Summary. An improved technique for measuring electric potential differences on the etiolated Avena .coleoptile is described. An electric wave has been ...
ELECTRIC POTENTIALS AND AUXIN TRANSLOCATION IN AVENA By 1. A.
NEWMAN-
[Manuscript received December 24, 1962] Summary An improved technique for measuring electric potential differences on the etiolated Avena .coleoptile is described. An electric wave has been observed to travel down the coleoptile at a speed of up to 14 mm/hr. The wave is observed either follo-wing illumination of the intact coleoptile or following application of 3·indolylacetic acid to the cut top. The wave appears to be associated-intimately with the stream of auxin being translocated. The mechanism of auxin translocation is considered in relation to the wave. It is suggested that the electric wave may be useful as a bioassay for auxin.
1.
INTRODUCTION
The mechanisms of translocation and redistribution of growth hormones have for long presented a challenge to plant physiologists. The hormones, 3-indolylacetic acid (IAA) in particular, act not ouly to produce extension growth nnder appropriate conditions but they also serve to transmit information which produces bending and other responses in the plant. The translocation is characteristically polar and at a speed many times greater than can be accounted for by diffusion. Went and White (1939) determined the speed of translocation of IAA in the Avena coleoptile to be about 12 mm/hr. No temperature was given as they apparently assumed, however, following van der Weij (1932), that the speed of translocation of 1AA is independent of temperature. Van der Weij's conclusion has been seriously criticized by Gregory and Hancock (1955) who have shown that his results do in fact demonstrate a temperature variation in the speed of translocation of 1AA in Avena. Gregory and Hancock also report a speed of translocation of 7 mm/hr for natural auxin in apple shoots at the optimum temperature, 27°C. The temperature coefficient of the speed at temperatures below 27°C is about 2. Tropic stimuli are known to cause alterations in the growth hormone stream moving from the apical regions of the plant (Went and Thimann 1937). Briggs, Tocher, and Wilson (1957) and Gillespie and Briggs (1961) have given clear evidence for - redistribution of auxin in the apex of corn coleoptiles following unilateral illumination by white light or following a geotropic stimulus. The hypothesis that lateral redistribution of auxin, to cause tropic responses, is mediated by an electric field was proposed by Went (1932) and has been considered by others since. Backus and Schrank (1952) and Grahm and Hertz (1962) have observed electric potential difference changes on the Avena coleoptile following phototropic or geotropic stimuli. These electric changes were found to occur, moreover, even when bending in response to the stimuli was suppressed by a lack of auxin . • Department of Physics, University of Tasmania, Hobart.
Awt. J. Bioi. Sci., 1963, 16,
629-4~
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I. A. NEWMAN
Schrank and Backus (1951) have observed bonding of Avena following the passage of a transverse electric current. From these results it appears clear that there are electric potentials associated with plant tropisms. Hmvever, the mechanism relating the two is obscure. It is possible also that the normal longitudinal translocation of auxin is associated with electric fields. If this is so, appropriate electrioal investigations should provide information about the mechanism of auxin translocation. It is generally held that the results of electrical measurements on plants are difficult to interpret in terms of plant development. Unless special precautions are taken with liquid contacts, variation in concentrations of salts in the liquid in contact with the plant may cause the measured values of the electric potentials of the plant under investigation to differ considerably from the potentials which it was designed to measure (Scott, McAulay, and Jeyes 1955).
The techniques developed in this Laboratory for measuring bioelectric potentials on coleoptiles and shoots have been designed to maintain a known constant concentration of solution at the points of electrical contact with the plant. It is found that reliable and repeatable measurements of bioelectric potentials on the plant surface can be made. This permits measurements to be made of variations in the potentials due to biological causes only. Other workers have avoided the difficulties inherent in the -liquid contacts by using electrostatic methods of measuring potential differences (e.g. Grahm and Hertz 1962). The electrostatic methods, however, suffer from two dis~dvantages: (1) they do not discriminate against electric fields set up by distributions of static charge which frequently occur on waxy surfaces, but which are unlikely to have much biological significance; (2) they are relatively insensitive for localized measurements-for example, the electrode .used by Grahm and Hertz (1962) had dimensions of 5 mm by 10 mm.
Clark (1937) has shown that electric fields of the magnitude of those measured on the surface of plants are insufficient to cause bulk movement of IAA through agar, or presumably through plant tissue. However, electric fields of much greater magnitude may exist in small regions of plants; for example, Walker (1955), among others, has shown that potential differences of over 100 m V exist across membranes of individual cells of some species of algae. Such fields may playa significant part in biological processes; they might, for example, be associated with 1AA translocation if membranes over which the fields act are sites of regulation of1AA movement. Although direct observation of such fields would be most difficult in multicellular plants, they would contribute to the overall field observable on the surface of the plant. Surface potential measurements may therefore provide information about the strong localized fields and hence about related biological processes. A previous publication (Newman 1959) has shown the existence of a well-defined electric potential wave which moves down the Avena coleoptile apparently in close association with the stream of 1AA. The further investigation of this wave, and its relationship to 1AA translocation, is the subject of the present paper.
ELECTRIC POTENTIALS AND AUXIN TRANSLOCATION IN AVENA
II.
631
:MATERIALS AND TECHNIQUES·
Avena sativa cv. Blythe Oats, kindly supplied by the Tasmanian Department of Agriculture, was the material used. The seeds were germinated and grown in tap water in darkness at 25'0 and with their distal end held in individual small plastic tubes suspen(led against moist filter paper from a rack in a closed dish. First internodal growth was suppressed by illumination by a red safelight "for 90 sec every hour during growth. The experimental cabinet was thermostatically controlled to within ±t'0 of the desired temperature. Humidity was not measured but was presumably near 100% as there was always ample free water within the cabinet and the agar block did not appear to decrease in size during the course of an experiment, except at the lowest temperatures when condensation occurred on the cooling coils, thereby lowering the humidity in the rest of the cabinet. Operations in the experimental cabinet, except where indicated below, were made in the dark or under a weak green safelight similar to that used by Nitsch and Nitsch (1956). Straight 75-85-hr-old seedlings, 33±3 mm high, were selected for the experiments. When IAA was to be subsequently applied the apical 2 mm was removed. Two hours later the primary leaf was pulled out and the seedling placed in a holder, which lightly clamped the base of the coleoptile, in the experimental cabinet at 25°0. For experiments in which isolated coleoptile segments were used, each 10 rum long sement was supported by having 1 mm of its lower end inserted into a thin plastic tube which was clamped in the holder. A far-red photographic safelight (Wratten No.1) provided the illumination for these operations. 165 minutes after the first decapitation the electrical contacts were placed on the coleoptile (see below). Fifteen minutes later a further 2-mm segment was removed from the top of the coleoptile and in the subsequent 10 min the temperature was adjusted if necessary to the value required for the experiment. Ten minutes after the second decapitation a 5 mm 3 block of agar was placed on top of the coleoptile. The agar had been soaking for between 2 and 5 hr in a solution of IAA of the required concentration. An incandescent lamp with heat filter and neutral density filters was used as the (unilateral) light source for experiments on the effect of illumination on potential differences. For Sections III(b)(i) and III(b)(ii) the intensity was 3000 metre candles (m.c.) and the whole of one side of the coleoptile was illuminated. For Section III(b)(iii) the intensity was 50 m.c. on one side oLthe apical 2 mm of the coleoptile. A black paper shield ensured that the intensity on the rest of the coleoptile was less than 5 m.c., most of this being due to light scattered from the illuminated region.
Electrical contact with the coleoptile was made by one or other of two methods, both of which involved flowing 10-2M potassium chloride solution at the points of electrical contact. The value of this technique is that the flowing solution maintains steady state conditions at the points of contact, despite evaporation or salt uptake. The first method was used for the measurements on intact coleoptiles (Sections III(a)· and III(b)). The probe system for this is shown diagrammatically in Figure 1. The contact solution was forced by a pressure of about t atm through the fine
632
I. A. NEWMAN
central tube to the tip of the probe where it formed a small liquid bridge (of diameter 0·5 ±0·1 mm) with the coleoptile before flo,,~ng away through the annular space and then past the electrode. It dripped from the outlet, 20 mm below the probe tip, at a rate of several cubic centimetres per hour. The probe was mounted on a micromanipulator and the probe tip could be placed at any level on one side of the coleoptile. Measurements could then be made of the potential difference between the probe and a reference probe at the base of the coleoptile. For transverse potential difference measurements an additional movable probe was used on the other side of the coleoptile.
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The second method is a semi-automatic Oile for which six absorbent cotton threads, along which the potassium chloride solution is flowing, are placed in contact with the coleoptile at 2-mm intervals. Evaporation from the solution flowing along the threadl:? is negligible. The solution from each of the six reservoirs flows along its thread at a rate of about t ml/hr and passes down a thin plastic tube to one of six mercury-calomel half-cells. Pairs of these cells are automatically connected in succession to the electrometer input (impedance about 1012.0) of a six-channel recorder (described by Scott 1957). Measurements of potential differences over five successive 2-mm segments near the tip of the coleoptile are thus recorded. To ensure good electrical contact with the coleoptile for either method it was found necessary to scrape the cuticle gently at the desired positions. This did not produce much electrical stimulus and served to reduce the contact resistance from more than lO'.Q to about 3 X 10'.Q.
ELECTRIC POTENTIALS .AND AUXIN TRANSLOCATION IN AVENA
III.
633
RESULTS
(a) Electric Potentials on the Intact, Uwtimulated Coleoptile The potential distribution along the surface of each of six coleoptiles was measured at 25°0 under the red safelight. Figure 2 shows the potential as a function of distance from the apex. The mean and 95% confidence limits for the mean of the observations are shown at each point, 2 mm apart. The point at 1 mm below the apex was taken as the zero of potential for each coleoptile. The base is electrically positive to the apical region. The region about 5 mm below the apex is significantly (at the 95% level) negative to the apex.
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Fig. 2.-Electric potential (V) plotted against distance below the apex of the intact coleoptile. The point at 1 mm below the apex was taken as the zero of potential for each coleoptile. Means of results from six plants are shown with 95 % confidence limits.
(b) Electric Potential Changes following Illumination of the Intact Colwptile (i) Transverse Potential Differences.-Measurements were made of the potential difference between probes placed on opposite ends of the narrow axis of the coleoptile at 1 mm below the apex. For a sample of 12 coleoptiles the mean transverse potential difference in red light alone was not significantly different from zero. The mean potential difference remained zero for the first 23 min after the white light of 3000 m.c. intensity, and which shone on to the side of the coleoptile contacted by one of the probes, was turned on. The shaded side of the coleoptile then became positive, reaching a maximum of +3·5±1·5 mV (95% confidence limits) at 33 min. Mter 43 min of illumination the mean potential difference had returned to zero where it remained until the end of the measurements at 55 min. In four experiments with the probes only O· 2 mm below the apex on either end of the wide axis of the coleoptile no consistent change in transverse potential difference was apparent. The probes in this case also were on illuminated and shaded sides of the coleoptile respectively. (il) Longitudinal Potential Differences.-Potential differences between the base of the coleoptile and points near the apex, measured at the same time as the transverse potential differences above, showed variations of large magnitnde. The potential at a point 1 mm below the apex rose to a peak of about 20 m V above its resting value and then fell again. The mean time of occurrence of this peak was
634
I. A. NEWMAN
35·9±1·1 min after the start of the white illumination of 3000 m.c. intensity (sample size = 12). Bending of the coleoptiles towards the light began at 38·9±2·5 min. At 4 mm below the apex the peak height was about 30 m V and it occurred 40·9±1·2 min after the illumination began (sample size = 6 in this case.) Bending began for this sample at 37 ±4 min. The peak occurs significantly later at the lower level. (iii) Potential Differences over 2-mm Intervals.-To determine the site of the large longitudinal potential change, potential differences were measured over 2-mm intervals between the points 0, 2, 4, 6, and 8 mm below the apex. To limit the light stimulus the illumination was decreased in intensity to 50 m.C. and restricted to the +, ,
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Fig. a.-Potential difference (V) over each of four successive zones below the coleoptile apex is plotted against time after the commencement of apical illumination. V o- V 2 , for example, is the potential difference between 0 and 2 mm below the top of the coleoptile. 95 % confidence limits for the means of six coleoptiles are shown at two times for the graphs V 2 - V 4 and V 6 - V s. Confidence limits at other points are of similar magnitude.
upper 2 mm of the coleoptile. Some light was scattered from the illuminated region down to lower levels of the coleoptiIe, but it is estimated that this was less than 10% of the incident intensity, i.e. less than 5 m.c. Illumination of the apical 2 mm by white light of 5 m.C. intensity did not produce any significant potential difference vari'ati9ns. The changes occurring in these four potential differences, measured on the shaded side of the coleoptiIe, are shown in Figure 3. The white light was turned on at the zero of time in the figure. V2 - V4 is the potential difference between the points at 2 mm and 4 mm below the top of the coleoptile. Each of the six plants treated showed tills behaviour. The points on the figure are the means of the results from the six coleoptiles. The fall in potential difference from peak to trough is, for each of the four graphs in the figure, statistically significant at tne 1 % level at least. A similar fall was observed, but not recorded, with diminished magnitude
ELECTRIC POTENTIALS AND AUXIN TRANSLOCATION IN AVENA
635
in the potential difference V s-'V10 ' No measurements were made below 10 mm from the apex. A few measurements were made concurrently of potentials on the side of the coleoptile from which the illumination was coming. These, in agreement with Section III(b)(i), showed no great difference from those on the shaded side. The coleoptile was observed under a dissecting microscope. Bending of the coleoptile towards the light was noted to begin at each level at about the time of the fall from peak to trough in the potential difference in that region. The spacing down Figure 3 of the four time-axes represents -2-mm intervals down the coleoptile, and it is seen that the times on these axes of the peaks form a straight line on the figure. Such a line may be drawn on the graphs of results from 1-10
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Fig. 4.-Potential difference (V) over each of five successive zones below the cut top of the coleoptile is plotted against time after application of lAA (2 mgjl) to the cut top. V 2 - V 4' for example, is the potential difference between 2 and 4 nun below the cut top of the coleoptile. 95% confidence limits for the means of the six plants are shown at two times for each graph.
each of the six coleoptiles. The mean slope of this line is 16·8±2·7 mmJhr (95% confidence limits for the mean). Thus this is the rate of downward movement of the electrical wave initiated by the illumination. The magnitude of the changes in potential difference may conveniently be represented by the mean change from the peak to trongh in Vo- V,. This is 16 mY. (c) Electric Wave Induced by the Application of IAA
(i) Basic Experiment at 25°C, using ColeoptUes Attached to the Beed.-Potential differences, were measured over 2-mm intervals beginning 2-mm from the top of a doubly decapitated coleoptile. The second decapitation was performed 15 min
636
I. A. NEWMAN
after the beginning of measurements. The electrical stimulus caused by decapitation was usually localized in the upper 6 mm and it died away after about 10 min. Ten minutes after the second decapitation a block of agar containing IAA at a coucentration of 2 mg/l was placed on the top of the coleoptile. Figure 4 shows the electrical behaviour following such treatment of six coleoptiles at 25°0. The time "zero" is the instant of application of the agar block. These measurements were made manually with the single movable probe. Other measurements, made with the wet cotton contacts and recorded automatically, conform to the behaviour shown +,
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in Figure 4. As was the case with the illuminated coleoptiles an electric wave is observed to move down the coleoptile. The rate of movement of the wave for each coleoptile can be measured as described in Section III(b)(iii). The mean magnitude of the changes in potential difference may be represented by the initial fall in V 2 - V 4 • It is 17 mV. It is seen that damped oscillations in potential difference occur in each experiment after the initial part of the disturbance. These oscillations may be seen more clearly in the results of measurements made on a few additional coleoptiles over longer periods of time. A representative example of changes in potential difference on one such coleoptile is shown in Figure 5. In control experi~ ments for which agar blocks without any IAA were used, no significant changes in potential difference were observed. (ii) Using Coleoptile Segments.-The above experiment was repeated using, instead of the coleoptile attached to the seed, the apical 15 mm of the coleoptile
ELECTRIC POTENTIALS AND AUXIN TRANSLOOATION IN AVENA
637
supported by its basal end in a thin plastic tube. An electric wave was observed as before. Its rate of movement, in this case for a sample of eight coleoptiIes, was l3·9±1·9 mm/hr, the same rate as was found for the whole coleoptile. The mean magnitude of the initial fall in V,- V, was 12 mY. (iii) Using the Central Region of the Coleoptile.-The basic experiment (see Section II1(c)(i)), was repeated with a different modification: the second decapitation was of 10 mm instead of 2 mm. This was to test for entry of 1AA and initiation
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Fig. 6.-Potential differences plotted against time as for Figure 4. In this case the agar contained IAA at a concentration of O· 2 mg/l and remained on top of the coleoptile for 10 min only". Representative 95% confidence limits are shown at two times on the graph V 2. - V 4'
of the electric wave at about 12 mm from the apex. Of 11 coleoptiles, one showed no significant wave; this was neglected. Six showed indistinct waves; these were given half weight in the statistical treatment. The mean rate of movement of the wave in this case was 1O±3 mm/hr. This is significantly slower (1 % level) than in the apical region and there is significantly more variability in the results (variance ratio test at the 0·1 % level). The mean magnitude of the inital fall in V,- V, is 4 m V, which is one-quarter the corresponding change stated in Section II1(c) (i). (iv) fAA Application for Short Time Only.-Figure 6 shows the mean behaviour of four coleoptiles on which agar blocks containing IAA at a concentration 0·2 mg/l were placed for only 10 min. The lower concentration of 1AA was necessary to avoid large passive absorption of the hormone by the top layer of tissue. Oscillations in the potential difference do normally occur with this concentration (see Section
638
I. A. NEWMAN
III(c)(vi)). In this case just one cycle of the oscillation occurs, without subsequent oscillations of decreasing magnitude. The mean rate of movement of the wave in this case is 1O·6±0·7 mm/hr. The mean magnitude of the initial fall in V2 - V. is 26 m V. This is larger than the fall (17 m V) which was observed when the concentration of 1M was 2·0 mg/I (see Section III(c)(i)). (v) The Electric Wave at Different Temperatures.-The basic experiment (see Section III(b)(i)), was repeated at five different temperatures. Figure 7 shows the mean speed of movement of the wave (with 95% confidence limits) and sample sizes at the six temperatures from 12 to 33°0. In each of five experiments at 7°0
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