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Effect of chloramphenicol, ethanol, kinetin and oligomycin on salt sensitivity and adenosine triphosphate content of wheat roots
Institute of Physiological Botany, University of Uppsala, Uppsala, Sweden
Effect of Chloramphenicol, Ethanol, Kinetin and Oligomycin on Salt Sensitivity and Adenosine Triphosphate Content of Wheat Roots Die Einwirkung von Chloramphenicol, Xthanol, Kinetin und Oligomycin auf Salzempfindlichkeit und A TP-Gehalt von Weizenwurzeln INGRID SKOGQVIST Received October 1, 1973
Summary Chloramphenicol, ethanol, kinetin and oligomycin protected wheat roots from lethal effects of salt treatment. This protection of wheat roots does not seem to have a direct correlation with ATP, since the ATP content of wheat root apex decreased in the presence of ethanol and kinetin and increased with the addition of chloramphenicol.
Zusammenfassung Chloramphenicol, Athanol, Kinetin und Oligomycin schiitzen Weizenwurzeln gegen die lethale Einwirkung von hohen Salzkonzentrationen im Medium. Diese Schutzwirkung scheint nicht direkt mit dem ATP-Gehalt der Wurzel korreliert zu sein, wei! Athanol und Kinetin eine Abnahme, Chloramphenicol aber eine Zunahme des ATP-Gehaltes der Wurzelspitze hervorrufen.
Introduction In previous papers (SKOGQVIST and FRIES, 1970; SKOGQVIST, 1973) it has been shown that heat-shocked wheat roots, in comparison to unshocked roots, are more sensitive to high salt concentrations. Chloramphenicol, kinetin and ethanol were used to prevent the heat-treated roots from dying at a supraoptimal incubation temperature. In this paper results are presented from experiments on the ability of chloramphenicol, ethanol, kinetin and oligomycin to prevent salt sensitivity in wheat roots, which had not been previously subjected to heat treatment. The content of adenosine triphosphate of the wheat roots was also determined, as some of the compounds under test have been reported to inhibit ATP production in various plant cells (ADEDIPE and FLETCHIER, 1970; JACOBY and PLESSNER, 1970).
z. Pjlanzenphysiol. Bd. 72. S. 297-304. 1974.
298
I. SKOGQVIST
Material and Methods Wheat seeds (Triticum aestivum L. "Svenno Varvete» from Weibullsholm, Landskrona, Sweden) were germinated at 25° C in the dark. The seedlings, at the three root stage were placed in 250 ml nutrient medium (SKOGQVIST, 1973). The medium was in a container (250 ml) and each container had 19 plants. The incubation temperature was 25° C in all experiments. When the effect of D-threo chloramphenicol (Park, Davis & Company), ethanol (99,5 0/0, v/v) , kinetin (Calbiochem) or oligomycin (Sigma: oligomycin A 15 % , oligomycin B 85 0/0) was to be studied, these compounds were dissolved prior to placing the plants in the nutrient medium, after which the wheat roots were salt-treated or their content of adenosine triphosphate was determined. The root systems were salt-treated by holding them for 5 minutes in nutrient solution to which an inorganic salt had been incorporated as given in Tables 1-3. The roots were then washed and transferred to fresh medium. The shoots were always kept in continuous light during the experiments. The number of living root tips was determined four days after the salt treatment. In this paper a dead root indicates a root with a visibly dead apical meristem. In each experimental series 57 roots were examined. Every experiment was performed at least three times. For more details of methods the reader is referred to SKOGQVIST (1973). The adenosine triphosphate (ATP) content of root tips was determined 16-20 hours after incubation of the plants in nutrient solution. Pieces of the central roots, 0-10 mm from the apex, were used. Single sections were ground in a glass homogenizer during 30 seconds with 2 ml of ice-cold arsenate buffer. In a Beckman scintillation counter No. 200, 0.1 ml of the homogenate was analysed for ATP by the firefly method (RASMUSSEN and NIELSEN, 1968; STENLID, 1970, 1971). A standard curve with known amounts of ATP was used to calculate the ATP content of the root section. Control extractions with buffer containing ATP showed that ATP was not destroyed during the preparation.
Results
Effect of chloramphenicol in relation to salt sensitivity. All roots in the experiment survived the salt shock when placed in a chloramphenicol solution prior to the salt treatment (table 1). The salt concentration was as high as 0.29 M. The D-threo isomer of chloramphenicol used in this investigation interferes with 50S-70S ribosomes and is an inhibitor of protein synthesis in mitochondria (LAMB et aI., 1968; MOORE et aI., 1971; SAKANO and ASAHI, 1971 a, b). MACDoNALD et a1. (1966) demonstrated that this isomer inhibits the development of CI- absorption capacity in beet disks. The 80S cytoplasmic ribosomes are insensitive to chloramphenicol both in vitro (ELLIS and MACDoNALD, 1967) and in vivo (ELLIS, 1964) but chloramphenicol inhibits salt uptake in several types of plant cells (SUTCLIFFE, 1960; ELLIS et aI., 1964). Therefore, if the D-threo isomer produces its effect through an inhibition of ribosome activity, it could be by an interference with protein synthesis in the mitochondria. This should result in an inhibition of the oxidative phosphorylation, since proteins of the electron transport chain enzymes are synthesized with mitochondrial ribosomes (SAKANO and ASAHI, 1971 a, b). The inhibitory effects of chloramphenicol on oxidative phosphorylation have been demonstrated
z. Pjlanzenphysiol. Bd. 72. S. 297-304. 1974.
Salt Sensitivity and Adenosine Triphosphate Content of Wheat Roots
299
Table 1: The effect of 10-3 M chloramphenicol on salt sensitivity of wheat roots. The values denote per cent surviving roots. Chloramphenicol was added to the nutrient medium 16 hours before the roots were placed in the salt solutions. After this the plants were incubated at 25° C in only nutrient medium. - Similar results were obtained with 10-4 and 5 X 10-4 M chloramphenicol.
Salt NaCI KCl NaBr KBr NaI KI
Chloramphenicol
+ + + + + +
0
0.19
0.20
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 99 100 100 100 99 100 95 100
100 100
0.21
Salt concentration M 0.23 0.24 0.25 55 100 50 100 80 100
100 100
40 100 80 100
30 100 35 100
0.29
100 0 100 0 100 0 100
75 100
100 100 98 100
0.27
0 100
a
100
both in mitochondria and in whole cells from beet disks (HANSON and HODGES, 1963; STONER et aI., 1964; HANSON and KREUGER, 1966; MACDoNALD et aI., 1966). Applied to the present study, this would mean that oxidative phosphorylation should be suppressed in the wheat roots which had been placed in chloramphenicol solution about 16 hours before salt treatment. Consequently the uptake of ions ought to be inhibited allowing for survival of the wheat roots. This possibility was tested in experiments to be described later. Effect of kinetin in relation to salt sensitivity. As with chloramphenicol, the wheat roots survived when kinetin was added to the medium prior to the salt shock (table 2). The protective effect was clear, but not so manifest as with chloramphenicol. Pretreatment with kinetin has earlier been employed to enhance incorporation of 14C-L-Ieucine in both salinity-stressed and non-stressed tobacco leaves (BEN-ZIONl et aI., 1967). In non-stressed leaves optimum incorporation was observed at a kinetin concentration of 5 X 10-7 M while stressed tissue had its optimum at 5 X 10-6 M. ITAl et al. (1968) showed that NaCI gave a marked decrease in the cytokinin activity of root exudates from sunflower, bean and tobacco plants. Salinity-stressed roots brought about a decline in the protein synthesis of the leaves, which could be partially corrected by pretreatment of the leaf disks with kinetin. Kinetin protects the wheat roots against salt treatment when the wheat plants are placed for an 18 hour period before exposure to salt shock in a kinetin solution.
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300
I. SKOGQVIST
Table 2: The effect of 5 X 10-6 M kinetin on salt sensitivity of wheat roots. Kinetin was added to the nutrient medium 18 hours before the roots were placed in the salt solution. Otherwise as table 1. - Similar results were obtained with 10-6 and 10-s M kinetin. Salt
Kinetin
NaCl KCl NaBr KBr NaI KI
+ + + + + +
0
0.19
0.20
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 99 100 100 100 100 100 100 100
100 100
Salt concentration M 0.21 0.23 0.24 55 100 40 90 80 100
100 100
100 100 100 100
0.27
90 100
0.29
1 75 5 75 30 75 0 75
75 95 70 100
95 100
0.25
1 80 50 95
Some workers have reported an effect of cytokinins on A TP synthesis. ADEDIPE and (1970) showed that the ATP content was lowered in leaves of intact bean plants which had been treated with benzyladenine (chemically related to kinetin), although the incorporation of 32p into ATP was higher. BERRIDGE and RALPH (1971), working with cabbage leaf discs found that kinetin increased the rate of synthesis of ATP. Effects 0/ oligomycin and ethanol in relation to salt sensitivity. When wheat plant roots were placed in an oligomycin solution where the oligomycin was dissolved directly at 5,ug/ml prior to the salt treatment, many roots survived the salt shock. Solution of oligomycin in absolute ethanol and subsequent incorporation into the nutrient medium brought about a survival of all roots (except in NaCI and KCI, where a few roots died). Simihrly, many wheat roots survived if only ethanol was added to the nutrient medium prior to the salt treatment (table 3). In many investigations ethanol has been used as a solvent for oligomycin (BLACKMON and MORELAND, 1971; FISHER and HODGES, 1969; HODGES, 1966; JACOBY and PLESSNER, 1970), the final concentration of the solvent in all assays and controls being 10f0 by volume. This concentration of ethanol exhibited no detectable effect on mitochondrial responses (BLACKMON and MORELAND, 1971). In the present investigation the ethanol concentration was only 0.2 0/0 by volume, the same concentration as JACOBY (1966) used. Surprisingly enough this concentration of ethanol alone protected the wheat roots against salts. JACOBY and PLESSNER (1970) found that chloride absorption by excised barley roots from dilute solutions (up to about 0.2 mM) is more oligomycin-sensitive than its absorption from more concentrated solutions. It was also observed that the ATP FLETCHIER
z. Pflanzenphysiol. Bd. 72. S. 297-304. 1974.
Salt Sensitivity and Adenosine Triphosphate Content of Wheat Roots
301
Table 3: The results of three experiments demonstrating the effects of oligomycin and ethanol, alone and together, on the salt sensitivity of wheat roots. When added, these compounds were present in the following concentrations: oligomycin 5,ug/ml and ethanol 1.58 mg/ml (= 0.2 % v/v). The additions to the medium were made 16 hours before the roots were placed in the salt solution. Otherwise as table 1. Salt added (0.27 M)
Addition to the medium Expt. a: No addition With oligomycin Expt. b: No addition With ethanol Expt. c: No addition With oligomycin and ethanol
No
NaCl
KCl
NaBr
KBr
NaI
KI
100 100
0 40
0 45
15 90
15 85
20 100
45 95
100 100
8 90
5 75
15 90
20 90
20 85
45 90
100 100
0 95
0 95
15 100
15 100
25 100
45 100
content of root tissue decreased in the presence of oligomycin. The inhibitor penetrates the plasmalemma and action is at the level of oxidative phosphorylation. In conclusion, if the A TP content of wheat roots is lowered by placing the plants 16 hours prior to salt treatment in an oligomycin solution, this might also influence the survival of the root apex. Effects of chloramphenicol, ethanol and kinetin on the ATP content in wheat roots. Table 4: Effect of chloramphenicol 10-3 M, ethanol 0.2 Ofo (3 X 10-2 M) and kinetin 10-5 M on the ATP content in wheat roots (0-10 mm from apex). ATP content given as per cent of values for control roots growing in nutrient solution without inhibitor. Experiment No.
Duration of treatment h 16
2
20
3
19
Addition to medium during treatment No (Control) Chloramphenicol Ethanol Kinetin No (Control) Chloramphenicol Ethanol Kinetin No (Control) Chloramphenicol Ethanol Kinetin
ATP content found in root sections, 0/ 0 100 120 72
55 100 125 75 60 100 121 60 65
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302
I. SKOGQVIST
The ATP content of the control roots of intact wheat plants corresponded to a concentration of about 10-4 M in sections 0-10 mm from the apex. In three different experiments, comprised of ten measurements each of ATP, chloramphenicol caused a significant increase of ATP. Ethanol and kinetin, on the other hand, reduced the content of ATP (table 4). Discussion In contrast to several earlier studies referred to above, the experiments of the present investigation demonstrated that chloramphenicol caused an increase rather than a decrease of the ATP level in the treated material. However, even such a classical uncoupler as 2,4-dinitrophenol may, under certain circumstances, act that way (STENLID, 1971). Since the regulatory mechanisms for the ATP level are very complicated (ATKINSON et aI., 1966) and chloramphenicol interferes with metabolism in several different ways (STONER et aI., 1964; HANSON and KRUEGER, 1966; LEONARD and HANSON, 1972) any attempt to explain the observed effect of chloramphenicol appears meaningless until more experimental data are available. Kinetin and ethanol lowered the A TP level in the roots, although, like chloramphenicol, they were capable to give protection against the deleterious influence of a high salt concentration. There does not seem to be any obvious correlation between the capacity of a compound to protect against salt exposure and its effect upon the ATP level. The possible effects of the protective compounds upon the membranes have not been dealt with in the present paper. It is evident from literature, however, that particularly ethanol and kinetin are active in this respect. In corn roots kinetin caused a decrease in phosphate absorption (LEONARD and HANSON, 1972), and in epidermal onion cells the permeability was changed by affecting the membrane proteins without damaging the membrane (FENG and UNGER, 1972). Oligomycin has been found to affect ion absorption and A TP-ase activity and these phenomena are bound to the membrane (FISHER and HODGES, 1969; JACOBY and PLESSNER, 1970). A change in permeability of the cell membrane may also affect the ATP level by permitting a leakage of ATP from the root. The author wishes to thank Professor NILS FRIES, Head of the Institute of Physiological Botany, Uppsala, and Professor GORAN STENLID, Department of Plant Physiology, Agricultural College, for their interest, valuable help and stimulating discussions. The excellent technical assistance of Mrs. PIA ISAKSSON and Miss. MARGARETHA MELIN is gratefully acknowledged. Thanks are also due to Professor EDGAR DASILVA for revising the English text. This investigation was supported by grants from P. O. Lundells fond.
References ADEDIPE, N. 0., and R. A. FLETCHIER: Retardation of bean leaf senescence by benzyl adenine and its influence of phosphate metabolism. Plant Physiol. 46, 614-617 (1970). ATKINSON, M. R., G. ECKERMANN, M. GRANT, and R. N. ROBERTSON: Salt accumulation and ATP in carrot xylem tissue. Proc. Natl. Acad. Sci. U.S.A. 55, 560-564 (1966).
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Salt Sensitivity and Adenosine Triphosphate Content of Wheat Roots
303
BEN-ZIONI, A., C. ITAI, and Y. VAADIA: Water and salt stresses, kinetin and protein synthesis in tobacco leaves. Plant Physio!. 42, 361-365 (1967). BERRIDGE, M. V., and R. K. RALPH: Kinetin and carbohydrate metabolism in chinese cabbage. Plant Physio!. 47, 562-567 (1971). BLACKMON, W. J., and D. E. MORELAND: Adenosine triphosphatase activity associated with mung bean mitochondria. Plant Physio!. 47, 532-536 (1971). ELLiS, R. J.: Effects of D-serine and chloramphenicol on amino acid metabolism. Phytochemistry 3, 221-228 (1964). ELLiS, R. J., K. W. JOY, and J F. SUTCLiFFE: The inhibition of salt uptake by D-serine. Phytochemistry 3, 213-219 (1964). ELLiS, R. J., and J. R. MACDONALD: Activation of protein synthesis by microsomes from aging beet disks. Plant Physio!. 42,1297-1302 (1967). FENG, K. A., and J W. UNGER: Influence of kinetin on the membrane permeability of Allium cepa epidermal cells. Experientia 28,1310»1311 (1972). FISHER, J, and T. K. HODGES: Monovalent ion stimulated adenosine triphosphatase from oat roots. Plant Physio!. 44, 385-395 (1969). HANSON, J B., and T. K. HODGES: Uncoupling action of chloramphenicol as a basis for the inhibition of ion accumulation. Nature 200,1009 (1963). HANSON, J B., and W. A. KRUEGER: Impairment of oxidative phosphorylation by D-threoand L-threo-chloramphenico!. Nature 211,1322 (1966). HODGES, T. K.: Oligomycin inhibition of ion transport in plant roots. Nature 209, 425-426 (1966). ITAI, c., A. RICHMOND, and Y. VAADIA: The role of root cytokinins during water and salinity stress. Israel J Bot. 17, 187-195 (1968). JACOBY, B.: The influence of oligomycin on sodium and chloride uptake by beet root tissue. Plant Cell Physio!. 7,307-311 (1966). JACOBY, B., and o. E. PLESSNER: Oligomycin effect on ion absorption by excised barley roots and their ATP content. Planta 90, 215-221 (1970). LAMB, A. J., G. D. CLARK-WALKER, and A. W. LINNANE: The biogene&is of mitochondria. 4. The differentiation of mitochondrial and cytoplasmic protein synthesizing systems in vitro by antibiotics. Biochim. Biophys. Acta 161, 415-427 (1968). LEONARD, R. T., and J. B. HANSON: Induction and development of increased ion absorption in corn root tissue. Plant Physio!. 49, 430-435 (1972). MACDONALD, I. R., J S. D. BACON, D. VAUGHAN, and R. J ELLiS: The relation between ion absorption and protein synthesis in beet disks. J Exp. Bot. 17, 822-837 (1966). MOORE, A. L., K. M. BORCK, and R. BAXTER: The incorporation of amino acids into the protein of isolated soya bean mitochondria. Planta 97, 299-309 (1971). RASMUSSEN, H., and R. NIELSEN: An improved analysis of adenosine triphosphate by the luciferase method. Acta Chern. Scand. 22, 1745-1756 (1968). SAKANO, K., and T. ASAHI: Biochemical studies on biogenesis of mitochondria in wounded sweet potato root tissue. I. Time course analysis of increase in mitochondrial enzymes. Plant Cell Physio!. 12,417-426 (1971 a). Biochemical studies on biogenesis of mitochondria in sweet potato root tissue. II. Active synthesis of membranebound protein of mitochondria. Plant Cell Physio!. 12, 427-436 (1971 b). SKOGQVIST, I.: Induction of thermo sensitivity in wheat roots: salt sensitivity and effects of chloramphenicol and ethano!' Physio!. Plant. 28, 77-80 (1973). SKOGQVIST, I., and N. FRIES: Induction of thermosensitivity and salt sensitivity in wheat roots (Triticum aestivum) and the effect of kinetin. Experientia 26,1160-1162 (1970). STENLID, G.: Flavonoids as inhibitors of the formation of adenosine triphosphate in plant mitochondria. Phytochem. 9, 2251-2256 (1970).
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- The effect of some inhibitory sugars upon the content of adenosine triphosphate in wheat roots. Physiol. Plant. 25, 397-401 (1971). STONER, C. D., T. K. HODGES, and J. B. HANSON: Chloramphenicol as an inhibitor of energy-linked processes in maize mitomondria. Nature 203, 258-261 (1964). SUTCLIFFE, ]. F.: New evidence for a relationship between ion absorption and protein turnover in plant cells. Nature 188,294-297 (1960). INGRID SKOGQVIST, Institute of Physiological Botany, University of Uppsala, S-75121 Uppsala, Sweden.
z. P/lanzenphysiol. Bd. 72. S. 297-304. 1974.
Effect of Chloramphenicol, Ethanol, Kinetin and Oligomycin on Salt Sensitivity and Adenosine Triphosphate Content of Wheat Roots Die Einwirkung von Chloramphenicol, Xthanol, Kinetin und Oligomycin auf Salzempfindlichkeit und A TP-Gehalt von Weizenwurzeln INGRID SKOGQVIST Received October 1, 1973
Summary Chloramphenicol, ethanol, kinetin and oligomycin protected wheat roots from lethal effects of salt treatment. This protection of wheat roots does not seem to have a direct correlation with ATP, since the ATP content of wheat root apex decreased in the presence of ethanol and kinetin and increased with the addition of chloramphenicol.
Zusammenfassung Chloramphenicol, Athanol, Kinetin und Oligomycin schiitzen Weizenwurzeln gegen die lethale Einwirkung von hohen Salzkonzentrationen im Medium. Diese Schutzwirkung scheint nicht direkt mit dem ATP-Gehalt der Wurzel korreliert zu sein, wei! Athanol und Kinetin eine Abnahme, Chloramphenicol aber eine Zunahme des ATP-Gehaltes der Wurzelspitze hervorrufen.
Introduction In previous papers (SKOGQVIST and FRIES, 1970; SKOGQVIST, 1973) it has been shown that heat-shocked wheat roots, in comparison to unshocked roots, are more sensitive to high salt concentrations. Chloramphenicol, kinetin and ethanol were used to prevent the heat-treated roots from dying at a supraoptimal incubation temperature. In this paper results are presented from experiments on the ability of chloramphenicol, ethanol, kinetin and oligomycin to prevent salt sensitivity in wheat roots, which had not been previously subjected to heat treatment. The content of adenosine triphosphate of the wheat roots was also determined, as some of the compounds under test have been reported to inhibit ATP production in various plant cells (ADEDIPE and FLETCHIER, 1970; JACOBY and PLESSNER, 1970).
z. Pjlanzenphysiol. Bd. 72. S. 297-304. 1974.
298
I. SKOGQVIST
Material and Methods Wheat seeds (Triticum aestivum L. "Svenno Varvete» from Weibullsholm, Landskrona, Sweden) were germinated at 25° C in the dark. The seedlings, at the three root stage were placed in 250 ml nutrient medium (SKOGQVIST, 1973). The medium was in a container (250 ml) and each container had 19 plants. The incubation temperature was 25° C in all experiments. When the effect of D-threo chloramphenicol (Park, Davis & Company), ethanol (99,5 0/0, v/v) , kinetin (Calbiochem) or oligomycin (Sigma: oligomycin A 15 % , oligomycin B 85 0/0) was to be studied, these compounds were dissolved prior to placing the plants in the nutrient medium, after which the wheat roots were salt-treated or their content of adenosine triphosphate was determined. The root systems were salt-treated by holding them for 5 minutes in nutrient solution to which an inorganic salt had been incorporated as given in Tables 1-3. The roots were then washed and transferred to fresh medium. The shoots were always kept in continuous light during the experiments. The number of living root tips was determined four days after the salt treatment. In this paper a dead root indicates a root with a visibly dead apical meristem. In each experimental series 57 roots were examined. Every experiment was performed at least three times. For more details of methods the reader is referred to SKOGQVIST (1973). The adenosine triphosphate (ATP) content of root tips was determined 16-20 hours after incubation of the plants in nutrient solution. Pieces of the central roots, 0-10 mm from the apex, were used. Single sections were ground in a glass homogenizer during 30 seconds with 2 ml of ice-cold arsenate buffer. In a Beckman scintillation counter No. 200, 0.1 ml of the homogenate was analysed for ATP by the firefly method (RASMUSSEN and NIELSEN, 1968; STENLID, 1970, 1971). A standard curve with known amounts of ATP was used to calculate the ATP content of the root section. Control extractions with buffer containing ATP showed that ATP was not destroyed during the preparation.
Results
Effect of chloramphenicol in relation to salt sensitivity. All roots in the experiment survived the salt shock when placed in a chloramphenicol solution prior to the salt treatment (table 1). The salt concentration was as high as 0.29 M. The D-threo isomer of chloramphenicol used in this investigation interferes with 50S-70S ribosomes and is an inhibitor of protein synthesis in mitochondria (LAMB et aI., 1968; MOORE et aI., 1971; SAKANO and ASAHI, 1971 a, b). MACDoNALD et a1. (1966) demonstrated that this isomer inhibits the development of CI- absorption capacity in beet disks. The 80S cytoplasmic ribosomes are insensitive to chloramphenicol both in vitro (ELLIS and MACDoNALD, 1967) and in vivo (ELLIS, 1964) but chloramphenicol inhibits salt uptake in several types of plant cells (SUTCLIFFE, 1960; ELLIS et aI., 1964). Therefore, if the D-threo isomer produces its effect through an inhibition of ribosome activity, it could be by an interference with protein synthesis in the mitochondria. This should result in an inhibition of the oxidative phosphorylation, since proteins of the electron transport chain enzymes are synthesized with mitochondrial ribosomes (SAKANO and ASAHI, 1971 a, b). The inhibitory effects of chloramphenicol on oxidative phosphorylation have been demonstrated
z. Pjlanzenphysiol. Bd. 72. S. 297-304. 1974.
Salt Sensitivity and Adenosine Triphosphate Content of Wheat Roots
299
Table 1: The effect of 10-3 M chloramphenicol on salt sensitivity of wheat roots. The values denote per cent surviving roots. Chloramphenicol was added to the nutrient medium 16 hours before the roots were placed in the salt solutions. After this the plants were incubated at 25° C in only nutrient medium. - Similar results were obtained with 10-4 and 5 X 10-4 M chloramphenicol.
Salt NaCI KCl NaBr KBr NaI KI
Chloramphenicol
+ + + + + +
0
0.19
0.20
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 99 100 100 100 99 100 95 100
100 100
0.21
Salt concentration M 0.23 0.24 0.25 55 100 50 100 80 100
100 100
40 100 80 100
30 100 35 100
0.29
100 0 100 0 100 0 100
75 100
100 100 98 100
0.27
0 100
a
100
both in mitochondria and in whole cells from beet disks (HANSON and HODGES, 1963; STONER et aI., 1964; HANSON and KREUGER, 1966; MACDoNALD et aI., 1966). Applied to the present study, this would mean that oxidative phosphorylation should be suppressed in the wheat roots which had been placed in chloramphenicol solution about 16 hours before salt treatment. Consequently the uptake of ions ought to be inhibited allowing for survival of the wheat roots. This possibility was tested in experiments to be described later. Effect of kinetin in relation to salt sensitivity. As with chloramphenicol, the wheat roots survived when kinetin was added to the medium prior to the salt shock (table 2). The protective effect was clear, but not so manifest as with chloramphenicol. Pretreatment with kinetin has earlier been employed to enhance incorporation of 14C-L-Ieucine in both salinity-stressed and non-stressed tobacco leaves (BEN-ZIONl et aI., 1967). In non-stressed leaves optimum incorporation was observed at a kinetin concentration of 5 X 10-7 M while stressed tissue had its optimum at 5 X 10-6 M. ITAl et al. (1968) showed that NaCI gave a marked decrease in the cytokinin activity of root exudates from sunflower, bean and tobacco plants. Salinity-stressed roots brought about a decline in the protein synthesis of the leaves, which could be partially corrected by pretreatment of the leaf disks with kinetin. Kinetin protects the wheat roots against salt treatment when the wheat plants are placed for an 18 hour period before exposure to salt shock in a kinetin solution.
z. Pjlanzenphysiol. Bd. 72. S. 297-304. 1974.
300
I. SKOGQVIST
Table 2: The effect of 5 X 10-6 M kinetin on salt sensitivity of wheat roots. Kinetin was added to the nutrient medium 18 hours before the roots were placed in the salt solution. Otherwise as table 1. - Similar results were obtained with 10-6 and 10-s M kinetin. Salt
Kinetin
NaCl KCl NaBr KBr NaI KI
+ + + + + +
0
0.19
0.20
100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 99 100 100 100 100 100 100 100
100 100
Salt concentration M 0.21 0.23 0.24 55 100 40 90 80 100
100 100
100 100 100 100
0.27
90 100
0.29
1 75 5 75 30 75 0 75
75 95 70 100
95 100
0.25
1 80 50 95
Some workers have reported an effect of cytokinins on A TP synthesis. ADEDIPE and (1970) showed that the ATP content was lowered in leaves of intact bean plants which had been treated with benzyladenine (chemically related to kinetin), although the incorporation of 32p into ATP was higher. BERRIDGE and RALPH (1971), working with cabbage leaf discs found that kinetin increased the rate of synthesis of ATP. Effects 0/ oligomycin and ethanol in relation to salt sensitivity. When wheat plant roots were placed in an oligomycin solution where the oligomycin was dissolved directly at 5,ug/ml prior to the salt treatment, many roots survived the salt shock. Solution of oligomycin in absolute ethanol and subsequent incorporation into the nutrient medium brought about a survival of all roots (except in NaCI and KCI, where a few roots died). Simihrly, many wheat roots survived if only ethanol was added to the nutrient medium prior to the salt treatment (table 3). In many investigations ethanol has been used as a solvent for oligomycin (BLACKMON and MORELAND, 1971; FISHER and HODGES, 1969; HODGES, 1966; JACOBY and PLESSNER, 1970), the final concentration of the solvent in all assays and controls being 10f0 by volume. This concentration of ethanol exhibited no detectable effect on mitochondrial responses (BLACKMON and MORELAND, 1971). In the present investigation the ethanol concentration was only 0.2 0/0 by volume, the same concentration as JACOBY (1966) used. Surprisingly enough this concentration of ethanol alone protected the wheat roots against salts. JACOBY and PLESSNER (1970) found that chloride absorption by excised barley roots from dilute solutions (up to about 0.2 mM) is more oligomycin-sensitive than its absorption from more concentrated solutions. It was also observed that the ATP FLETCHIER
z. Pflanzenphysiol. Bd. 72. S. 297-304. 1974.
Salt Sensitivity and Adenosine Triphosphate Content of Wheat Roots
301
Table 3: The results of three experiments demonstrating the effects of oligomycin and ethanol, alone and together, on the salt sensitivity of wheat roots. When added, these compounds were present in the following concentrations: oligomycin 5,ug/ml and ethanol 1.58 mg/ml (= 0.2 % v/v). The additions to the medium were made 16 hours before the roots were placed in the salt solution. Otherwise as table 1. Salt added (0.27 M)
Addition to the medium Expt. a: No addition With oligomycin Expt. b: No addition With ethanol Expt. c: No addition With oligomycin and ethanol
No
NaCl
KCl
NaBr
KBr
NaI
KI
100 100
0 40
0 45
15 90
15 85
20 100
45 95
100 100
8 90
5 75
15 90
20 90
20 85
45 90
100 100
0 95
0 95
15 100
15 100
25 100
45 100
content of root tissue decreased in the presence of oligomycin. The inhibitor penetrates the plasmalemma and action is at the level of oxidative phosphorylation. In conclusion, if the A TP content of wheat roots is lowered by placing the plants 16 hours prior to salt treatment in an oligomycin solution, this might also influence the survival of the root apex. Effects of chloramphenicol, ethanol and kinetin on the ATP content in wheat roots. Table 4: Effect of chloramphenicol 10-3 M, ethanol 0.2 Ofo (3 X 10-2 M) and kinetin 10-5 M on the ATP content in wheat roots (0-10 mm from apex). ATP content given as per cent of values for control roots growing in nutrient solution without inhibitor. Experiment No.
Duration of treatment h 16
2
20
3
19
Addition to medium during treatment No (Control) Chloramphenicol Ethanol Kinetin No (Control) Chloramphenicol Ethanol Kinetin No (Control) Chloramphenicol Ethanol Kinetin
ATP content found in root sections, 0/ 0 100 120 72
55 100 125 75 60 100 121 60 65
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The ATP content of the control roots of intact wheat plants corresponded to a concentration of about 10-4 M in sections 0-10 mm from the apex. In three different experiments, comprised of ten measurements each of ATP, chloramphenicol caused a significant increase of ATP. Ethanol and kinetin, on the other hand, reduced the content of ATP (table 4). Discussion In contrast to several earlier studies referred to above, the experiments of the present investigation demonstrated that chloramphenicol caused an increase rather than a decrease of the ATP level in the treated material. However, even such a classical uncoupler as 2,4-dinitrophenol may, under certain circumstances, act that way (STENLID, 1971). Since the regulatory mechanisms for the ATP level are very complicated (ATKINSON et aI., 1966) and chloramphenicol interferes with metabolism in several different ways (STONER et aI., 1964; HANSON and KRUEGER, 1966; LEONARD and HANSON, 1972) any attempt to explain the observed effect of chloramphenicol appears meaningless until more experimental data are available. Kinetin and ethanol lowered the A TP level in the roots, although, like chloramphenicol, they were capable to give protection against the deleterious influence of a high salt concentration. There does not seem to be any obvious correlation between the capacity of a compound to protect against salt exposure and its effect upon the ATP level. The possible effects of the protective compounds upon the membranes have not been dealt with in the present paper. It is evident from literature, however, that particularly ethanol and kinetin are active in this respect. In corn roots kinetin caused a decrease in phosphate absorption (LEONARD and HANSON, 1972), and in epidermal onion cells the permeability was changed by affecting the membrane proteins without damaging the membrane (FENG and UNGER, 1972). Oligomycin has been found to affect ion absorption and A TP-ase activity and these phenomena are bound to the membrane (FISHER and HODGES, 1969; JACOBY and PLESSNER, 1970). A change in permeability of the cell membrane may also affect the ATP level by permitting a leakage of ATP from the root. The author wishes to thank Professor NILS FRIES, Head of the Institute of Physiological Botany, Uppsala, and Professor GORAN STENLID, Department of Plant Physiology, Agricultural College, for their interest, valuable help and stimulating discussions. The excellent technical assistance of Mrs. PIA ISAKSSON and Miss. MARGARETHA MELIN is gratefully acknowledged. Thanks are also due to Professor EDGAR DASILVA for revising the English text. This investigation was supported by grants from P. O. Lundells fond.
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