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The inhibition of the development of photosystem II-mediated electron transport in greening bean leaves by D-threo-chloramphenicol
Plant Science Letters, 7 (1976) 95--100 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
95
THE INHIBITION O F THE D E V E L O P M E N T OF PHOTOSYSTEM IIMEDIATED E L E C T R O N T R A N S P O R T IN G R E E N I N G BEAN LEAVES BY D - T H R E O - C H L O R A M P H E N I C O L
O N N O P W A R A - A S W A P A T I * and J.W. B R A D B E E R Department of Plant Sciences, King's College, 68 Half M o o n Lane, London 8E24 9JF (Great Britain) (Received January 5th, 1976) (Revision received March 19th, 1976) (Accepted April 13th, 1976)
SUMMARY
Plastids, prepared from the primary leaves of 14-day-old dark-grown bean plants which had been allowed to green for 50 h in the presence of 250 ~g/ml of D.threo.chlorarnphenicol, showed no detectable p h o t o s y s t e m II-mediated electron transport although p h o t o s y s t e m I-mediated electron transport occurred at normal rates. It is deduced that at least one c o m p o n e n t of either p h o t o s y s t e m II or its associated electron transport p a t h w a y was absent from the etioplasts and that its formation was d e p e n d e n t on polypeptide synthesis on 70S ribosomes. Cycloheximide showed some inhibition of the development of electron transport m e d i a t e d b y each photosystem.
INTRODUCTION
Electron microscopy has shown that when D-threo-chloramphenicol was supplied to greening bean leaves thylakoid synthesis de novo was completely inhibited [1]. The plastids developed under this treatment contained only a few large grana and a few stroma thylakoids which altogether accounted for the same a m o u n t of membrane as that present in the etioplasts from which they had developed. As leaves, which greened in the presence Of L-threochloramphenicol, contained chloroplasts which showed no structural differences from those of the water control it was deduced that the synthesis of one or more essential thylakoid c o m p o n e n t s was dependent on polypeptide synthesis on the 70S ribosomes of the plastids. So far there is evidence that D.threo-chloramphenicol inhibits the synthesis of 11 o f the major SDS*Present address: Department of Biology, Chiang Mai University, Chiang Mai, (Thailand). Abbreviations: D C M U , 3-(3,4-dichlorophenyl)-l,l-dimethylurea;SDS, sodium dodecyl sulphate.
96 polypeptides of Vicia faba thylakoids [2] and of at least 7 of those of Phaseolus vulgaris [ 3]. Investigations in a number of laboratories have established that some thylakoid proteins are synthesized on 70S ribosomes and some on 80S ribosomes (see e.g. ref. 4). In this laboratory the development of photosynthetic CO2 fixation in Phaseolus vulgaris has been found to be virtually completely inhibited by D-threo-chloramphenicol but unaffected by L-threo-chloramphenicol [5], thus confirming an earlier finding [6]. In the present paper the photochemical properties of plastids obtained from bean leaves greened in the presence of chloramphenicol and of cycloheximide are reported. As it proved difficult to obtain bean chloroplast preparations which showed coupled photophosphorylation, this study has been confined to electron transport mediated by photosystems I and II. MATERIALS AND METHODS
Phaseolusvulgaris L. cv. Alabaster seedlings were grown in the dark for 14 days as described previously [7]. The hypocotyls were cut 13 cm below the cotyledons and the e
97 reduced by ascorbate as electron donor. The reaction mixture contained in a total volume of 2 ml: 100 ~moles N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) pH 7.8, 660 pmoles sorbitol, 8 umoles MgC12, 20 pmoles NaC1, 2/~moles MnC12, 3.2 pmoles EDTA, 20 pmoles Na2HPO+, 0.4 pmoles methylviologen, 4 pmoles NAN3, 20/~moles dichlorophenolindophenol, 20 pmoles sodium ascorbate, 0.01 pmoles DCMU and chloroplasts equivalent to 20--50 ~g chlorophyll. Photosystem II-mediated electron transport was measured as an oxygen evolution with ferricyanide as electron acceptor and water as electron donor. The reaction mixture was the same as for Photosystem I, except that 5 pmoles potassium ferricyanide and 40 ~g catalase were substituted for methylviologen, NAN3, dichlorophenolindophenol, sodium ascorbate and DCMU. The linked activity of photosystems I and II was measured as an oxygen uptake with methylviologen as electron acceptor and water as electron donor. The reaction mixture was the same as for photosystem I except that dichlorophenolindophenol, sodium ascorbate and DCMU were omitted. Chloramphenicol was obtained from Parke, Davis and Co., cycloheximide from Calbiochem Ltd., catalase from Boehringer and DCMU from the Du Pont Co. All other chemicals used were of the highest grade commercially available from BDH Chemicals. RESULTS AND DISCUSSION The chloroplast preparations obtained from bean leaves, by the method described above, showed uncoupled electron transport and as osmotic shock did not increase their permeability to ferricyanide it was evident that they did not possess a functional envelope. Table I shows that plastids from leaves greened in the presence of D-threochloramphenicol had no detectable photosystem II-mediated electron transport but that electron transport mediated by photosystem I occurred at about the same rate as that of the control. As the small amount of electron transport detected with the linked photosystems I and II was not inhibited by including 2.5.10-6M DCMU in the reaction mixture this small amount of activity would not seem to involve photosystem II. L-threo-Chloramphenicol had no effect on photosystem I electron transport and gave only slight inhibition of electron transport mediated by photosystem II and the linked photosystems. Neither isomer of chloramphenicol nor cycloheximide had any direct effect on electron transport when included in the electron transport assays of chloroplasts obtained from glasshouse grown beans. However, we have previously reported that for coupled preparations of broken spinach chloroplasts, type C chloroplasts, both isomers of chloramphenicol were equally effective as energy transfer inhibitors in that they inhibited both phosphorylation and phosphorylating electron transport, but that after adding an uncoupler no inhibition of the uncoupled electron transport was found [11]. If chloram-
396 141 317 158
Water
aNot inhibited by 2.5 - 1 0 - ' M DCMU.
Cycloheximide ( 6 ~ g/ml)
D-threo-chloramphenicol (250 ~g/ml) L-threo-chloramphenicol (250 ~g/ml)
Chlorophyll c o n t e n t after 50 h illumination (nmoles/leaf)
Solution supplied to greening shoots
32.6 31.7 32.2 6.1
Photosystem I
24.2 0 20.9 11.1
Photosystem II
O5 uptake or evolution of plastids (~moles OJh/leaf)
9.6 1.7 a 6.6 3.3
Photosystems I and II.
14-day-old dark-grown shoots of Phaseolus vulgaris were placed in the test solution and illuminated at 1.6 mW/cm 2 for 50 h. Plastids were obtained from the leaves and tested for electron transport mediated by photosystems I and II as described in the text.
THE EFFECTS OF CHLORAMPHENICOL AND CYCLOHEXIMIDE ON THE DEVELOPMENT OF ELECTRON TRANSPORT MEDIATED BY PHOTOSYSTEMS I AND II IN PLASTIDS OF GREENING BEAN LEAVES
TABLE I
¢~ (30
99
phenicol acts as an energy transfer inhibitor in bean chloroplasts in vivo it is possible that this may have some influence on chloroplast development. However, as both isomers of chloramphenicol are equally effective as energy transfer inhibitors they would be equally effective in inhibiting chloroplast development by this mechanism. Since L-threo.chloramphenicol gave only a small inhibition of the development of photosystem II-mediated electron transport it may be assumed that only a small part of the inhibition by D-threochloramphenicol can be accounted for by this mechanism. As protein synthesis on 70S ribosomes is the only biological system which is known to be inhibited by D-threo-chloramphenicol but not by L-threo.chloramphenicol it may be concluded that the development of either photosystem II or its associated electron transport chain requires the synthesis of at least one polypeptide on 70S ribosomes. Furthermore although many chloroplast components are present in the etioplasts of dark-grown leaves it is probable that the missing photosystem II component(s) does not occur in etioplasts. It is of interest to note for example that a number of the SDS-polypeptides of the thylakoids of Avena chloroplasts were not detected in the etioplasts [12]. As the chlorophyll a : b ratio of the D-threo-chloramphenicol treated leaves was not abnormal and as the development of the photosystem II protein : pigment complex, as detected by SDS-polyacrylamide gel electrophoresis, was not inhibited by D-threo-chloramphenicol in Vicia faba and Phaseolus vulgaris leaves [2,3], it would seem that the deficient component(s) was not part of the protein: pigment complex of photosystem II. Earlier work in this laboratory established that the development of cytochrome b 559Hp in greening Phaseolus vulgaris was totally inhibited by D-threo-chloramphenicol, but since L-threo-chloramphenicol was equally effective the absence of this substance cannot account for the lack of photosystem II-mediated electron transport [13]. D-threo-Chloramphenicol shows what is presumably an indirect inhibition of chlorophyll synthesis [6,8] although it did not inhibit the development of photosystem I-mediated electron transport as expressed on a per leaf basis. If the photosystem I electron transport is expressed on a chlorophyll basis the rate for D.threo-chloramphenicol treated leaves was 251 ~moles 02 uptake/ h/mg chlorophyll as compared with a value of 92 for the water control. Similar results have been obtained for Euglena but not for Chlamydomonas [14]. In both organisms D-threo-chloramphenicol was an effective inhibitor of the development of photosystem II-mediated electron transport while the development of the photosystem I-mediated system was inhibited in Chlamydomonas but not in Euglena. Cycloheximide has been used frequently as an inhibitor of protein synthesis on 80S ribosomes. However, in plants it shows other inhibitory effects [15] and in higher plants it is not possible to make a clear distinction between the results of the inhibition of protein synthesis and the results of the other inhibitory effects. Table I shows that, when cycloheximide was supplied to greening bean leaves, chlorophyll synthesis and the development of electron transport were considerably inhibited with the development of photosystem
100
I-mediated electron transport showing the greatest inhibition. Although it is not clear to what extent these inhibitions result from the inhibition of polypeptide synthesis on 80S ribosomes, the results m a y be of some interest in relation to the results of cycloheximide exper'.nnents carried out in other laboratories, for example [3,16--18]. ACKNOWLEDGEMENTS
Grateful acknowledgements are made to theColombo Plant (scholarship to Onnop Wam-Aswapati) and to Dr. M.C.W. Evans, Professor D.O. Hall and Dr. K.K. Rao for helpful discussions. REFERENCES 1 J.W. Bradbeer, A.O. Gyldenholm, H.M.M. Ireland, J.W. Smith, J. Rest and H.J.W. Edge, New Phytol., 73 (1974) 271. 2 0 . Machold and O. Aurich, Biochim. Biophys. Acta, 281 (1972) 103. 3 A. Herrera, (1976) (pers. comm.). 4 J.P. Thornber, Ann. Rev. Plant Physiol., 26 (1975) 127. 5 0 n n o p Wara-Aswapati, Ph.D. Thesis, University of London, 1973. 6 M.K. Nikolaeva, O.P. Osipova and Yu.V. Krylov, Dokl. Akad. Nauk SSSR, 175 (1967) 487. 7 J.W. Bradbeer, New Phytol., 68 (1969) 233. 8 H.M.M. Ireland and J.W. Bradbeer, Planta, 96 (1971) 254. 9 D.A. Walker, Biochem. J., 92 (1964) 22C. 10 T. Delieu and D.A. Walker, New Phytol., 71 (1972) 201. 11 Onnop Wara-Aswapati and J.W. Bradbeer, Plant Physiol., 53 (1974) 691. 12 A.H. Cobb and A.R. Wellburn, Planta, 114 (1973) 131. 13 P. Gregory and J.W. Bradbeer, Planta, 109 (1973) 317. 14 I. Ohad, (1976) (pers. comm.). 15 D. McMahon, Plant Physiol., 55 (1975) 815. 16 J.K. Hoober, J. Biol. Chem., 245 (1970) 4327. 17 H. Liebers and B. Parthier, Biochem. Physiol. Pflanzen, 165 (1974) 517. 18 R.M. Smfllie, D.G. Bishop, G.C. Gibbons, D. Graham, A.M. Grieve, J.K. Raison and B.J. Reger, Determination of the sites of synthesis of proteins and lipids of the chloroplast using chloramphenicol and cycloheximide, in N.K. Boardman, A.W. Linnane and R.M. Smillie (Eds.), Autonomy and Biogenesis of Mitochondria and Chloroplasts, North Holland, Amsterdam, 1971, p. 422.
95
THE INHIBITION O F THE D E V E L O P M E N T OF PHOTOSYSTEM IIMEDIATED E L E C T R O N T R A N S P O R T IN G R E E N I N G BEAN LEAVES BY D - T H R E O - C H L O R A M P H E N I C O L
O N N O P W A R A - A S W A P A T I * and J.W. B R A D B E E R Department of Plant Sciences, King's College, 68 Half M o o n Lane, London 8E24 9JF (Great Britain) (Received January 5th, 1976) (Revision received March 19th, 1976) (Accepted April 13th, 1976)
SUMMARY
Plastids, prepared from the primary leaves of 14-day-old dark-grown bean plants which had been allowed to green for 50 h in the presence of 250 ~g/ml of D.threo.chlorarnphenicol, showed no detectable p h o t o s y s t e m II-mediated electron transport although p h o t o s y s t e m I-mediated electron transport occurred at normal rates. It is deduced that at least one c o m p o n e n t of either p h o t o s y s t e m II or its associated electron transport p a t h w a y was absent from the etioplasts and that its formation was d e p e n d e n t on polypeptide synthesis on 70S ribosomes. Cycloheximide showed some inhibition of the development of electron transport m e d i a t e d b y each photosystem.
INTRODUCTION
Electron microscopy has shown that when D-threo-chloramphenicol was supplied to greening bean leaves thylakoid synthesis de novo was completely inhibited [1]. The plastids developed under this treatment contained only a few large grana and a few stroma thylakoids which altogether accounted for the same a m o u n t of membrane as that present in the etioplasts from which they had developed. As leaves, which greened in the presence Of L-threochloramphenicol, contained chloroplasts which showed no structural differences from those of the water control it was deduced that the synthesis of one or more essential thylakoid c o m p o n e n t s was dependent on polypeptide synthesis on the 70S ribosomes of the plastids. So far there is evidence that D.threo-chloramphenicol inhibits the synthesis of 11 o f the major SDS*Present address: Department of Biology, Chiang Mai University, Chiang Mai, (Thailand). Abbreviations: D C M U , 3-(3,4-dichlorophenyl)-l,l-dimethylurea;SDS, sodium dodecyl sulphate.
96 polypeptides of Vicia faba thylakoids [2] and of at least 7 of those of Phaseolus vulgaris [ 3]. Investigations in a number of laboratories have established that some thylakoid proteins are synthesized on 70S ribosomes and some on 80S ribosomes (see e.g. ref. 4). In this laboratory the development of photosynthetic CO2 fixation in Phaseolus vulgaris has been found to be virtually completely inhibited by D-threo-chloramphenicol but unaffected by L-threo-chloramphenicol [5], thus confirming an earlier finding [6]. In the present paper the photochemical properties of plastids obtained from bean leaves greened in the presence of chloramphenicol and of cycloheximide are reported. As it proved difficult to obtain bean chloroplast preparations which showed coupled photophosphorylation, this study has been confined to electron transport mediated by photosystems I and II. MATERIALS AND METHODS
Phaseolusvulgaris L. cv. Alabaster seedlings were grown in the dark for 14 days as described previously [7]. The hypocotyls were cut 13 cm below the cotyledons and the e
97 reduced by ascorbate as electron donor. The reaction mixture contained in a total volume of 2 ml: 100 ~moles N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES) pH 7.8, 660 pmoles sorbitol, 8 umoles MgC12, 20 pmoles NaC1, 2/~moles MnC12, 3.2 pmoles EDTA, 20 pmoles Na2HPO+, 0.4 pmoles methylviologen, 4 pmoles NAN3, 20/~moles dichlorophenolindophenol, 20 pmoles sodium ascorbate, 0.01 pmoles DCMU and chloroplasts equivalent to 20--50 ~g chlorophyll. Photosystem II-mediated electron transport was measured as an oxygen evolution with ferricyanide as electron acceptor and water as electron donor. The reaction mixture was the same as for Photosystem I, except that 5 pmoles potassium ferricyanide and 40 ~g catalase were substituted for methylviologen, NAN3, dichlorophenolindophenol, sodium ascorbate and DCMU. The linked activity of photosystems I and II was measured as an oxygen uptake with methylviologen as electron acceptor and water as electron donor. The reaction mixture was the same as for photosystem I except that dichlorophenolindophenol, sodium ascorbate and DCMU were omitted. Chloramphenicol was obtained from Parke, Davis and Co., cycloheximide from Calbiochem Ltd., catalase from Boehringer and DCMU from the Du Pont Co. All other chemicals used were of the highest grade commercially available from BDH Chemicals. RESULTS AND DISCUSSION The chloroplast preparations obtained from bean leaves, by the method described above, showed uncoupled electron transport and as osmotic shock did not increase their permeability to ferricyanide it was evident that they did not possess a functional envelope. Table I shows that plastids from leaves greened in the presence of D-threochloramphenicol had no detectable photosystem II-mediated electron transport but that electron transport mediated by photosystem I occurred at about the same rate as that of the control. As the small amount of electron transport detected with the linked photosystems I and II was not inhibited by including 2.5.10-6M DCMU in the reaction mixture this small amount of activity would not seem to involve photosystem II. L-threo-Chloramphenicol had no effect on photosystem I electron transport and gave only slight inhibition of electron transport mediated by photosystem II and the linked photosystems. Neither isomer of chloramphenicol nor cycloheximide had any direct effect on electron transport when included in the electron transport assays of chloroplasts obtained from glasshouse grown beans. However, we have previously reported that for coupled preparations of broken spinach chloroplasts, type C chloroplasts, both isomers of chloramphenicol were equally effective as energy transfer inhibitors in that they inhibited both phosphorylation and phosphorylating electron transport, but that after adding an uncoupler no inhibition of the uncoupled electron transport was found [11]. If chloram-
396 141 317 158
Water
aNot inhibited by 2.5 - 1 0 - ' M DCMU.
Cycloheximide ( 6 ~ g/ml)
D-threo-chloramphenicol (250 ~g/ml) L-threo-chloramphenicol (250 ~g/ml)
Chlorophyll c o n t e n t after 50 h illumination (nmoles/leaf)
Solution supplied to greening shoots
32.6 31.7 32.2 6.1
Photosystem I
24.2 0 20.9 11.1
Photosystem II
O5 uptake or evolution of plastids (~moles OJh/leaf)
9.6 1.7 a 6.6 3.3
Photosystems I and II.
14-day-old dark-grown shoots of Phaseolus vulgaris were placed in the test solution and illuminated at 1.6 mW/cm 2 for 50 h. Plastids were obtained from the leaves and tested for electron transport mediated by photosystems I and II as described in the text.
THE EFFECTS OF CHLORAMPHENICOL AND CYCLOHEXIMIDE ON THE DEVELOPMENT OF ELECTRON TRANSPORT MEDIATED BY PHOTOSYSTEMS I AND II IN PLASTIDS OF GREENING BEAN LEAVES
TABLE I
¢~ (30
99
phenicol acts as an energy transfer inhibitor in bean chloroplasts in vivo it is possible that this may have some influence on chloroplast development. However, as both isomers of chloramphenicol are equally effective as energy transfer inhibitors they would be equally effective in inhibiting chloroplast development by this mechanism. Since L-threo.chloramphenicol gave only a small inhibition of the development of photosystem II-mediated electron transport it may be assumed that only a small part of the inhibition by D-threochloramphenicol can be accounted for by this mechanism. As protein synthesis on 70S ribosomes is the only biological system which is known to be inhibited by D-threo-chloramphenicol but not by L-threo.chloramphenicol it may be concluded that the development of either photosystem II or its associated electron transport chain requires the synthesis of at least one polypeptide on 70S ribosomes. Furthermore although many chloroplast components are present in the etioplasts of dark-grown leaves it is probable that the missing photosystem II component(s) does not occur in etioplasts. It is of interest to note for example that a number of the SDS-polypeptides of the thylakoids of Avena chloroplasts were not detected in the etioplasts [12]. As the chlorophyll a : b ratio of the D-threo-chloramphenicol treated leaves was not abnormal and as the development of the photosystem II protein : pigment complex, as detected by SDS-polyacrylamide gel electrophoresis, was not inhibited by D-threo-chloramphenicol in Vicia faba and Phaseolus vulgaris leaves [2,3], it would seem that the deficient component(s) was not part of the protein: pigment complex of photosystem II. Earlier work in this laboratory established that the development of cytochrome b 559Hp in greening Phaseolus vulgaris was totally inhibited by D-threo-chloramphenicol, but since L-threo-chloramphenicol was equally effective the absence of this substance cannot account for the lack of photosystem II-mediated electron transport [13]. D-threo-Chloramphenicol shows what is presumably an indirect inhibition of chlorophyll synthesis [6,8] although it did not inhibit the development of photosystem I-mediated electron transport as expressed on a per leaf basis. If the photosystem I electron transport is expressed on a chlorophyll basis the rate for D.threo-chloramphenicol treated leaves was 251 ~moles 02 uptake/ h/mg chlorophyll as compared with a value of 92 for the water control. Similar results have been obtained for Euglena but not for Chlamydomonas [14]. In both organisms D-threo-chloramphenicol was an effective inhibitor of the development of photosystem II-mediated electron transport while the development of the photosystem I-mediated system was inhibited in Chlamydomonas but not in Euglena. Cycloheximide has been used frequently as an inhibitor of protein synthesis on 80S ribosomes. However, in plants it shows other inhibitory effects [15] and in higher plants it is not possible to make a clear distinction between the results of the inhibition of protein synthesis and the results of the other inhibitory effects. Table I shows that, when cycloheximide was supplied to greening bean leaves, chlorophyll synthesis and the development of electron transport were considerably inhibited with the development of photosystem
100
I-mediated electron transport showing the greatest inhibition. Although it is not clear to what extent these inhibitions result from the inhibition of polypeptide synthesis on 80S ribosomes, the results m a y be of some interest in relation to the results of cycloheximide exper'.nnents carried out in other laboratories, for example [3,16--18]. ACKNOWLEDGEMENTS
Grateful acknowledgements are made to theColombo Plant (scholarship to Onnop Wam-Aswapati) and to Dr. M.C.W. Evans, Professor D.O. Hall and Dr. K.K. Rao for helpful discussions. REFERENCES 1 J.W. Bradbeer, A.O. Gyldenholm, H.M.M. Ireland, J.W. Smith, J. Rest and H.J.W. Edge, New Phytol., 73 (1974) 271. 2 0 . Machold and O. Aurich, Biochim. Biophys. Acta, 281 (1972) 103. 3 A. Herrera, (1976) (pers. comm.). 4 J.P. Thornber, Ann. Rev. Plant Physiol., 26 (1975) 127. 5 0 n n o p Wara-Aswapati, Ph.D. Thesis, University of London, 1973. 6 M.K. Nikolaeva, O.P. Osipova and Yu.V. Krylov, Dokl. Akad. Nauk SSSR, 175 (1967) 487. 7 J.W. Bradbeer, New Phytol., 68 (1969) 233. 8 H.M.M. Ireland and J.W. Bradbeer, Planta, 96 (1971) 254. 9 D.A. Walker, Biochem. J., 92 (1964) 22C. 10 T. Delieu and D.A. Walker, New Phytol., 71 (1972) 201. 11 Onnop Wara-Aswapati and J.W. Bradbeer, Plant Physiol., 53 (1974) 691. 12 A.H. Cobb and A.R. Wellburn, Planta, 114 (1973) 131. 13 P. Gregory and J.W. Bradbeer, Planta, 109 (1973) 317. 14 I. Ohad, (1976) (pers. comm.). 15 D. McMahon, Plant Physiol., 55 (1975) 815. 16 J.K. Hoober, J. Biol. Chem., 245 (1970) 4327. 17 H. Liebers and B. Parthier, Biochem. Physiol. Pflanzen, 165 (1974) 517. 18 R.M. Smfllie, D.G. Bishop, G.C. Gibbons, D. Graham, A.M. Grieve, J.K. Raison and B.J. Reger, Determination of the sites of synthesis of proteins and lipids of the chloroplast using chloramphenicol and cycloheximide, in N.K. Boardman, A.W. Linnane and R.M. Smillie (Eds.), Autonomy and Biogenesis of Mitochondria and Chloroplasts, North Holland, Amsterdam, 1971, p. 422.