Planta (1985)164:534-539
P l a n t a 9 Springer-Verlag 1985
Ribulose-l,5-bisphosphate carboxylase and fruit set or degeneration of unpollinated ovaries of Pisum sativum L. J. Carbonell and J.L. Garcia-Martinez Instituto de Agroquimica y Tecnologia de Alimentos, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain
The polypeptide patterns obtained by sodium dodecylsulphate-polyacrylamide gel electrophoresis of undigested and autodigested extracts from pea (P&um sativum L.) ovaries at the early stages of development or degeneration have been studied. Development of unpollinated ovaries was stimulated by application of different plant growth regulators (gibberellic acid, 2,4-dichlorophenoxyacetic acid, and N6-benzyladenine) or by plant topping. Polypeptide bands of similar mobility to ribulose-l,5-bisphosphate carboxylase (RuBPCase) subunits (16 and 55 kDa) could be detected in all types of autodigested extracts from stimulated ovaries. However these bands were absent in electrophoretic patterns of autodigested extracts from unstimulated ovaries after 3 d post anthesis and in patterns of autodigested mixtures of these extracts with either those from stimulated ovaries or those from unstimulated ovaries before day 3. These observations indicate that a proteolytic activity which promotes the hydrolysis of RuBPCase appears in unstimulated ovaries about 3 d after anthesis. This event coincides with the loss of the capacity of unpollinated ovaries to develop in response to gibberellic acid and with the degeneration of the ovary wall. Abstract.
-
Key words: Fruit development- Fruit set - Ovary Pisum (RuBPCase and fruit set) - Proteolysis - Ribulose-l,5-bisphosphate carboxylase.
Introduction
Fruit set and development of unpollinated pea ovaries can be induced by applying several types Abbreviations: BA=N6-benzyladenine; 2,4-D=2,4-dichloro-
phenoxyacetic acid; GA 3= gibberellic acid; RuBPCase = ribulose-1,5-bisphosphate carboxylase; SDS-PAGE=sodium dodecylsulphate-polyacrylamide gel electrophoresis
of plant growth regulators (auxin, gibberellin, and cytokinin) to ovaries (Garcia-Martinez and Carbonell 1980) or by topping the plants (Carbonell and Garcia-Martinez 1980). Unstimulated ovaries grow only slightly after anthesis and, approx. 3 d post anthesis their capacity to develop in response to gibberellic acid (GAg) is lost. Simultaneously the endocarp of the ovary wall degenerates (Vercher et al. 1984). The absence of biochemical studies on the degenerative process of unstimulated ovaries prompted us to investigate proteolytic activity in the ovaries by autodigestion and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude extracts (Ragster and Chrispeels 1981). The results show that the degeneration of unstimulated ovaries is associated with the presence of a new proteolytic activity which hydrolyzes ribulose-l,5-bisphosphate-carboxylase (RuBPCase). Some interesting correlations between morphological and biochemical observations are also shown. M a t e r i a l and m e t h o d s Plant material. Seeds of Pisum sativum L. cv. Alaska No. 7
(harvested from plants grown from seeds purchased from Asgrow, Complejo Agricola Semillas, Madrid) were sterilized with 1% NaC10 for 20 rain and selected seedlings were grown in vermiculite. Other culture conditions were as described previously (Carbonell and Garcia-Martinez 1980). Usually only the first and-or the second flowers were used. Unpollinated ovaries were obtained by removing petals and stamens 2 d before anthesis (day -2). Development of unpoUinated ovaries was induced on the day of anthesis (day 0) either by topping the plant by cutting the internode just below the second flower (Carbonell and Garcia-Martinez 1980) or by applying plant growth regulators to the ovary (Garcia-Martlnez and Carbonell 1980). The rate of development of the ovaries and fruits as well as the rate of degeneration of unstimulated ovaries apparently depend on the temperature in the greenhouse (Carbonell and Garcia-Martinez 1980). However, we systematically observed that the biochemical changes concerning unstimulated
J. Carbonell and J.L. Garcla-Martinez: Ribulose-l,5-bisphosphate carboxylase and fruit set
535
ovaries were initiated on day 3, although their extent depended on the environmental temperature. Therefore, the average weight of the ovaries as well as their age is included to define their stage of development at sampling. Samples consisted usually of 20 to 30 ovaries on days 0 and 1, 10 to 20 ovaries on days 2 and 3, and 15 to 20 unstimulated ovaries or 4 to 10 stimulated ovaries on days 4 and 5.
Application of plant growth regulators. Plant growth regulators were applied by dipping the emasculated flowers in a 0.1% aqueous solution of Tween 80 (pH7) containing 100 gg GA 3 ml-1, 10 gg 2,4-dichlorophenoxyacetic acid (2,4-D) ml-1 or 100 gg N6-benzyladenine (BA) m l - 1. These compounds were from Fluka AG, Buchs, Switzerland. Unpollinated ovaries dipped in 0.1% Tween 80 were used as unstimulated ovaries. Autodigestion in crude extracts. Ovaries were homogenized in a mortar with 50 mmol 1 1 2-amino-2-(hydroxymethyl)-l,3propanediol (Tris)-HC1 pH 7.5 (22 °C) containing 10 mmol lmercaptoethanol (5 ml buffer g-1 fresh weight) and homogenares were centrifuged at 30000 g for 15 min. These operations were carried out at about 4 °C. Aliquots of supernatants were prepared immediately for electrophoresis and other aliquots were incubated in capped tubes at 30 °C for 20 h, then boiled for electrophoresis as indicated below. Purification and assay of RuBPCase. The purification procedure of Goldthwaite and Bogorad (1975) was basically followed. Ovaries were homogenized in a Polytron (3 x 0.5 rain, set 5) with 100mmol1-1 Tris-sulphate pH7.5 (22°C) containing 10 mmol 1-1 MgSO4, 20 mmol 1-1 mercaptoethanol and 2%
GA3 1OO pg/ml
2,4-D 1Q jig/m[
BA
1QOJJg/ml
Fig. 1. Seeded and parthenocarpic fruits of Pisum sativum L. obtained from ovaries stimulated in different ways. Fruits were collected on day 18 after anthesis (w/v) polyvinylpolypyrrolidone (2 ml buffer g - 1 fresh weight) and homogenates were centrifuged at 30000 g for 30 min. Aliquots (1 ml) of the extract were placed on the top of a 16-ml linear sucrose gradient (0.2-0.8 mol 1-1) prepared in 10 mmol 1-1 Tris-HC1 pH 8.0 (22 °C) containing 10 mmol 1-1
Fig. 2a, h. Analysis by SDS-PAGE of extracts from unstimulated ovaries (U) and from ovaries stimulated by plant topping (7-) or by application of BA (B), G A 3 (G), and 2,4-D (D). a Fresh extracts, b Autodigested extracts. Ovaries were collected on day 4. The average weight (mg) of the ovaries in this experiment was: U=33, T = 168, B = 104, G=222, and D=212. Arrows indicate bands of 85, 55, and 16 kDa
536
J. Carbonell and J.L. Garcia-Martinez: Ributose-l,5-bisphosphate carboxylase and fruit set
MgSO,~, 10 mmol 1-1 NaHCO3 ' and 1 mmol 1-1 mercaptoethanol. Centrifugation was carried out in an MSE-65 ultracentrifuge with a fixed-angle rotor (8 x 25 ml) at 40000 rpm for 4 h. All these operations were carried out at about 4 ~ Gradients were fractionated (0.6mi per fraction) and fractions were assayed for protein with Coomassie Blue reagent (Bradford 1976) and for RuBPCase activity by fixation of NaHI~CO3 (Amersham International Bucks., UK) essentially as described by Lorimer et al. (1977). Electrophoresis. i) Sodium dodecyl sulphate-PAGE with discontinuous buffers was carried out in slab gels according to Conejero and Semancik (1977) except that the solutions used to prepare gels were at room temperature and Coomassie Brilliant Blue R-250 (E. Merck, Darmstadt, F R G ) was used for staining. Aliquots (20 gl) of samples prepared as described by these authors as well as standards prepared from an electrophoresis calibration kit of low-molecular-weight proteins (Pharmacia Fine Chemicals, Uppsala, Sweden) were used. ii) Nondissociating-PAGE was carried out in 5% acrylamide slab gels prepared in 0.375 tool 1-1 Tris-HC1 pH 8.9 (22 ~ Electrode buffer was 50 mmol 1-1 Tris-glycine pH 8.3 (22 ~ Aliquots (20 gl) of samples prepared by mixing 100 gl of sucrose-gradient fractions with 42.8 gl of 0.2 m o l t - 1 Tris-HC1 pH 8.0 (22 ~ containing 25% (v/v) glycerol were run. Both types of electrophoresis were run in a 4 ~ air-cooled chamber at constant current (20 or 24 mA for 10 or 12 samples, respectively).
Results and discussion
The application of different plant growth regulators (GA3, 2,4-D, or BA) or the topping of pea plants on the day of anthesis promoted the development of unpollinated ovaries. Only the parthenocarpic fruits obtained with GA 3 had a shape similar to that of seeded fruits obtained from self-pollinated ovaries (Fig. 1) as reported previously (Garcia-Martinez and Carbonell 1980). This similarity between GA3-treated and pollinated ovaries has also been observed at the histological level during early stages of development (Vercher et al. 1984). Parthenocarpic fruits obtained with 2,4-D showed more variability in their shape, were usually deformed and only occasionally showed a light swelling. Similar effects of different gibberellins and naphthylacetic acid on the development of pea fruits with killed seeds have been reported by Sponsel (1982). Parthenocarpic fruits obtained by plant topping or by application of BA remained flat and were a darker green colour than the other differently treated fruits (Fig. 1). Interesting correlations between morphological observations and electrophoretic patterns of autodigested extracts of stimulated and unstimulated ovaries were observed. During the early stages of development (up to day 4) the pattern of polypeptides in extracts of stimulated and unstimulated ovaries was quite similar (Fig. 2a). Only minor quantitative, but not qualitative, differences were
Fig. 3. a Profile of RuBPCase activity and protein after sucrosegradient separation of an extract of GA3-treated ovaries. Ovaries collected on day 4. Average weight of the ovaries was 500 rag. b Analysis by SDS-PAGE of some fractions corresponding to the sucrose gradient, r/ = refractive index; arrows indicate bands of 55 and 16 kdalton
observed. However, after autodigestion of extracts for 20 h at 30 ~ and pH 7.5 the pattern of polypeptides showed important changes (Fig. 2b). In general clearer patterns were observed in all cases and, in particular, the pattern corresponding to unstimulated ovaries was appreciably different from that corresponding to stimulated ovaries, which showed similar patterns regardless of the type of stimulus. Several polypeptide bands present in the latter were absent in the former, the most remarkable being the 85- and 55-kDa bands. The 85-kDa band was more prominent in patterns cor-
J. Carbonell and J.L. Garcia-Martinez: Ribulose-l,5-bisphosphate carboxylase and fruit set
537
Fig. 4a-e. Analysis by SDS-PAGE of autodigested extracts from unstimulated ovaries (a) or from ovaries stimulated with GA 3 (b) or by plant topping (c) at early stages of development (days 0, 1, 2, 3, and 4). The average weight (mg) of the ovaries was: unstimulated, day 0=17, day 1 =21, day 2=24, day 3= 26, and day4=26; GA3-treated, day0=21, day1=38, day 2 = 75, day 3 = 121, and day 4 = 233 ; plant topping, day 0 = 18, day 1=28, day 2=48, day 3=82, and day 4=139. Arrows indicate bands of 85, 55, and 16 kdalton
responding to ovaries stimulated by plant topping or by application of BA. This result and the morphological observations indicate that the induction of development of ovaries in topped plants may be promoted by cytokinins. This hypothesis is also supported by experiments on leaf senescence in topped plants (data not shown). The hypothesis that the polypeptide band of 55 kDa as well as that of 16 kDa (a band that is practically absent in patterns of unstimulated ovaries (Fig. 2b)) correspond, respectively, to the
large and small subunits of RuBPCase was tested by sucrose-gradient fractionation of GA3-treated ovary extracts. Two major peaks of protein were observed, the peak with higher mobility being assodated with a peak of RuBPCase activity (Fig. 3 a). Nondissociating-PAGE of sucrose-gradient fractions corresponding to this peak showed essentially one major band (data not shown) and only two major bands at 55 and 16 kDa were observed after SDS-PAGE (Fig. 3 b). These results were not observed after PAGE and SDS-PAGE in the patterns
538
J. Carbonell and J.L. Garcia-Martinez: Ribulose-l,5-bisphosphate carboxylase and fruit set
Fig. 5a, b. Analysis by SDS-PAGE of autodigested mixtures of extracts from unstimulated ovaries (U) and GA3-treated ovaries (G). a Mixtures of extracts of unstimulated and GA 3treated ovaries of the same age (days 1, 3, 5). b Mixtures of extracts with the same treatment and different age (days 0, 5). The average weight (rag) of the ovaries was : U, day 0 = 22, day 1 =33, day 3=32, and day 5 = 1 0 ; G, day 0=22, day 1 =51, day 3=257, and day 5 = 513. Arrows indicate bands of 55 and 16 kDa
corresponding to fractions without RuBPCase activity. Appreciable changes in patterns corresponding to autodigested extracts from unstimulated ovaries had already appeared by day 3 and only a few
faint polypeptide bands remained in patterns from day 4 ovaries. Bands corresponding to RuBPCase were apparently absent (Fig. 4a). On the other hand, patterns corresponding to autodigested extracts from ovaries stimulated by GA 3 or by plant
J. Carbonell and J.L. Garcia-Martinez: Ribulose-l,5-bisphosphate carboxylase and fruit set
topping showed only a few differences between day 0 and day 4 (Figs. 4b, c). One of the most important differences was the disappearance of the 85kdalton band around day 3 in patterns corresponding to GA3-treated ovaries whereas that band remained in patterns corresponding to ovaries from topped plants. Therefore, the degradation of polypeptides corresponding to the 55- and 16-kDa bands in unstimulated but not in stimulated ovary extracts (Figs. 2b, 4) indicates the presence of a proteolytic activity in the former which hydrolyzes the RuBPCase protein. The appearance of a proteolytic activity hydrolyzing RuBPCase in unstimulated ovaries on day 3 after anthesis is coincident with the loss of sensitivity of the pea ovaries to GA 3 (Garcia-Martinez and Carbonell 1980) and with the begining of the degeneration of the ovary wall (Vercher etal. 1984). The hydrolysis of RuBPCase has been associated with senescence in other systems (Wittenbach 1979; Thomas and Stoddart 1980; Miller and Huffaker 1982). The hypothesis concerning the appearance of new proteolytic activity is also supported by mixing experiments. Polypeptide patterns of autodigested mixtures of extracts from unstimulated and GA3-treated ovaries were the same as those corresponding to the original extracts when made on day l, but different from them when made on days 3 and 5 (Fig. 5a). In these latter mixtures, the 55-kDa polypeptide band appeared to move to lower-molecular-weight zones. Autodigested mixtures of extracts from unstimulated ovaries of days 0 and 5 showed appreciable differences in relation to the original extracts, but no differences were observed in mixtures corresponding to GA 3treated ovaries (Fig. 5 b). Whether or not the detected proteolytic activity is a consequence either of the appearance of a (some) new protease(s) by synthesis or activation of a precursor or of the disappearance of an (some) inhibitor(s) of proteolytic activity cannot be decided in the absence of direct evidence on enzyme fractionation and characterization. However, we can conclude that the hydrolysis of RuBPCase in extracts of pea ovaries could serve as an indicator
539
of the begining of a senescence process in unstimulated ovaries whereas its stability is an indicator of fruit set and development. The technical assistance of Ms. Sabater in the laboratory and of Mr. R. Martinez-Pardo in the greenhouse are gratefully acknowledged. Thanks are also given to Dr. R. Marco (Instituto de Enzimologia, CSIC, Madrid) for his critical reading of the manuscript. This work was supported by a grant from the Comisi6n Asesora de Investigaci6n Cientifica y T6cnica (Spain).
References Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 Carbonell, J., Garcia-Martinez, J.L. (1980) Fruit-set of unpollinated ovaries of Pisum sativum L. Influence of vegetative parts. Planta 147, 444-450 Conejero, V., Semancik, J.S. (1977) Analysis of the proteins in crude plant extracts by polyacrylamide slab gel electrophoresis. Phytopathology 67, 1424-1426 Garcia-Martinez, J.L., Carbonell, J. (1980) Fruit-set of unpollinated ovaries of Pisum sativum L. Influence of plant growth regulators. Planta 147, 451-456 Goldthwaite, J., Bogorad, L. (1975) Ribulose-l,5-diphosphate carboxylase from leaf. Methods Enzymol. 42, 481-484 Lorimer, G.H., Badger, M.R., Andrews, T.J. (1977) D-Ribulose-l,5-bisphosphate carboxylase-oxygenase. Improved methods for the activation and assay of catalytic activites. Anal. Biochem. 78, 66-75 Miller, B.L., Huffaker, R.C. (1982) Hydrolysis of ribulose-l,5bisphosphate carboxylase by endoproteinases from senescing barley leaves. Plant Physiol. 69, 58-62 Ragster, L.E., Chrispeels, M.J. (1981) Autodigestion in crude extracts of soybean leaves and isolated chloroplasts as a measure of proteolytic activity. Plant Physiol. 67, 104-109 Sponsel, V.M. (1982) Effects of applied gibberellins and naphthylacetic acid on pod development in fruits of Pisum sat# rum L. cv. Progress no. 9. J. Plant Growth Regul. 1,147-152 Thomas, H., Stoddart, J.L. (1980) Leaf senescence. Annu. Rev. Plant Physiol. 31, 83-111 Vercher, Y., Molowny, A., L6pez, C., Garcia-Martinez, J.L., Carbonell, J. (1984) Structural changes in the ovary of Pisum sativum L. induced by pollination and gibberellic acid. Plant Sci. Lett. 36, 87-91 Wittenbach, V.A. (1979) Ribulose bisphosphate carboxylase and proteolytic activity in wheat leaves from anthesis through senescence. Plant Physiol. 64, 884-887 Received 21 August; accepted 14 November 1984
P l a n t a 9 Springer-Verlag 1985
Ribulose-l,5-bisphosphate carboxylase and fruit set or degeneration of unpollinated ovaries of Pisum sativum L. J. Carbonell and J.L. Garcia-Martinez Instituto de Agroquimica y Tecnologia de Alimentos, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain
The polypeptide patterns obtained by sodium dodecylsulphate-polyacrylamide gel electrophoresis of undigested and autodigested extracts from pea (P&um sativum L.) ovaries at the early stages of development or degeneration have been studied. Development of unpollinated ovaries was stimulated by application of different plant growth regulators (gibberellic acid, 2,4-dichlorophenoxyacetic acid, and N6-benzyladenine) or by plant topping. Polypeptide bands of similar mobility to ribulose-l,5-bisphosphate carboxylase (RuBPCase) subunits (16 and 55 kDa) could be detected in all types of autodigested extracts from stimulated ovaries. However these bands were absent in electrophoretic patterns of autodigested extracts from unstimulated ovaries after 3 d post anthesis and in patterns of autodigested mixtures of these extracts with either those from stimulated ovaries or those from unstimulated ovaries before day 3. These observations indicate that a proteolytic activity which promotes the hydrolysis of RuBPCase appears in unstimulated ovaries about 3 d after anthesis. This event coincides with the loss of the capacity of unpollinated ovaries to develop in response to gibberellic acid and with the degeneration of the ovary wall. Abstract.
-
Key words: Fruit development- Fruit set - Ovary Pisum (RuBPCase and fruit set) - Proteolysis - Ribulose-l,5-bisphosphate carboxylase.
Introduction
Fruit set and development of unpollinated pea ovaries can be induced by applying several types Abbreviations: BA=N6-benzyladenine; 2,4-D=2,4-dichloro-
phenoxyacetic acid; GA 3= gibberellic acid; RuBPCase = ribulose-1,5-bisphosphate carboxylase; SDS-PAGE=sodium dodecylsulphate-polyacrylamide gel electrophoresis
of plant growth regulators (auxin, gibberellin, and cytokinin) to ovaries (Garcia-Martinez and Carbonell 1980) or by topping the plants (Carbonell and Garcia-Martinez 1980). Unstimulated ovaries grow only slightly after anthesis and, approx. 3 d post anthesis their capacity to develop in response to gibberellic acid (GAg) is lost. Simultaneously the endocarp of the ovary wall degenerates (Vercher et al. 1984). The absence of biochemical studies on the degenerative process of unstimulated ovaries prompted us to investigate proteolytic activity in the ovaries by autodigestion and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude extracts (Ragster and Chrispeels 1981). The results show that the degeneration of unstimulated ovaries is associated with the presence of a new proteolytic activity which hydrolyzes ribulose-l,5-bisphosphate-carboxylase (RuBPCase). Some interesting correlations between morphological and biochemical observations are also shown. M a t e r i a l and m e t h o d s Plant material. Seeds of Pisum sativum L. cv. Alaska No. 7
(harvested from plants grown from seeds purchased from Asgrow, Complejo Agricola Semillas, Madrid) were sterilized with 1% NaC10 for 20 rain and selected seedlings were grown in vermiculite. Other culture conditions were as described previously (Carbonell and Garcia-Martinez 1980). Usually only the first and-or the second flowers were used. Unpollinated ovaries were obtained by removing petals and stamens 2 d before anthesis (day -2). Development of unpoUinated ovaries was induced on the day of anthesis (day 0) either by topping the plant by cutting the internode just below the second flower (Carbonell and Garcia-Martinez 1980) or by applying plant growth regulators to the ovary (Garcia-Martlnez and Carbonell 1980). The rate of development of the ovaries and fruits as well as the rate of degeneration of unstimulated ovaries apparently depend on the temperature in the greenhouse (Carbonell and Garcia-Martinez 1980). However, we systematically observed that the biochemical changes concerning unstimulated
J. Carbonell and J.L. Garcla-Martinez: Ribulose-l,5-bisphosphate carboxylase and fruit set
535
ovaries were initiated on day 3, although their extent depended on the environmental temperature. Therefore, the average weight of the ovaries as well as their age is included to define their stage of development at sampling. Samples consisted usually of 20 to 30 ovaries on days 0 and 1, 10 to 20 ovaries on days 2 and 3, and 15 to 20 unstimulated ovaries or 4 to 10 stimulated ovaries on days 4 and 5.
Application of plant growth regulators. Plant growth regulators were applied by dipping the emasculated flowers in a 0.1% aqueous solution of Tween 80 (pH7) containing 100 gg GA 3 ml-1, 10 gg 2,4-dichlorophenoxyacetic acid (2,4-D) ml-1 or 100 gg N6-benzyladenine (BA) m l - 1. These compounds were from Fluka AG, Buchs, Switzerland. Unpollinated ovaries dipped in 0.1% Tween 80 were used as unstimulated ovaries. Autodigestion in crude extracts. Ovaries were homogenized in a mortar with 50 mmol 1 1 2-amino-2-(hydroxymethyl)-l,3propanediol (Tris)-HC1 pH 7.5 (22 °C) containing 10 mmol lmercaptoethanol (5 ml buffer g-1 fresh weight) and homogenares were centrifuged at 30000 g for 15 min. These operations were carried out at about 4 °C. Aliquots of supernatants were prepared immediately for electrophoresis and other aliquots were incubated in capped tubes at 30 °C for 20 h, then boiled for electrophoresis as indicated below. Purification and assay of RuBPCase. The purification procedure of Goldthwaite and Bogorad (1975) was basically followed. Ovaries were homogenized in a Polytron (3 x 0.5 rain, set 5) with 100mmol1-1 Tris-sulphate pH7.5 (22°C) containing 10 mmol 1-1 MgSO4, 20 mmol 1-1 mercaptoethanol and 2%
GA3 1OO pg/ml
2,4-D 1Q jig/m[
BA
1QOJJg/ml
Fig. 1. Seeded and parthenocarpic fruits of Pisum sativum L. obtained from ovaries stimulated in different ways. Fruits were collected on day 18 after anthesis (w/v) polyvinylpolypyrrolidone (2 ml buffer g - 1 fresh weight) and homogenates were centrifuged at 30000 g for 30 min. Aliquots (1 ml) of the extract were placed on the top of a 16-ml linear sucrose gradient (0.2-0.8 mol 1-1) prepared in 10 mmol 1-1 Tris-HC1 pH 8.0 (22 °C) containing 10 mmol 1-1
Fig. 2a, h. Analysis by SDS-PAGE of extracts from unstimulated ovaries (U) and from ovaries stimulated by plant topping (7-) or by application of BA (B), G A 3 (G), and 2,4-D (D). a Fresh extracts, b Autodigested extracts. Ovaries were collected on day 4. The average weight (mg) of the ovaries in this experiment was: U=33, T = 168, B = 104, G=222, and D=212. Arrows indicate bands of 85, 55, and 16 kDa
536
J. Carbonell and J.L. Garcia-Martinez: Ributose-l,5-bisphosphate carboxylase and fruit set
MgSO,~, 10 mmol 1-1 NaHCO3 ' and 1 mmol 1-1 mercaptoethanol. Centrifugation was carried out in an MSE-65 ultracentrifuge with a fixed-angle rotor (8 x 25 ml) at 40000 rpm for 4 h. All these operations were carried out at about 4 ~ Gradients were fractionated (0.6mi per fraction) and fractions were assayed for protein with Coomassie Blue reagent (Bradford 1976) and for RuBPCase activity by fixation of NaHI~CO3 (Amersham International Bucks., UK) essentially as described by Lorimer et al. (1977). Electrophoresis. i) Sodium dodecyl sulphate-PAGE with discontinuous buffers was carried out in slab gels according to Conejero and Semancik (1977) except that the solutions used to prepare gels were at room temperature and Coomassie Brilliant Blue R-250 (E. Merck, Darmstadt, F R G ) was used for staining. Aliquots (20 gl) of samples prepared as described by these authors as well as standards prepared from an electrophoresis calibration kit of low-molecular-weight proteins (Pharmacia Fine Chemicals, Uppsala, Sweden) were used. ii) Nondissociating-PAGE was carried out in 5% acrylamide slab gels prepared in 0.375 tool 1-1 Tris-HC1 pH 8.9 (22 ~ Electrode buffer was 50 mmol 1-1 Tris-glycine pH 8.3 (22 ~ Aliquots (20 gl) of samples prepared by mixing 100 gl of sucrose-gradient fractions with 42.8 gl of 0.2 m o l t - 1 Tris-HC1 pH 8.0 (22 ~ containing 25% (v/v) glycerol were run. Both types of electrophoresis were run in a 4 ~ air-cooled chamber at constant current (20 or 24 mA for 10 or 12 samples, respectively).
Results and discussion
The application of different plant growth regulators (GA3, 2,4-D, or BA) or the topping of pea plants on the day of anthesis promoted the development of unpollinated ovaries. Only the parthenocarpic fruits obtained with GA 3 had a shape similar to that of seeded fruits obtained from self-pollinated ovaries (Fig. 1) as reported previously (Garcia-Martinez and Carbonell 1980). This similarity between GA3-treated and pollinated ovaries has also been observed at the histological level during early stages of development (Vercher et al. 1984). Parthenocarpic fruits obtained with 2,4-D showed more variability in their shape, were usually deformed and only occasionally showed a light swelling. Similar effects of different gibberellins and naphthylacetic acid on the development of pea fruits with killed seeds have been reported by Sponsel (1982). Parthenocarpic fruits obtained by plant topping or by application of BA remained flat and were a darker green colour than the other differently treated fruits (Fig. 1). Interesting correlations between morphological observations and electrophoretic patterns of autodigested extracts of stimulated and unstimulated ovaries were observed. During the early stages of development (up to day 4) the pattern of polypeptides in extracts of stimulated and unstimulated ovaries was quite similar (Fig. 2a). Only minor quantitative, but not qualitative, differences were
Fig. 3. a Profile of RuBPCase activity and protein after sucrosegradient separation of an extract of GA3-treated ovaries. Ovaries collected on day 4. Average weight of the ovaries was 500 rag. b Analysis by SDS-PAGE of some fractions corresponding to the sucrose gradient, r/ = refractive index; arrows indicate bands of 55 and 16 kdalton
observed. However, after autodigestion of extracts for 20 h at 30 ~ and pH 7.5 the pattern of polypeptides showed important changes (Fig. 2b). In general clearer patterns were observed in all cases and, in particular, the pattern corresponding to unstimulated ovaries was appreciably different from that corresponding to stimulated ovaries, which showed similar patterns regardless of the type of stimulus. Several polypeptide bands present in the latter were absent in the former, the most remarkable being the 85- and 55-kDa bands. The 85-kDa band was more prominent in patterns cor-
J. Carbonell and J.L. Garcia-Martinez: Ribulose-l,5-bisphosphate carboxylase and fruit set
537
Fig. 4a-e. Analysis by SDS-PAGE of autodigested extracts from unstimulated ovaries (a) or from ovaries stimulated with GA 3 (b) or by plant topping (c) at early stages of development (days 0, 1, 2, 3, and 4). The average weight (mg) of the ovaries was: unstimulated, day 0=17, day 1 =21, day 2=24, day 3= 26, and day4=26; GA3-treated, day0=21, day1=38, day 2 = 75, day 3 = 121, and day 4 = 233 ; plant topping, day 0 = 18, day 1=28, day 2=48, day 3=82, and day 4=139. Arrows indicate bands of 85, 55, and 16 kdalton
responding to ovaries stimulated by plant topping or by application of BA. This result and the morphological observations indicate that the induction of development of ovaries in topped plants may be promoted by cytokinins. This hypothesis is also supported by experiments on leaf senescence in topped plants (data not shown). The hypothesis that the polypeptide band of 55 kDa as well as that of 16 kDa (a band that is practically absent in patterns of unstimulated ovaries (Fig. 2b)) correspond, respectively, to the
large and small subunits of RuBPCase was tested by sucrose-gradient fractionation of GA3-treated ovary extracts. Two major peaks of protein were observed, the peak with higher mobility being assodated with a peak of RuBPCase activity (Fig. 3 a). Nondissociating-PAGE of sucrose-gradient fractions corresponding to this peak showed essentially one major band (data not shown) and only two major bands at 55 and 16 kDa were observed after SDS-PAGE (Fig. 3 b). These results were not observed after PAGE and SDS-PAGE in the patterns
538
J. Carbonell and J.L. Garcia-Martinez: Ribulose-l,5-bisphosphate carboxylase and fruit set
Fig. 5a, b. Analysis by SDS-PAGE of autodigested mixtures of extracts from unstimulated ovaries (U) and GA3-treated ovaries (G). a Mixtures of extracts of unstimulated and GA 3treated ovaries of the same age (days 1, 3, 5). b Mixtures of extracts with the same treatment and different age (days 0, 5). The average weight (rag) of the ovaries was : U, day 0 = 22, day 1 =33, day 3=32, and day 5 = 1 0 ; G, day 0=22, day 1 =51, day 3=257, and day 5 = 513. Arrows indicate bands of 55 and 16 kDa
corresponding to fractions without RuBPCase activity. Appreciable changes in patterns corresponding to autodigested extracts from unstimulated ovaries had already appeared by day 3 and only a few
faint polypeptide bands remained in patterns from day 4 ovaries. Bands corresponding to RuBPCase were apparently absent (Fig. 4a). On the other hand, patterns corresponding to autodigested extracts from ovaries stimulated by GA 3 or by plant
J. Carbonell and J.L. Garcia-Martinez: Ribulose-l,5-bisphosphate carboxylase and fruit set
topping showed only a few differences between day 0 and day 4 (Figs. 4b, c). One of the most important differences was the disappearance of the 85kdalton band around day 3 in patterns corresponding to GA3-treated ovaries whereas that band remained in patterns corresponding to ovaries from topped plants. Therefore, the degradation of polypeptides corresponding to the 55- and 16-kDa bands in unstimulated but not in stimulated ovary extracts (Figs. 2b, 4) indicates the presence of a proteolytic activity in the former which hydrolyzes the RuBPCase protein. The appearance of a proteolytic activity hydrolyzing RuBPCase in unstimulated ovaries on day 3 after anthesis is coincident with the loss of sensitivity of the pea ovaries to GA 3 (Garcia-Martinez and Carbonell 1980) and with the begining of the degeneration of the ovary wall (Vercher etal. 1984). The hydrolysis of RuBPCase has been associated with senescence in other systems (Wittenbach 1979; Thomas and Stoddart 1980; Miller and Huffaker 1982). The hypothesis concerning the appearance of new proteolytic activity is also supported by mixing experiments. Polypeptide patterns of autodigested mixtures of extracts from unstimulated and GA3-treated ovaries were the same as those corresponding to the original extracts when made on day l, but different from them when made on days 3 and 5 (Fig. 5a). In these latter mixtures, the 55-kDa polypeptide band appeared to move to lower-molecular-weight zones. Autodigested mixtures of extracts from unstimulated ovaries of days 0 and 5 showed appreciable differences in relation to the original extracts, but no differences were observed in mixtures corresponding to GA 3treated ovaries (Fig. 5 b). Whether or not the detected proteolytic activity is a consequence either of the appearance of a (some) new protease(s) by synthesis or activation of a precursor or of the disappearance of an (some) inhibitor(s) of proteolytic activity cannot be decided in the absence of direct evidence on enzyme fractionation and characterization. However, we can conclude that the hydrolysis of RuBPCase in extracts of pea ovaries could serve as an indicator
539
of the begining of a senescence process in unstimulated ovaries whereas its stability is an indicator of fruit set and development. The technical assistance of Ms. Sabater in the laboratory and of Mr. R. Martinez-Pardo in the greenhouse are gratefully acknowledged. Thanks are also given to Dr. R. Marco (Instituto de Enzimologia, CSIC, Madrid) for his critical reading of the manuscript. This work was supported by a grant from the Comisi6n Asesora de Investigaci6n Cientifica y T6cnica (Spain).
References Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 Carbonell, J., Garcia-Martinez, J.L. (1980) Fruit-set of unpollinated ovaries of Pisum sativum L. Influence of vegetative parts. Planta 147, 444-450 Conejero, V., Semancik, J.S. (1977) Analysis of the proteins in crude plant extracts by polyacrylamide slab gel electrophoresis. Phytopathology 67, 1424-1426 Garcia-Martinez, J.L., Carbonell, J. (1980) Fruit-set of unpollinated ovaries of Pisum sativum L. Influence of plant growth regulators. Planta 147, 451-456 Goldthwaite, J., Bogorad, L. (1975) Ribulose-l,5-diphosphate carboxylase from leaf. Methods Enzymol. 42, 481-484 Lorimer, G.H., Badger, M.R., Andrews, T.J. (1977) D-Ribulose-l,5-bisphosphate carboxylase-oxygenase. Improved methods for the activation and assay of catalytic activites. Anal. Biochem. 78, 66-75 Miller, B.L., Huffaker, R.C. (1982) Hydrolysis of ribulose-l,5bisphosphate carboxylase by endoproteinases from senescing barley leaves. Plant Physiol. 69, 58-62 Ragster, L.E., Chrispeels, M.J. (1981) Autodigestion in crude extracts of soybean leaves and isolated chloroplasts as a measure of proteolytic activity. Plant Physiol. 67, 104-109 Sponsel, V.M. (1982) Effects of applied gibberellins and naphthylacetic acid on pod development in fruits of Pisum sat# rum L. cv. Progress no. 9. J. Plant Growth Regul. 1,147-152 Thomas, H., Stoddart, J.L. (1980) Leaf senescence. Annu. Rev. Plant Physiol. 31, 83-111 Vercher, Y., Molowny, A., L6pez, C., Garcia-Martinez, J.L., Carbonell, J. (1984) Structural changes in the ovary of Pisum sativum L. induced by pollination and gibberellic acid. Plant Sci. Lett. 36, 87-91 Wittenbach, V.A. (1979) Ribulose bisphosphate carboxylase and proteolytic activity in wheat leaves from anthesis through senescence. Plant Physiol. 64, 884-887 Received 21 August; accepted 14 November 1984
