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An anomalous effect of isopropanol on lettuce germination
Plant Science Letters, 15 (1979) 25--28 © Elsevier/North-Holland Scientific Publishers Ltd.
25
AN ANOMALOUS EFFEC'r OF ISOPROPANOL ON LETTUCE GERMINATION T. REYNOLDS
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey (United Kingdom) (Received October 3rd, 1978) (Accepted December 12th, 1978)
SUMMA~Y 1
Isopropanol inhibited the germination of lettuce fruits with EDs0 of 35 -+ 12 mM. However as the concentration of the inhibitor in the medium was increased above 300 mM the percentage germination started to rise reaching a maximum of 62% at 440 mM after which it again decreased. This effect was not shown by closely-related alcohols. Growth of the hypocotyl and to a lesser extent the root showed similar behaviour although at no time did it exceed the control, so that stimulation as such cannot be claimed. It is suggested that isopropanol may produce the effect by altering the permeability of some membrane involved in regulation of germination although why this compound should be unique in showing this effect is not clear from its known properties.
INTRODUCTION
During the course of an investigation on the comparative effects of different concentrations of aliphatic alcohols on germination [ 1] an unusual and so far unique effect of isopropanol was noted. Reports in the literature on the biological activity of isopropanol describe it as being of the same order and type as other aliphatic alcohols of similar molecular weight. The effect was sufficiently prominent to warrant reporting it although explanations at present must remain tentative. MATERIALS AND METHODS
Fruits of Lactuca sativa L. cv. Great Lakes were incubated at 30°C for 3 days on 0.5% agar containing various concentrations of isopropanoI [2]. Measurements of hypocotyl and root growth were made by methods described previously [3], except that normal bench level fluorescent lighting (approx. 1 W/m 2) rather than high-'mtensity fluorescent lighting (17 W/m 2) was used to allow greater hypocotyl growth in the controls.
26 RESULTS AND DISCUSSION
As the concentration o f isopropanol in the medium rose above 20 mM the percentage of fruits germinating fell, reach~lg 50% at 35 -+ 12 mM. However when the concentration rose above 300 mM a proportion o f fruits were able to germinate, to the extent o f 62% at 440 mM, after which the number once again decreased (Fig. 1). This effect was not shown by the closely related alcohols, ethanol, 1-propanol, 1-butanol, 2-butanol, 2-methylpropan-l-ol and 2-methylpropan-2-ol. It was thought that the inhibition might be caused by a stimulatory effect o f the alcohol on the axis at these higher concentrations, as
IN
N
g
40 O
i" !"
103
~
I
-
L 0
' "~_
200
Concentration
b
N4 400
600
SOil
(raM)
Fig. 1. Effects of various concentrations of isopropanol on (a) germination after 3 days at 30°C, (b) hypocotyl growth after 6 days, (c) rootlet growth after 6 days of lettuce (Lactuca sativa L. cv. Great Lakes). Vertical bars show confidence limits for P = 0.05, for means of 5 replicates at each point.
2.7 reported for cucumber hypocotyls [4], acting in opposition to the inhibitory effect. However measurements of stimulation of lettuce hypocotyl growth by isopropanol shows that while a similar course of events as detailed above takes place (Fig. 1) at no concentration is the level of growth higher than control so that the compound cannot be said to stimulate. Measurements of root growth show a progressive inhibition as the concentration is raised, with a much smaller promotion just significant at P = 0.05 (Fig. 1). These results indicate that while the response to isopropanol is shown by both the hypocotyl and the intact seed it is not the result of the opposing interactions of an inhibition and a stimulation. A survey of several chemical and physical properties of isopropanol [ 5--7 ] failed to reveal other anomalies peculiar to this compound while other reports speak of its biological effects as differing in degree but not in kind from ethanOl. Isopropanol forms liquid crystals with cholesterol and it is implied that this behaviour is unique among the lower alcohols [8]. It has been found that for a non-permeating undissociated solute germination is inhibited by osmotic pressures produced by concentrations of approx. 140 mM [2]. The effects described here occur at much higher concentrations implying that isopropanol is penetrating the cell barriers. This raises the question as to whether the alcohol is metabolized and if this metabolism is in any way responsible for the phenomenon observed. Membrane effects are frequently invoked in explanation of biological reactions of organic solvents. It may be in this instance that when the concentration of isopropanol in the lettuce fruits rose to some level critical for any particular fruit, then permeability of a membrane was altered in such a way that germination was penn~tted in spite of the high ambient concentration of the compound. As the site of inhibition is also unknown explanations can only be speculative. It could be that isopropanol at the effective level increases permeability of a cell compartment as was suggested by Guinn [9] allowing enhanced transport of a promotor sufficient to overcome the inhibitory effects. It is noteworthy that at the height of the secondary germination 'peak' only approx. 60% of the population responded by germinating, although 8 0 - 9 0 % germinated [10] in water at the temperature of the experiment. Also once germination had taken place, the EDso for inhibition of shoot and root growth was much greater than that for germination. ACKNOWLEDGEMENTS
I am grateful for technical assistance from Mr. F. Jowah, Miss A. Clydesdale and Miss J. Moore. REFERENCES
1 T. Reynolds, Ann. Bot., 41 (1977) 637. 2 T. Reynolds, Ann. Bot., 39 (1975) 791. 3 T. Reynolds, J. Exp. Bot., 21 (1970) 702.
9.8
4 5 6 7
W.K. Purvcs and C.H. Fricker, Z. Pflanzenphysiol., 54 (1966) 203. FLS. Garrick, Ind. Chem., 3 (1927) 392. L.F. Hatch, Isopropyl Alcohol, McGraw-Hill, New York, 1961. R.C. West (Ed.) Handbook of Chemistry and Physics, 54th edition, C.R.C. Press, Cleveland, OH, 1973. 8 W.E. Acree and G.L. Bertrand, Nature, 9.69 (1977) 450. 9 G. Guinn, Plant Physiol., 60 (1977) 446. I0 T. Reynolds, Planta, 113 (1973) 327.
25
AN ANOMALOUS EFFEC'r OF ISOPROPANOL ON LETTUCE GERMINATION T. REYNOLDS
Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey (United Kingdom) (Received October 3rd, 1978) (Accepted December 12th, 1978)
SUMMA~Y 1
Isopropanol inhibited the germination of lettuce fruits with EDs0 of 35 -+ 12 mM. However as the concentration of the inhibitor in the medium was increased above 300 mM the percentage germination started to rise reaching a maximum of 62% at 440 mM after which it again decreased. This effect was not shown by closely-related alcohols. Growth of the hypocotyl and to a lesser extent the root showed similar behaviour although at no time did it exceed the control, so that stimulation as such cannot be claimed. It is suggested that isopropanol may produce the effect by altering the permeability of some membrane involved in regulation of germination although why this compound should be unique in showing this effect is not clear from its known properties.
INTRODUCTION
During the course of an investigation on the comparative effects of different concentrations of aliphatic alcohols on germination [ 1] an unusual and so far unique effect of isopropanol was noted. Reports in the literature on the biological activity of isopropanol describe it as being of the same order and type as other aliphatic alcohols of similar molecular weight. The effect was sufficiently prominent to warrant reporting it although explanations at present must remain tentative. MATERIALS AND METHODS
Fruits of Lactuca sativa L. cv. Great Lakes were incubated at 30°C for 3 days on 0.5% agar containing various concentrations of isopropanoI [2]. Measurements of hypocotyl and root growth were made by methods described previously [3], except that normal bench level fluorescent lighting (approx. 1 W/m 2) rather than high-'mtensity fluorescent lighting (17 W/m 2) was used to allow greater hypocotyl growth in the controls.
26 RESULTS AND DISCUSSION
As the concentration o f isopropanol in the medium rose above 20 mM the percentage of fruits germinating fell, reach~lg 50% at 35 -+ 12 mM. However when the concentration rose above 300 mM a proportion o f fruits were able to germinate, to the extent o f 62% at 440 mM, after which the number once again decreased (Fig. 1). This effect was not shown by the closely related alcohols, ethanol, 1-propanol, 1-butanol, 2-butanol, 2-methylpropan-l-ol and 2-methylpropan-2-ol. It was thought that the inhibition might be caused by a stimulatory effect o f the alcohol on the axis at these higher concentrations, as
IN
N
g
40 O
i" !"
103
~
I
-
L 0
' "~_
200
Concentration
b
N4 400
600
SOil
(raM)
Fig. 1. Effects of various concentrations of isopropanol on (a) germination after 3 days at 30°C, (b) hypocotyl growth after 6 days, (c) rootlet growth after 6 days of lettuce (Lactuca sativa L. cv. Great Lakes). Vertical bars show confidence limits for P = 0.05, for means of 5 replicates at each point.
2.7 reported for cucumber hypocotyls [4], acting in opposition to the inhibitory effect. However measurements of stimulation of lettuce hypocotyl growth by isopropanol shows that while a similar course of events as detailed above takes place (Fig. 1) at no concentration is the level of growth higher than control so that the compound cannot be said to stimulate. Measurements of root growth show a progressive inhibition as the concentration is raised, with a much smaller promotion just significant at P = 0.05 (Fig. 1). These results indicate that while the response to isopropanol is shown by both the hypocotyl and the intact seed it is not the result of the opposing interactions of an inhibition and a stimulation. A survey of several chemical and physical properties of isopropanol [ 5--7 ] failed to reveal other anomalies peculiar to this compound while other reports speak of its biological effects as differing in degree but not in kind from ethanOl. Isopropanol forms liquid crystals with cholesterol and it is implied that this behaviour is unique among the lower alcohols [8]. It has been found that for a non-permeating undissociated solute germination is inhibited by osmotic pressures produced by concentrations of approx. 140 mM [2]. The effects described here occur at much higher concentrations implying that isopropanol is penetrating the cell barriers. This raises the question as to whether the alcohol is metabolized and if this metabolism is in any way responsible for the phenomenon observed. Membrane effects are frequently invoked in explanation of biological reactions of organic solvents. It may be in this instance that when the concentration of isopropanol in the lettuce fruits rose to some level critical for any particular fruit, then permeability of a membrane was altered in such a way that germination was penn~tted in spite of the high ambient concentration of the compound. As the site of inhibition is also unknown explanations can only be speculative. It could be that isopropanol at the effective level increases permeability of a cell compartment as was suggested by Guinn [9] allowing enhanced transport of a promotor sufficient to overcome the inhibitory effects. It is noteworthy that at the height of the secondary germination 'peak' only approx. 60% of the population responded by germinating, although 8 0 - 9 0 % germinated [10] in water at the temperature of the experiment. Also once germination had taken place, the EDso for inhibition of shoot and root growth was much greater than that for germination. ACKNOWLEDGEMENTS
I am grateful for technical assistance from Mr. F. Jowah, Miss A. Clydesdale and Miss J. Moore. REFERENCES
1 T. Reynolds, Ann. Bot., 41 (1977) 637. 2 T. Reynolds, Ann. Bot., 39 (1975) 791. 3 T. Reynolds, J. Exp. Bot., 21 (1970) 702.
9.8
4 5 6 7
W.K. Purvcs and C.H. Fricker, Z. Pflanzenphysiol., 54 (1966) 203. FLS. Garrick, Ind. Chem., 3 (1927) 392. L.F. Hatch, Isopropyl Alcohol, McGraw-Hill, New York, 1961. R.C. West (Ed.) Handbook of Chemistry and Physics, 54th edition, C.R.C. Press, Cleveland, OH, 1973. 8 W.E. Acree and G.L. Bertrand, Nature, 9.69 (1977) 450. 9 G. Guinn, Plant Physiol., 60 (1977) 446. I0 T. Reynolds, Planta, 113 (1973) 327.