Planta (Berl.) 73, 324--327 (1967)
THE EFFECTS OF INHIBITORS ON THE DIFFERENTIATION OF
OF PROTEIN SYNTHESIS PLASTIDS IN ETIOLATED
BEAN SEEDLINGS MERCEDES WRISCI-IER Laboratory of Electron Microscopy, Department of Biology, Institute "Rudjer Boikovi5", Zagreb, Yugoslavia Received September 21, 1966
Summary. It has been shown that inhibitors of protein synthesis do not influence the breakdown of the crystal-lattice-like structure of the prolamellar bodies in the plastids when etiolated plants are exposed to light. The formation of grana and the greening of leaves are however considerably inhibited, depending on the concentration of the inhibitor used. I n s t u d y i n g the effects of g a m m a rays on the differentiation of plastids a delay or even i n h i b i t i o n of g r a n a f o r m a t i o n has been observed (W~IscHER a n d DEVID]~, 1966). As this delay m a y have been caused b y a n i n h i b i t i o n of p r o t e i n synthesis, the effect of some i n h i b i t o r s of p r o t e i n synthesis on the d e v e l o p m e n t of the chloroplast has been investigated. Materials and Methods The experiments were carried out with 8-day-old etiolated bean seedlings (Phaseolus vulgaris cv. Butterfisole) which were grown and prepared as described previously (W~IscHER and DEVID]~, 1966). The shoot apices of the etiolated seedlings (with cotyledons attached) were infiltrated in complete darkness with solutions of the inhibitors. After the infiltration the stems of the plants were dipped in flasks containing the same solution and left standing for 12 hours. Then the solution in the flask was replaced by tap water. The treated material was either exposed to white light (4000 Lux) immediately after the infiltration, or kept at first for some time in darkness. The following inhibitors were used: chloramphenicol (0,1---0,5 %), 2-thiouracil (0,01--0,2%), D,L-5-methyltryptophan (10-4--10-2 M) and D,L-ethionine (10-2--5 910-2 M). At certain intervals, during the exposure of the plants to light, small pieces of leaf lamina were cu~ out, fixed with OsO4 or KMnO4 and embedded in araldlte.
Results and Discussion I n t r e a t e d p l a n t s exposed for some t i m e to strong light the crystallattice-like a r r a n g e m e n t of t u b u l i i n the prolamellar bodies of the plastids was altered i n the same w a y as i n the u n t r e a t e d plants. The complete disappearance of the prolamellar bodies was however s o m e w h a t r e t a r d e d w h e n v e r y high c o n c e n t r a t i o n s of the i n h i b i t o r s were
Effects of Inhibitors on Differentiation of Plastids
325
applied. I n such cases it was possible to find the remainders of prolamellar bodies even after 12 hours of illumination (Fig. 2), whereas they disappeared as a rule already after 6 hours in the controls. The formation of grana, in contrast, was always considerablyinhibited. While the plastids of the untreated plants, after 12 hours of illumination, contained a considerable number of grana (Fig. 1), in the treated material this number was very much reduced, the degree of reduction depending on the concentration of the inhibitor applied (Fig. 3). The leaves of
Fig. 1--4. Phaseolus vulgaris cv. Butterfisole. Details of plastids. KMnO4-fixation, araldite, 45000:1 Fig. 1. Untreated. 12 hours of illumination. No prolamellar bodies are present. Grana are already well developed Fig. 2. 2-thiouraeil (0,1% solution). Exposed to light for 12 hours. Prolamellar bodies are still present. No grana are developed Fig, 3. D,L-5-methyltryptophan (5.10 -~ M). Exposed to light for 12 hours. Grana are rare and poorly developed Fig, 4. ChloramphenicoI (0,5%) 12 hours. Thereafter rinsed, transferred to tap water and exposed to light. Fixed 6 days after the treatment, intergrana thylakoids are hardly developed
treated plants started to green much later than those of the control plants, and even after several days the treated plants did not reach the controls in their development, their leaves remaining as a rule less green than those of the latter. Their chloroplasts sometimes appeared to be less numerous and smaller, usually containing also a smaller number
326
M. WRISCHER:
of grana. I n some cases, e.g. after t r e a t m e n t with chloramphenicol, the intergrana thylakoids were only weakly developed (Fig. 4). If after a previous infiltration with D,L-5-methyltryptophan (concentration 5.10 `8 M), the plants were infiltrated - - i m m e d i a t e l y or after 1 - - 2 hours of illumination - - with an equimolar solution of D,n-tryptophan, the inhibitory effect of D,L-5-methyltryptophan was almost completely overcome. However, when the infiltration with the D,nt r y p t o p h a n solution occurred 12 hours after the beginning of the illumination or later, the differentiation of the plastids was retarded, or even inhibited, almost to the same extent as in plants treated only with D,L-5-methyltryptophan. The inhibitor seemed thus to have already been incorporated into the structures of the cells. Similar effects were obtained with combined t r e a t m e n t with D,n-ethionine and D,n-methionine (concentration 10-2M). I t was, however, not possible to eliminate completely the effect of ethionine, even when the concentration of D,L-methionine was increased five-fold. All inhibitors used in the present work are known to interfere directly or indirectly with the synthesis of proteins necessary for the normal differentiation of plastids (HEsLoP-HAI~mSON, 1962; DSB]~r~, 1963; MAnGVLIES, 1964; BEI~IDZE et al., 1966), and for the normal morphogenesis of cells as well as of whole plants (BERGF~LD, 1964; L~VAI~r 1965). Judging from the grade of differentiation of the plastids in our experiments protein synthesis seems to have been only partially inhibited. MA~GULIES (1964) who measured the amount of chlorophyll synthetized, and DOBEL (1963) who observed a partial greening of treated leaves, came to the same conclusion. The formation of grana seems to be the most labile step in the differentiation of plastids, being strongly affected by inhibitors of protein synthesis, as well as b y other factors with presumably similar effects, e.g. b y ionizing radiation (Wmsc~E~ and DEVID~, 1966) and b y inhibition of respiration (DEvID]~ and W~ISC~EI~, 1966). The effect of chloramphenicol on chloroplast structure and protein formation during the greening of etiolatcd bean leaves has been recently studied b y MAnGVLIES (1966). His results are in good agreement with the observations in the present work. References BERGFELD, l:~.: Der EinfluB roter und blauer Strahlung auf die Ausbildung der Ch]oroplasten bei gehemmter Proteinsynthese. Z. Naturforsch. 19b, 1076--1078 (1964). BERIDZE, T. G., M. S. ODINTSOVA,N. A. C~ERKASn~rA, and N. M. SlSSAI~IAN: The effect or nucleic acid synthesis inhibitors on the chlorophyll formation by etiolated bean leaves. Bioehem. biophys. Res. Commun. 23, 683--690 (1966).
Effects of Inhibitors on Differentiation of Plastids
327
DEWD]~, Z., u. M. WRISCHER: ]~ber den Einflu8 von AtmungsstSrungen auf die Differenzierung der Plastiden im Blattgewebe etiolierter Bohnenkeimlinge. Z. Natufforschg. 1966. (In press.) DSBEL, P.: Untersuchungen der Wirkung yon Streptomycin-, Chloramphenicolund 2-Thiouracil-Behandlung auf die Plastidenentwieklung yon Lycopersicum esculentum MILLEm Biol. Zbl. 82, 275---295 (1963). HESLO]e-H~ml~SON, J.: Evanescent and persistent modifications of chloroplast ultrastructure induced by an unnatural pyrimidine. Planta (Berl.) ~8, 237---256 (1962). LIbyaN, N. : Les effets de la dl-~thionine sur la croissanee et la morphogen~se des jeunes plantes de Sinapis alba L. (Crucif~res). C. l~. Acad. Sci. (Paris) 260, 6675--6678 (1965). MARGVLInS, M. M.: Effect of chloramphenicol on light-dependent synthesis of proteins and enzymes of ]eaves and chloroplasts of Phaseolus vulgaris. Plant Physiol. 39, 579--585 (1964). - - Effect of chloramphenicol on formation of chloroplast structure and protein during greening of etiolated leaves of Phaseolus vulgaris. Plant Physiol. 41, 992--1003 (1966). WRISCHER,~/[., U. Z. DEu Uber die Wirkung von y-Strahlen auf die Entwicklung der Plastiden etiolierter Bohnenkeimlinge. Z. Naturforschg. 1966. (In press.) l)r. MERCEDES WRISCttER
Institut ,,Rudier Bo~kovi6" Zagreb, Bi]eni6ka c. 54, Jugoslavija
THE EFFECTS OF INHIBITORS ON THE DIFFERENTIATION OF
OF PROTEIN SYNTHESIS PLASTIDS IN ETIOLATED
BEAN SEEDLINGS MERCEDES WRISCI-IER Laboratory of Electron Microscopy, Department of Biology, Institute "Rudjer Boikovi5", Zagreb, Yugoslavia Received September 21, 1966
Summary. It has been shown that inhibitors of protein synthesis do not influence the breakdown of the crystal-lattice-like structure of the prolamellar bodies in the plastids when etiolated plants are exposed to light. The formation of grana and the greening of leaves are however considerably inhibited, depending on the concentration of the inhibitor used. I n s t u d y i n g the effects of g a m m a rays on the differentiation of plastids a delay or even i n h i b i t i o n of g r a n a f o r m a t i o n has been observed (W~IscHER a n d DEVID]~, 1966). As this delay m a y have been caused b y a n i n h i b i t i o n of p r o t e i n synthesis, the effect of some i n h i b i t o r s of p r o t e i n synthesis on the d e v e l o p m e n t of the chloroplast has been investigated. Materials and Methods The experiments were carried out with 8-day-old etiolated bean seedlings (Phaseolus vulgaris cv. Butterfisole) which were grown and prepared as described previously (W~IscHER and DEVID]~, 1966). The shoot apices of the etiolated seedlings (with cotyledons attached) were infiltrated in complete darkness with solutions of the inhibitors. After the infiltration the stems of the plants were dipped in flasks containing the same solution and left standing for 12 hours. Then the solution in the flask was replaced by tap water. The treated material was either exposed to white light (4000 Lux) immediately after the infiltration, or kept at first for some time in darkness. The following inhibitors were used: chloramphenicol (0,1---0,5 %), 2-thiouracil (0,01--0,2%), D,L-5-methyltryptophan (10-4--10-2 M) and D,L-ethionine (10-2--5 910-2 M). At certain intervals, during the exposure of the plants to light, small pieces of leaf lamina were cu~ out, fixed with OsO4 or KMnO4 and embedded in araldlte.
Results and Discussion I n t r e a t e d p l a n t s exposed for some t i m e to strong light the crystallattice-like a r r a n g e m e n t of t u b u l i i n the prolamellar bodies of the plastids was altered i n the same w a y as i n the u n t r e a t e d plants. The complete disappearance of the prolamellar bodies was however s o m e w h a t r e t a r d e d w h e n v e r y high c o n c e n t r a t i o n s of the i n h i b i t o r s were
Effects of Inhibitors on Differentiation of Plastids
325
applied. I n such cases it was possible to find the remainders of prolamellar bodies even after 12 hours of illumination (Fig. 2), whereas they disappeared as a rule already after 6 hours in the controls. The formation of grana, in contrast, was always considerablyinhibited. While the plastids of the untreated plants, after 12 hours of illumination, contained a considerable number of grana (Fig. 1), in the treated material this number was very much reduced, the degree of reduction depending on the concentration of the inhibitor applied (Fig. 3). The leaves of
Fig. 1--4. Phaseolus vulgaris cv. Butterfisole. Details of plastids. KMnO4-fixation, araldite, 45000:1 Fig. 1. Untreated. 12 hours of illumination. No prolamellar bodies are present. Grana are already well developed Fig. 2. 2-thiouraeil (0,1% solution). Exposed to light for 12 hours. Prolamellar bodies are still present. No grana are developed Fig, 3. D,L-5-methyltryptophan (5.10 -~ M). Exposed to light for 12 hours. Grana are rare and poorly developed Fig, 4. ChloramphenicoI (0,5%) 12 hours. Thereafter rinsed, transferred to tap water and exposed to light. Fixed 6 days after the treatment, intergrana thylakoids are hardly developed
treated plants started to green much later than those of the control plants, and even after several days the treated plants did not reach the controls in their development, their leaves remaining as a rule less green than those of the latter. Their chloroplasts sometimes appeared to be less numerous and smaller, usually containing also a smaller number
326
M. WRISCHER:
of grana. I n some cases, e.g. after t r e a t m e n t with chloramphenicol, the intergrana thylakoids were only weakly developed (Fig. 4). If after a previous infiltration with D,L-5-methyltryptophan (concentration 5.10 `8 M), the plants were infiltrated - - i m m e d i a t e l y or after 1 - - 2 hours of illumination - - with an equimolar solution of D,n-tryptophan, the inhibitory effect of D,L-5-methyltryptophan was almost completely overcome. However, when the infiltration with the D,nt r y p t o p h a n solution occurred 12 hours after the beginning of the illumination or later, the differentiation of the plastids was retarded, or even inhibited, almost to the same extent as in plants treated only with D,L-5-methyltryptophan. The inhibitor seemed thus to have already been incorporated into the structures of the cells. Similar effects were obtained with combined t r e a t m e n t with D,n-ethionine and D,n-methionine (concentration 10-2M). I t was, however, not possible to eliminate completely the effect of ethionine, even when the concentration of D,L-methionine was increased five-fold. All inhibitors used in the present work are known to interfere directly or indirectly with the synthesis of proteins necessary for the normal differentiation of plastids (HEsLoP-HAI~mSON, 1962; DSB]~r~, 1963; MAnGVLIES, 1964; BEI~IDZE et al., 1966), and for the normal morphogenesis of cells as well as of whole plants (BERGF~LD, 1964; L~VAI~r 1965). Judging from the grade of differentiation of the plastids in our experiments protein synthesis seems to have been only partially inhibited. MA~GULIES (1964) who measured the amount of chlorophyll synthetized, and DOBEL (1963) who observed a partial greening of treated leaves, came to the same conclusion. The formation of grana seems to be the most labile step in the differentiation of plastids, being strongly affected by inhibitors of protein synthesis, as well as b y other factors with presumably similar effects, e.g. b y ionizing radiation (Wmsc~E~ and DEVID~, 1966) and b y inhibition of respiration (DEvID]~ and W~ISC~EI~, 1966). The effect of chloramphenicol on chloroplast structure and protein formation during the greening of etiolatcd bean leaves has been recently studied b y MAnGVLIES (1966). His results are in good agreement with the observations in the present work. References BERGFELD, l:~.: Der EinfluB roter und blauer Strahlung auf die Ausbildung der Ch]oroplasten bei gehemmter Proteinsynthese. Z. Naturforsch. 19b, 1076--1078 (1964). BERIDZE, T. G., M. S. ODINTSOVA,N. A. C~ERKASn~rA, and N. M. SlSSAI~IAN: The effect or nucleic acid synthesis inhibitors on the chlorophyll formation by etiolated bean leaves. Bioehem. biophys. Res. Commun. 23, 683--690 (1966).
Effects of Inhibitors on Differentiation of Plastids
327
DEWD]~, Z., u. M. WRISCHER: ]~ber den Einflu8 von AtmungsstSrungen auf die Differenzierung der Plastiden im Blattgewebe etiolierter Bohnenkeimlinge. Z. Natufforschg. 1966. (In press.) DSBEL, P.: Untersuchungen der Wirkung yon Streptomycin-, Chloramphenicolund 2-Thiouracil-Behandlung auf die Plastidenentwieklung yon Lycopersicum esculentum MILLEm Biol. Zbl. 82, 275---295 (1963). HESLO]e-H~ml~SON, J.: Evanescent and persistent modifications of chloroplast ultrastructure induced by an unnatural pyrimidine. Planta (Berl.) ~8, 237---256 (1962). LIbyaN, N. : Les effets de la dl-~thionine sur la croissanee et la morphogen~se des jeunes plantes de Sinapis alba L. (Crucif~res). C. l~. Acad. Sci. (Paris) 260, 6675--6678 (1965). MARGVLInS, M. M.: Effect of chloramphenicol on light-dependent synthesis of proteins and enzymes of ]eaves and chloroplasts of Phaseolus vulgaris. Plant Physiol. 39, 579--585 (1964). - - Effect of chloramphenicol on formation of chloroplast structure and protein during greening of etiolated leaves of Phaseolus vulgaris. Plant Physiol. 41, 992--1003 (1966). WRISCHER,~/[., U. Z. DEu Uber die Wirkung von y-Strahlen auf die Entwicklung der Plastiden etiolierter Bohnenkeimlinge. Z. Naturforschg. 1966. (In press.) l)r. MERCEDES WRISCttER
Institut ,,Rudier Bo~kovi6" Zagreb, Bi]eni6ka c. 54, Jugoslavija
![[The effect of pre-illumination on the development of plastids in primary leaves of γ-irradiated etiolated bean seedlings].](/assets/img/hold.png)
