (1973). LAMPUGNANI, M. G., P. MARTELLINI, O. SERVETTAZ, and C. P. LONGO: Interaction between benzyladenine and white light on excised watermelon cotyledons. Plant Sci. Lett. 18, 351- 358 (1980). LONGO, G. P., M. PEDRETII, G. ROSSI, and C. P. LONGO: Effect of benzyladenine on the development of plastids and microbodies in excised watermelon cotyledons. Planta 145, 209-217 {1979 a). - - - - Benzyladenine stimulates the development of mitochondria in watermelon cotyledons. Plant Sci. Lett. 14,213-223 (1979 b). LONGO, G. P., G. ROSSI, R. FANTELLI, M. G. BUSSOLATI, and C. P. LONGO: Expansion growth is not the main driving force for cytokinin-promoted development of cotyledons. Plant Sci. Lett. 25, 271-279 (1982). MARRIOTT, K. M. and D. H. NORTHCOTE: The induction of enzyme activity in the endosperm of germinating castor bean seeds. Biochern. J. 152, 65-70 (1975). MOROHASHI, Y.: Development of mitochondrial activity in pea cotyledons following imbibition: Influence of the embryonic axis. J. Exp. Bot. 31,805-812 (1980). - Patterns of mitochondrial development in reserve tissues of germinated seeds. Physiol. Plantarum 66,653-658 (1986). NAWA, Y. and T. ASAHI: Rapid development of mitochondria in pea cotyledons during the early state of germination. Plant Physiol. 48,671-674 (1971). OVERATH, P., E. M. RAUFUSS, W. STOFFEL, and W. ECKER: The induction of enzymes of fatty acid degradation in E. coli. Biochem. Biophys. Res. Commun. 29, 28 - 33 (1967). PETERSON, L. W. and R. C. HUFFAKER: Loss of ribulose 1,5-diphosphate carboxylase and increase in proteolytic activity during senescence of detached primary barley leaves. Plant Physiol. 55, 1009-1015 (1975). PIERPOINT, W. S.: The distribution of succinate dehydrogenase and malate dehydrogenase among components of tobacco-leaf extracts. Biochem. J. 88, 120-125 (1963). SCHOPFER, P. and K. APEL: Intracellular photomorphogenesis. In: SCHROPSHIRE, W. and H. MOHR (eds.): Encyclopedia of Plant Physiology, New Series, Vol. 16A, 258-288. Springer-Verlag, New York (1983). THEIMER, R. R., G. ANDING, and P. MATZNER: Kinetin action on the development of microbody enzymes in sunflower cotyledons in the dark. Planta 128,41-47 (1976). VAN STADEN, J.: Seeds and cytokinins. Physiol. Plantarum 58, 340-346 (1983).
Effect of the Embryonic Axis on the Development of Glyoxysomes, Plastids and Mitochondria in Cotyledons of Germinated Cucumber Seeds 1 YUKIO MOROHASHI )
and HISASHI MATSUSHIMA2)
I) Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183 2) Department of Regulation Biology, Faculty of Science, Saitama University, Urawa 337 Oapan) Received March 31 , 1987 . Accepted August 19, 1987
Summary The effect of the presence of the embryonic axis was examined on the following parameters of activities of cellular organelles in dark-grown cucumber cotyledons: activities of malate synthase, isocitrate lyase and 3-hydroxy-acyl-CoA dehydrogenase with glyoxysomes, ribulose bis-phosphate carboxylase activity and rate of chlorophyll formation with plastids, and activities of some respiratory enzymes (succinate and malate dehydrogenase and cytochrome oxidase) and respiratory activities (0 2 uptake and its cyanide-sensitivity, and respiratory control) with mitochondria. The development of these parameters depends upon the presence of the axis, and benzyladenine can bring about a full replacement of the action of the axis on the development of all the parameters except the two, respiratory control and cyanide-sensitivity of mitochondrial respiration. As to these two parameters, benzyladenine showed little effect on their development. On the basis of these results, the role of the axis in the development of the organelles in cotyledons is discussed.
Key words: CucumlS sativus, axis-effect, henzyladenine, glyoxysome, mitochondrion, plastid. Abbreviations: BA, benzyladenine; CN, cyanide; Rubisco, ribulose bisphosphate carboxylase_ Introduction The cotyledons of oil-storing seeds of some plant species, such as cucumber, watermelon and sunflower, undergo drastic changes in function and in cell structure after germination in the light; the storage function is gradually replaced by the photosynthetic one (Kagawa et al. 1973). The development or transformation of plastids and glyoxysomes is deeply involved in this process. But the understanding of the mechanism controlling these changes is still rather incomplete. It is known that the phytochrome system is involved (Schopfer and Apel 1983). In sunflower or watermelon cotyledons, however, asubstantial increase in activity of enzymes of plastids and microbodies has been observed even in the absence of light, if cytokinins are supplied to the cotyledons (Theimer et al. 1976, Longo et al. 1979 a, b, Lampugnani et al. 1980). In addition to plastids and microbodies, r'§"1
1988 by Gust av F Ischer Verlag, Stuttgart
activities of mitochondria in excised watermelon cotyledons also increase when the cotyledons are treated with cytokinins (Longo et al. 1979 a, b). On the other hand, it has been suggested that the greening of dark-grown cucumber cotyledons after the transfer to light is stimulated by cytokinins which are transported from the axis (presumably from the radicle) (Dei 1978). Taking these observations into consideration, it is possible that the embryonic axis has critical effects on the development of the organelles in cotyledons through supplying cytokinins. However, most of the studies on the role of the axis in the development of the organelles, though not many in number, have dealt mainly with glyoxysomes in realtion to mobilization of reserve lipids (Davies and Slack 1981) and have paid little attention to the other organelles. In the present study, we examined the effects of the axis on the development of glyoxysomes, plastids and mitochondria in cucumber cotyledons.
280
YUKIO MOROHASHI and HISASHI MArSUSHIMA
Materials and Methods
bisphosphate and [14C]NaHC0 3 (Amersham) at 30°C according to Peterson and Huffaker (1975). Chlorophyll was determined by the method of Arnon (1949). Protein was estimated according to Breidenbach et al. (1966).
Plant materials Cucumber seeds (Cucumis sativus L., cv. Shinfushinarijibae) were soaked in water for 15 h at 25°C, then put on filter paper in Petri dishes wetted with water, and incubated at 28 °C in the dark. The cotyledons were excised from the embryonic axis on the 2nd day after imbibition and further incubated on filter paper wetted with water, or BA-solution (0.3 mM). In some experiments, a part (1110 or less) of the seed coat was stripped off from the cotyledons when the axis was removed, and incubated on wet filter paper. The BAtreated, or husked, cotyledons were subjected to analysis one day after the treatments. When activities of chlorophyll formation and of Rubisco were examined, cotyledons were transferred to light (white fluorescent lamp, 2000 lux) at indicated times, kept for 4 h at 25 oC, and then subjected to analyses.
Preparation ofenzymes and mitochondnal fractions Washed mitochondrial fractions were prepared from cotyledons as described previously (Morohashi 1980), except that the bovine serum albumin concentration was raised to 0.3 % (w/v). The glyoxysomal enzymes were extracted as mentioned before (Morohashi 1986). For the extraction of Rubisco, cotyledons were disrupted in a Polytron homogenizer in Tris-S0 4 buffer (0.2M, pH 8.0). The homogenate was centrifuged at 35,000 g for 15 min and the resulting supernatant was used as a source of the enzyme.
Results Effect ofaxis-removal Glyoxysomal enzymes: Malate synthase, 3-hydroxyacylCoA dehydrogenase and isocitrate lyase activities in darkgrown, attached cucumber cotyledons greatly increased during the experimental period (Fig. 1). When the axis was removed, the development of malate synthase and isocitrate lyase activities was retarded. These results are in good agreement with the findings of Davies and Chapman (1979). On the other hand, as to 3-hydroxyacyl-CoA dehydrogenase, excision of the axis showed little effects on the development of its activity. Thus, enzymes housed in the same organelle respond differently to the removal of the axis. Similar situations have been reported with castor bean endosperm; the development of isocitrate lyase was slower in embryo-less endosperm, while the development of 3-hydroxyacyl-CoA dehydrogenase was unaffected by the removal of the embryo (Marriott and Northcote 1975). At present there is no definite explanation for such different responses to the axis-removal.
Assays The rate of respiration of mitochondria was assayed polarographically with succinate as substrate (10 mM) as previously mentioned (Morohashi 1980). The activities of enzymes, except for Rubisco, were determined spectrophotometrically at 30°C. The methods employed were essentially the same as described in the literature: succinate dehydrogenase (Pierpoint 1963); malate dehydrogenase and cytochrome oxidase (Nawa and Asahi 1971); 3-hydroxyacyl-CoA dehydrogenase (Overath et al. 1967); malate synthase (Cooper and Beevers 1969); isocitrate lyase (Dixon and Kornberg 1959). Rubisco activity was determined by measuring the acid-stable radioactivity produced in the reaction between ribulose
Rubisco activity and chlorophyll formation Cotyledons from dark-grown, 2-day-old seedlings could produce little chlorophyll when transferred to light, but in older seedlings the ability to form chlorophyll markedly increased (Fig. 2). In detached cotyledons, the development of the ability of chlorophyll synthesis was severely retarded. The increase in Rubisco activity, which was observed in attached cotyledons, also was delayed when the axis was excised (Fig. 3).
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Fig. 1: Changes in activities of malate synthase (A), 3-hydroxyacyl-CoA dehydrogenase (B) and isocitrate lyase (C) in darkgrown cucumber cotyledons. The mean of, and the difference between (vertical bar), two replicates are shown .• , attached cotyledons; 0, detached cotyledons; A, detached and BA-treated cotyledons; 6, detached and husked cotyledons.
Organelle development in cucumber cotyledons
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Fig. 2: Chlorophyll content after 4 h treatment with white light in cucumber cotyledons grown in the dark for 2, 3 and 4 days. Symbols are the same as in Fig. 1.
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Fig. 4: Changes in state 3 respiration rate (A) and respiratory control (RC) ratio (B) of washed mitochondria isolated from dark-grown cucumber cotyledons. Shown are the mean and the SE of the mean (vertical bar) of 3 replicates. Substrate, 10 mM succinate. Symbols are the same as in Fig. 1. The specific rates of respiration for washed mitochondria from 3-day-old attached, detached, detached and BAtreated, and detached and partially husked cotyledons were 121,69, 97 and 92 nmol O 2 min -\ (mg proteint \, respectively.
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Fig. 3: Changes in Rubisco activity in dark-grown cucumber cotyledons. The mean of, and the difference between (vertical bar), of two replicates are shown. Symbols are the same as in Fig. 1.
Mitochondrial respiration and enzymes Mitochondrial activities (state 3 respiration, respiratory control) markedly increased during the period from 2 to 4
days after imbibition in attached cotyledons, but in detached ones the increases were severely inhibited (Fig. 4). In Fig. 4 the respiration activity was expressed on a per cotyledon basis, because it is possible that washed mitochondria isolated from cotyledons treated in different ways are contaminated with other organelles and organelle fragments to various degrees and that this may have some effect on the specific activities of mitochondrial respiration. But, even if the activities were expressed on a per protein weight basis, situations were not much altered (see the legend for Fig. 4). Activities of mitochondrial enzymes examined (succinate and malate dehydrogenase, and cytochrome oxidase) also increased in attached cotyledons during the experimental period (Fig. 5). In detached cotyledons, the increase was more or less delayed (Fig. 5). The sensitivity of state 3 respiration to eN (1 mM) was lower in mitochondria isolated from detached cotyledons than those isolated from attached ones (Table 1). In the presence of eN plus salicylhydroxamate, an inhibitor of the alternative respiration, oxygen uptake was severely (93 -100 %) inhibited in all samples, indicating the eN-insensitive respiration is mainly due to the alternative pathway.
282
YUKIO MOROHASHI
and
HISASHI MATSUSHIMA
c
B
A
4
'0 E c:
)-
-
I-
->
Fig. 5: Changes in activities of succinate (A) and malate (B) dehydrogenase, and cytochrome oxidase (C) in dark-grown cucumber cotyledons. The mean of, and the difference between (vertical bar), of two replicates are shown. Symbols are the same as in Fig. 1.
I-
ul
o
2
3
4 TIM E
AFT E R I M BIB I T ION
(d a y s)
Table 1: Cyanide-sensitivity of washed mitochondria isolated from dark-grown cucumber cotyledons. The mean of, and the difference between (in parenthesis), two replicates are shown. Cotyledons 2-day-old, attached 3-day-old, attached detached detached, husked detached, BA-treated
Inhibition by CN (%) 81 (2) 89 (1) 76 (1)
72 (5)
Table 2: Fresh weight of dark-grown cucumber cotyledons. Cotyledons
Fresh weight (g/IOO cotyledons)
2-day-old, attached 3-day-old, attached detached detached, husked detached, BA-treated
1.65 1.85 1.63 1.83
2.01
73 (3)
Effect of BA-treatment and husking In BA-treated, or partially husked, detached cotyledons, the values of all parameters examined, except for respiratory control ratio and CN-sensitivity of mitochondrial respiration, were increased, although the degrees of increase were variable depending on the parameters and on the treatments (Figs. 1-5)_On the other hand, respiratory control ratio and CN-sensitivity were little changed irrespective of the treatments (Fig. 4 and Table I}. Discussion Attached cotyledons of cucumber grow well and the testa ruptures during the period between the 2nd and the 3rd day after imbibition, while detached cotyledons expand little and the testa remains unruptured. This is reflected in the changes in fresh weight of cotyledons (Table2). When a part of the testa is removed from detached cotyledons, the cotyledons can expand as well as attached ones (Table 2). It seems as if the testa is a mechanical barrier to the expansion growth of cotyledons. BA-treated cotyledons are endowed with the ability to expand (Hutton et al. 1982, Longo et al. 1982, Table 2), and the testa is actually ruptured in BA-treated axis-
removed seeds. It is thought that, in germinated seeds, cytokinins are transported to the cotyledons from the embryonic radicle and that they induce the growth of the cotyledons (van Staden 1983). This is supposed to be one of the reasons why attached cotyledons can grow well and why detached ones cannot. It has also been suggested that the testa of cucumber seeds is a barrier for oxygen permeability (Davies and Slack 1981). Therefore, the presence of the unruptured testa may result in inadequate supply of oxygen to cotyledons, in addition to the mechanical hindrance of expansiongrowth of cotyledons. One of, or both of, these factors might be responsible for the delay of growth of cotyledons and for the retardation of the development of physiological and biochemical activities in detached cotyledons. The development of glyoxysomal enzymes is accelerated in husked detached cotyledons (Fig. I). Davies and Chapman (1979) have also reported similar results. It seems, therefore, that under the conditions where the cotyledons can grow well the development of glyoxysomal enzymes occurs in cotyledons without the axis attached; in other words, if the cotyledons are allowed to expand and! or if they are adequately supplied with oxygen, cytokinins from the radicle are not necessarily required for the development of glyoxysomal enzymes. On the other hand, the removal of the testa could only partially restore the retardation of the development of activ-
Organelle development in cucumber cotyledons ities of chlorophyll synthesis and Rubisco (Figs. 2 and 3). But BA-treatment could almost fully restore these activities (Figs. 2 and 3). Therefore, it is supposed that the hindrance of the expansion-growth of cotyledons and/or the poor supply of oxygen are not the only cause for the retardation of the plastid development; the supply of cytokinins to cotyledons from the axis seems to be indispensable for the plastids development in cotyledons. Respiratory and enzymatic activities of mitochondria in husked, or BA-treated, detached cotyledons were increased to the levels of those in attached ones (Figs. 4 and 5). However, respiratory control ratio and eN-sensitivity of mitochondrial respiration in detached cotyledons remained unchanged after these treatments (Fig.4 and Table 1). Therefore, the retardation of the development of some qualitative properties of mitochondria in detached cotyledons cannot be explained only in terms of the absence of cytokinins. Other factor(s) controlling mitochondrial development in cotyledons must be taken into consideration. Some metabolites accumulate in detached cotyledons (Davies and Slack 1981). It is possible that some accumulated metabolites, such as free fatty acids (Baddeley and Hanson 1967), may have some inhibitory effect on mitochondrial activities when detached cotyledons are macerated. However, even though the concentration of bovine serum albumin was raised to 2 % in preparing mitochondria, it has no effect on the respiratory control ratio and on eN-sensitivity (data not shown). Furthermore, when mitochondria are prepared from a mixture consisting of the same number of attached and detached cotyledons, their respiratory control ratio lays between respective values for mitochondria prepared from the two cotyledons (data not shown). Therefore, the possibility is unlikely that the difference in mitochondrial properties between attached and detached cotyledons is due to any accumulated cytoplasmic factor(s). It is also possible that hormonal substances other than cytokinins are transported from the axis to the cotyledons and that these factors might be involved in the development of mitochondria in cotyledons. However, treatments of detached cotyledons with IAA, GA 3, or the combinations ofIAA, GA 3 , and BA, did not show any effect on respiratory control ratio and eN-sensitivity (data not shown). Further investigations are necessary to elucidate what factor(s) causes the difference in mitochondrial properties between attached and detached cotyledons. In conclusion, the axis-effects on the development of plastids and glyoxysomes in cotyledons of germinated cucumber seeds seem to be explained in terms of cytokininsupply by the axis to the cotyledons, although in the case of glyoxysomal development the involvement of cytokinins is supposed to be of an indirect nature. On the other hand, concerning mitochondrial development, factor(s) other than cytokinin-supply must be taken into consideration.
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