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The effect of streptomycin on the mitochondria and plastids of barley
621 THE
EFFECT
OF STREPTOMYCIN AND
PLASTIDS
J. T. 0. KIRK The Botany
ON THE
MITOCHONDRIA
OF BARLEY
and B. E. JUNIPER School, Oxford, England
ReceivedMarch 29, 1963 VON E ULER [5] found that barley seedlings germinated in solutions of streptomycin contain neither chlorophyll nor carotenoids. Chlorophyll synthesis in Pinus jeffreyi is inhibited by streptomycin both in the dark and the light [2]. When Euglena gracilis is grown in the presence of streptomycin it loses the ability to make chloroplasts [12]. In bacteria, streptomycin has been thought to affect respiration [7, 81, membrane formation [l] and protein synthesis [6, 181. The effects of streptomycin on the ultrastructure of algal chloroplasts and the leaf chloroplasts of higher plants have been described by a number of workers [4, 13, 14, 16, 171. This paper is concerned with the effects of streptomycin on the structure of cell organelles in non-photosynthetic as well as photosynthetic tissue. Barley seeds (Spratt Archer) were germinated on filter paper in an illuminated incubator at 25°C. The filter paper was moistened either with tap water or tap water containing streptomycin sulphate at 4 mg free base/ml. The percentage germination (30 per cent) was the same under both conditions. After four days the average shoot length of the control seedlings was 37 mm and of the treated 14 mm, while the average length of root material per seedling was 101 mm for the control and 23 mm for the treated. Root hair formation was almost completely inhibited by streptomycin. For the electron microscopy whole root tips 3 mm long were taken from the root and transverse sections & mm thick from the base of the shoot (this region was green in the control seedlings and white in the treated). The tissue was fixed in KMnO, and embedded and sectioned as already described [9]. In normal root cap cells (Fig. 1) the mitochondria are typically oval and smooth in outline and the cristae are distributed around the periphery. In root cap cells of streptomycin-treated seedlings (Fig. 2) the mitochondria (m) are highly variable in shape with irregular outlines; few normal cristae can be seen and the internal structure now consists of one, two or three independent lamellae or double-membraned vesicles (0) which do not appear to be connected to the inner membrane of the mitochondrion. The aberrant mitochondria are the same average length (0.89 p) as the normal mitochondria. The overall size and shape of the root plastids are similar in the treated and untreated seedlings but the electron density of the starch grains is markedly reduced in the streptomycin-treated roots. The mitochondria of the shoot and coleoptile are affected in exactly the same way as those of the root. However, the plastids of these tissues are more severely affected by the drug than those of the root. Instead of the highly developed system of granal and inter-granal lamellae of the normal chloroplasts (Fig. 3) they contain (Fig. 4) only a few vesicles (sometimes in rows) and short fragments of lamellae [l]. Experimental
Cell Research 30
622
Experimental
J. T. 0. Kirk and B. E. Juniper
Cell Research 30
Mitochondria
and plastids of barley
623
The appearance suggests that the drug arrests at an. early stage the development of the mature chloroplasts. However this could also represent a degenerative change. Ample amounts of starch (s) were present in the coleoptile plastids (Figs. 3, 4) of both the treated and untreated seedlings. The finding by Rubin and Ladygina [15] that germination of barley seedlings in solutions of streptomycin lowered their cytochrome oxidase activity could be explained by the effect of the drug on mitochondrial structure. De Deken-Grenson’s observations [3] that the drug inhibits the growth of barley seedlings in the dark as well as in the light also suggests that the effects of streptomycin are not confined to the chloroplasts. Moreover, Kirk [lo, 111 found that as well as inhibiting pigment synthesis in etiolated Euglena gracilis streptomycin has fundamental effects on biosynthesis in the dark and in the light. REFERENCES
1. 2. 3. 4. 5. 6.
ANAND, N. and DAVIS, B. D., Nature 185, 22 (1960). BOGORAD, L., Am. J. Rot. 37, 676 (1950). DE DEKEN-GRENSON, M., Biochim. Biophys. Acfa 17, 35 (1955). DRAWERT, H. and MIX, M., Planta 57, 51 (1961). EON EDLER, H., Kern. Arb. II. 9, 1 (1947). HAHN, F. E., CIAK, J., WOLFE, A. D., HARTMAN, R. E., ALLISON,
J. L. and HARTMAN,
R. S.,
Biochim.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Biophys. Acta 61, 741 (1962). HANCOCK, R., J. Gen. Microbiof. 25, 429 (1961). JACKSON, F. L., Nature 181, 281 (1958). JUNIPER, B. E., Nature 194, 1296 (1962). KIRK, J. T. O., Biochim. Biophys. Acfa 56, 139 (1962). ibid. 59, 476 (1962). PROVASOLI, L., HUTNER, S. H. and SCHATZ, A., Proc. Sot. Expfl. Biol. Med. 69, 279 (1948). ROSEN, W. G. and SIEGESMUND, K. A., J. Biophys. Biochem. Cyfol. 9, 910 (1961). ROSSNER, W., Protoplasma 52, 580 (1960). RUBIN, B. A. and LADYGINA, M. E., Biokhimiya 22, 984 (1957). SIEGESMUND, K. A., ROSEN, W. G. and GAWLIK, S. R., Am. J. Botany 49, 137 (1962). SIGNOL, M. M., Compf. Rend. Acad. Sci. Paris, 27, 1993 (1961). SPOTTS, C. R. and STANIER, R. Y., Nature 192, 632 (1961).
Fig.
l.-Mitochondria
of the root
cap of normal
Fig.
2.-Mitochondria
of the root
cap of streptomycin-treated
seedling.
Fig.
3.-Chloroplast
of coleoptile
of normal
Fig.
4.-Chloroplast
of coleoptile
of streptomycin-treated
seedling.
x 24,000. seedling.
x 24,000.
x 12,000. seedling.
x 12,000.
Experimental
Cell Research 30
EFFECT
OF STREPTOMYCIN AND
PLASTIDS
J. T. 0. KIRK The Botany
ON THE
MITOCHONDRIA
OF BARLEY
and B. E. JUNIPER School, Oxford, England
ReceivedMarch 29, 1963 VON E ULER [5] found that barley seedlings germinated in solutions of streptomycin contain neither chlorophyll nor carotenoids. Chlorophyll synthesis in Pinus jeffreyi is inhibited by streptomycin both in the dark and the light [2]. When Euglena gracilis is grown in the presence of streptomycin it loses the ability to make chloroplasts [12]. In bacteria, streptomycin has been thought to affect respiration [7, 81, membrane formation [l] and protein synthesis [6, 181. The effects of streptomycin on the ultrastructure of algal chloroplasts and the leaf chloroplasts of higher plants have been described by a number of workers [4, 13, 14, 16, 171. This paper is concerned with the effects of streptomycin on the structure of cell organelles in non-photosynthetic as well as photosynthetic tissue. Barley seeds (Spratt Archer) were germinated on filter paper in an illuminated incubator at 25°C. The filter paper was moistened either with tap water or tap water containing streptomycin sulphate at 4 mg free base/ml. The percentage germination (30 per cent) was the same under both conditions. After four days the average shoot length of the control seedlings was 37 mm and of the treated 14 mm, while the average length of root material per seedling was 101 mm for the control and 23 mm for the treated. Root hair formation was almost completely inhibited by streptomycin. For the electron microscopy whole root tips 3 mm long were taken from the root and transverse sections & mm thick from the base of the shoot (this region was green in the control seedlings and white in the treated). The tissue was fixed in KMnO, and embedded and sectioned as already described [9]. In normal root cap cells (Fig. 1) the mitochondria are typically oval and smooth in outline and the cristae are distributed around the periphery. In root cap cells of streptomycin-treated seedlings (Fig. 2) the mitochondria (m) are highly variable in shape with irregular outlines; few normal cristae can be seen and the internal structure now consists of one, two or three independent lamellae or double-membraned vesicles (0) which do not appear to be connected to the inner membrane of the mitochondrion. The aberrant mitochondria are the same average length (0.89 p) as the normal mitochondria. The overall size and shape of the root plastids are similar in the treated and untreated seedlings but the electron density of the starch grains is markedly reduced in the streptomycin-treated roots. The mitochondria of the shoot and coleoptile are affected in exactly the same way as those of the root. However, the plastids of these tissues are more severely affected by the drug than those of the root. Instead of the highly developed system of granal and inter-granal lamellae of the normal chloroplasts (Fig. 3) they contain (Fig. 4) only a few vesicles (sometimes in rows) and short fragments of lamellae [l]. Experimental
Cell Research 30
622
Experimental
J. T. 0. Kirk and B. E. Juniper
Cell Research 30
Mitochondria
and plastids of barley
623
The appearance suggests that the drug arrests at an. early stage the development of the mature chloroplasts. However this could also represent a degenerative change. Ample amounts of starch (s) were present in the coleoptile plastids (Figs. 3, 4) of both the treated and untreated seedlings. The finding by Rubin and Ladygina [15] that germination of barley seedlings in solutions of streptomycin lowered their cytochrome oxidase activity could be explained by the effect of the drug on mitochondrial structure. De Deken-Grenson’s observations [3] that the drug inhibits the growth of barley seedlings in the dark as well as in the light also suggests that the effects of streptomycin are not confined to the chloroplasts. Moreover, Kirk [lo, 111 found that as well as inhibiting pigment synthesis in etiolated Euglena gracilis streptomycin has fundamental effects on biosynthesis in the dark and in the light. REFERENCES
1. 2. 3. 4. 5. 6.
ANAND, N. and DAVIS, B. D., Nature 185, 22 (1960). BOGORAD, L., Am. J. Rot. 37, 676 (1950). DE DEKEN-GRENSON, M., Biochim. Biophys. Acfa 17, 35 (1955). DRAWERT, H. and MIX, M., Planta 57, 51 (1961). EON EDLER, H., Kern. Arb. II. 9, 1 (1947). HAHN, F. E., CIAK, J., WOLFE, A. D., HARTMAN, R. E., ALLISON,
J. L. and HARTMAN,
R. S.,
Biochim.
7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Biophys. Acta 61, 741 (1962). HANCOCK, R., J. Gen. Microbiof. 25, 429 (1961). JACKSON, F. L., Nature 181, 281 (1958). JUNIPER, B. E., Nature 194, 1296 (1962). KIRK, J. T. O., Biochim. Biophys. Acfa 56, 139 (1962). ibid. 59, 476 (1962). PROVASOLI, L., HUTNER, S. H. and SCHATZ, A., Proc. Sot. Expfl. Biol. Med. 69, 279 (1948). ROSEN, W. G. and SIEGESMUND, K. A., J. Biophys. Biochem. Cyfol. 9, 910 (1961). ROSSNER, W., Protoplasma 52, 580 (1960). RUBIN, B. A. and LADYGINA, M. E., Biokhimiya 22, 984 (1957). SIEGESMUND, K. A., ROSEN, W. G. and GAWLIK, S. R., Am. J. Botany 49, 137 (1962). SIGNOL, M. M., Compf. Rend. Acad. Sci. Paris, 27, 1993 (1961). SPOTTS, C. R. and STANIER, R. Y., Nature 192, 632 (1961).
Fig.
l.-Mitochondria
of the root
cap of normal
Fig.
2.-Mitochondria
of the root
cap of streptomycin-treated
seedling.
Fig.
3.-Chloroplast
of coleoptile
of normal
Fig.
4.-Chloroplast
of coleoptile
of streptomycin-treated
seedling.
x 24,000. seedling.
x 24,000.
x 12,000. seedling.
x 12,000.
Experimental
Cell Research 30