Furthermore, elongation rate was higher in roots with higher oscillation frequency. Oscillation frequency had a strong depend- ence on temperature; i.e. 010was ...
Plant Physiol. (1990) 93, 532-536 0032-0889/90/93/0532/05/$01 .00/0
Received for publication September 8, 1989 and in revised form January 15, 1990
Relationship between Growth and Electric Oscillations in Bean Roots Masaaki Souda, Kiyoshi Toko*, Kenshi Hayashi, Takanori Fujiyoshi, Shu Ezaki, and Kaoru Yamafuji Department of Electronics, Faculty of Engineering, Kyushu University 36, Fukuoka 812, Japan (M.S., K. T., K.H. T.F., K. Y.) Department of Electric Engineering, Faculty of Engineering, Kinki University in Kyushu, lizuka 820, Japan (S.E.) lation was propagated to the mature region inside the parenchyma. A mechanism for the oscillations has not, however, been elucidated especially from the viewpoint of a relation to growth. The purpose of the present paper is to study the relationship between growth and electric oscillation in roots. The surface electric potentials were measured together with a measurement of elongation.
ABSTRACT Extracellular and intracellular electric potentials in bean roots are known to show electric oscillations along the longitudinal axis with a period of several minutes. The relationship between growth and the electric oscillations was studied using roots of adzuki (Phaseolus chrysanthos). We measured surface electric potentials with a multielectrode apparatus while simultaneously measuring elongation using a CCD camera and monitor. Roots having an electric oscillation grew faster than roots with no oscillation. Furthermore, elongation rate was higher in roots with higher oscillation frequency. Oscillation frequency had a strong dependence on temperature; i.e. 010 was estimated at 1.7. These results suggest a correlation between electric oscillation and elongation.
MATERIALS AND METHODS Plant Material
Experiments were done using roots of adzuki bean (Phaseolus chrysanthos) seedlings which were 4 to 5 d old. Seeds were soaked in water at 40 ± 1°C for 3 h and were placed on filter papers wetted with 0.1 mm KCI plus 0.05 mM CaCl2 solution in darkness at 30 ± 1°C.
A periodic electrical pattern is formed near the surface of the internodal cell of Characean species (13). Electric current patterns appear along the surfaces of roots (1, 4, 20, 25). Oscillations of surface electric potential also occur (7, 8, 16, 19, 22). Bean roots show spontaneously electric oscillations without a stimulus along the root surface with about 5 min period, which continues constant for over a few hours (16, 19). The amplitude of the oscillation is maximal in the elongation region and the phase of the oscillation in that region differs by 1800 from that in the mature region. It is noticeable that the oscillation appears consistent (or coherent) in the mature region over several centimeters, because the phase and also the period are the same at any position (16, 19, 22). Resonance of oscillation occurs in such a way that the phase of osmotic pressure variation sets the phase of electric oscillation when an oscillation of osmotic pressure is applied to a root (7, 8). In a previous paper (22), the membrane potential within the root was measured with a microelectrode technique (2) while the surface electric potential was measured using a multielectrode measuring method (4, 19, 20). In the elongation region, the membrane potential of epidermal cells oscillated with the same period and the same phase as the surface potential. In the mature region, on the other hand, the membrane potential did not oscillate, although the surface potential oscillated. It was concluded from a theoretical analysis using an equivalent electrical circuit that the source ofsurfacepotential oscillations existed in the membrane at the xylem/ parenchyma interface in the elongation region and the oscil-
Measurement System Figure 1 shows the measurement system. An acrylic case was divided into two chambers by an acrylic sheet for measurements. A filter paper was laid on the bottom of the upper chamber and a seedling was laid horizontally on the filter paper. A seedling was submerged in 0.1 mm KCI and 0.05 mM CaCl2 . A root was fixed on the filter papers by applying a 0.5% agar plate on the elongation zone, because the root tended to float on the aqueous solution 5 mm in depth. Three to seven pipette electrodes were arranged near the root surface at about 1 mm intervals along the tip and elongation zone; a reference electrode was placed in the medium. Each electrode has a tip diameter of approximately 300 ,um and filled with 100 mM KCI and 1% agar, containing an Ag/AgCl wire. A waterproof transistor was placed in the solution to measure the temperature. It was connected to an amplifier, and hence the temperature was measured from a change of the electric current in the transistor. A CCD camera mounted on a microscope was set above the case for tracking the movement of the root tip. We illuminated the case with a miniature bulb (4.8 V, 0.5 A) from the bottom for the purpose of saving the intensity (300 lux) of light and increasing the contrast. The image of the root was recorded using a video-tape recorder and also displayed on a monitor. To control the temperature of the solution bathing the root, the water temperature in the lower chamber was adjusted using a heater and a thermistor con532
GROWTH AND ELECTRIC OSCILLATIONS IN ROOTS
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,um. The power spectra of electric potentials were calculated by means of a maximum entropy method in order to estimate the frequency of oscillations. This method (23) is useful and accurate in the case of not so many data points like this experiment. Because the root elongated substantially on the filter paper during measurements, the positions of electrodes changed relative to the position of root tip. However the oscillations near the elongation region, when they occurred, had similar wave forms at all electrode locations. Therefore, only representative data from one electrode will be shown in "Results."
RESULTS Miniature bulb
Figure 1. Measurement system. The acrylic case was divided into two chambers. The temperature of solution in the upper chamber was controlled by changing the water temperature in the lower chamber. A CCD camera was set above the case to measure the elongation, and at the same time the extracellular surface potentials were measured with pipette electrodes.
Relation between Elongation and Oscillation
Figure 2 shows examples of extracellular electric potentials in a root exhibiting an oscillation (A) and a root with no oscillation (B). The oscillation period is about 7.5 min and the oscillations were observed at all three electrode positions (A). In (B) very small fluctuations appear at all the electrodes. The elongation speeds were estimated at 667 and 429 um/30 min for (A) and (B), respectively.
nected to a temperature controller. The measured surface electric potentials were loaded into a personal computer after analog-digital conversion.
Measurement After positioning the root and electrodes in the upper chamber, the system was allowed to equilibrate for 1 h. Then, short-term measurements ofthe surface electric potential were made for 30 min while displacement of the root tip was recorded using the CCD camera. The temperature, which was measured by transistor in solution, was controlled with the heater and the air conditioner in room. In this experiment, three electrodes were arranged near the root surface in the elongation region. Removing the root after the measurement, the offset potential caused by each electrode itself in the aqueous medium was input to the computer and extracellular surface potential was obtained by subtraction of offset potential (19, 20). Elongation was evaluated from the change of the position of the root tip on the monitor. When the effect of temperature on the oscillation of surface potential was studied, water at 1 9°C was poured into the lower chamber before placing the seedling in the upper chamber. Potentials near the elongation region were measured after 1 h at a low temperature (-20°C) for 1.5 h at a middle temperature (-23°C) for 2 h, and then at a high temperature (-27°C) for 2 h. The temperature oflower chamber reached the desired one in 1 min and then the temperature of upper chamber reached the same in several minutes. In these long-term experiments, the elongation region was displaced more than 3 mm during measurements; hence, seven electrodes were arranged in a straight line ahead of the root tip and near the root-tip side, including the elongation region. The electrodes were placed about 400 jgm distant from the root surface; it is a distance enough for electrodes not to contact with the root if root extension occurs. The electric oscillations could be detected safely by this arrangement. Elongation per 10 min was calculated using the monitor, which could distinguish 5
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20 30 Time (min) Figure 2. Extracellular electric potentials at three points in elongation region of a root showing oscillations (A) and a root with no oscillations (B).
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period for a given temperature or narrow temperature range (also see Fig. 4). However, the period has a tendency to become shorter at higher temperatures as shown by a line to fit the data using the least squares method. The temperature coefficient, Qio, of the period can be calculated to be 1.7. Figure 6 shows the relationship between the logarithm of the frequency and the inverse of the absolute temperature. The data are from Figure 5, the line being obtained with the method of least squares. The slope of the line is -2031. Regarding the frequency as a measure of the reaction velocity, an activation energy can be calculated to be about 9249 (cal/ mol), which is close to the activation energy of typical enzymic reactions (26). This result along with the above Qlo suggests that the oscillation has some relation with enzymic reactions.
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Real-Time Correlation between Elongation and Oscillation
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Plant Physiol. Vol. 93, 1990
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Figure 3. Difference of the elongation per 30 min between oscillating (A) and nonoscillating roots (B). The total number of used roots was 118. Among them, 87 roots showed the electric oscillations.
Figure 3 shows the distribution of elongation rates for roots showing electric oscillations (A) and roots showing no oscillations (B) at temperatures between 18 and 30C. Of the 1 18 roots examined, 87 showed oscillations. The elongation rate of oscillating roots extends over a wide range between 300 and 1000 Mm/30 min and has two peaks around 500 and 700 ,gm/30 min. The average elongation rate was 668 Mm/30 min. The roots without oscillations, on the other hand, tended to elongate more slowly (the average, 501 ,um/30 min). Figure 4 shows the relationship between elongation rate and the frequency of oscillation at nearly a constant temperature (28-29°C). The data are from 13 roots from Figure 3. The line is drawn by means of a least squares method. Similar results were obtained for other temperatures. Whereas the data are widely scattered, we can see a tendency of the larger elongation with the higher oscillation frequency. Effect of Temperature on Oscillation Figure 5 shows a relationship between temperature and the oscillation period. The data are from the 87 roots showing oscillations from Figure 3A. There is a wide variation of
Statistical treatments were made in Figures 3 to 6 for about 100 roots. The behavior of electric oscillations was studied below for one root in Figures 7 and 8. Figure 7 shows one example of the change in period in a root when the temperature was changed. The data indicates the potential at one point in the elongation region. At 90 min, the temperature was changed from 20.5°C to 23°C. During the first 30 min of the experiment, the oscillation gradually became distinct with a period of 14.8 min. When the temperature was changed at 90 min, the oscillation became obscure. About 20 min later, however, the oscillation gradually reappeared with a period of 8.9 min, which is different from the period at 20.5°C. The period of oscillation was usually shorter at higher temperatures for a given root, as found in Figure 5 using many roots. Figure 8 indicates the variation of elongation rate and electric potential in the elongation region measured for over 5 h in one root. The temperature was changed at 90 min from 20 to 22.5°C, and then at 210 min to 27C. Initially, the oscillations were obscure. About 50 min later, an oscillation
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Figure 4. Relation between the elongation per 30 min and the frequency of oscillations at a nearly constant temperature (28-29°C). The data are from Figure 3.
GROWTH AND ELECTRIC OSCILLATIONS IN ROOTS
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Figure 5. Relation between the temperature and the oscillation period. The period has a tendency to become shorter at higher temperatures.
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Figure 6. Relationship between the logarithm of the oscillation frequency, f, and the inverse of the absolute temperature, T. The data are from Figure 5. The line was obtained by means of the method of
least squares and expressed by the analytic function: logf = -2031 /T + 4.17.
with
a period of 1 1.1 min appeared. When the temperature increased to 22.5°C, elongation was promoted. Although the period of oscillation did not change, the amplitude increased. For a while this period of oscillation continued. At 115 min its amplitude reached a maximum and then damped. A new period of oscillation appeared at 140 min. This oscillation had a period of 8.9 min, which was shorter than before. At the same time, the elongation was promoted further; this fact agrees with the result in Figure 4 obtained for the averaged behavior of 13 roots. The present results, therefore, show that elongation is related to the period of oscillation in a given root. Such a correlation, however, was not always obtained. For example, at 210 min (Fig. 8), the oscillation disappeared when the temperature was raised from 22.5°C to 27C. Elongation first increased and then gradually decreased. Whereas the elongation at 27°C was of the same order as that at 22.5°C, no oscillation was seen at 27°C.
was
generating oscillations have higher elongation speed than roots with no oscillations. This seems to agree with the results of Scott (16), who showed that concentrations of indoleacetic acid which inhibit root elongation also damp electric oscillation in the root. Nevertheless, Figure 8 shows that the electric oscillation does not always appear when the elongation is large (e.g. 200-300 min values) whereas a close connection between elongation and electric oscillation is often found, e.g. for the 50 to 200 min interval in Figure 8. It was reported (16) that the surface electric potential of roots does not oscillate when elongation oscillates. These results may imply that the electric oscillation is not directly related to the elongation in a real, short-time scale. At the present stage, there-
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DISCUSSION The electric oscillations studied here depend strongly on the temperature. This property is different from various plant circadian rhythms, where the Qlo is around 1 (18). The value of QIo (= 1.7) obtained for the oscillations observed in this study is close to those in a rhythm of protoplasmic streaming (9, 12). Elongation is also known to show oscillations or nutations (6, 11, 16, 18). The coleoptiles of the white mustard show an oscillation of elongation rate with a period of a few minutes. This period depends on the temperature; generally, at high temperatures the period is shorter than that at low temperatures (6, 11, 18). The Qlo is estimated at about 2.2 and 2.4. These facts suggest a close correlation between oscillation of electric potential and the elongation. Recently, biophoton emission, which may reflect some biochemical steps, has been shown to be associated with the electric oscillation (24). The electric oscillation is clearly related to the elongation rate measured as mm per 30 min. Figure 3 shows that roots
22 E
20
-E
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1
2
3
Time (h) Figure 7. Change in the oscillation period in a root when the temperature was changed. At 90 min the temperature in the solution bathing the root was changed from 20.50C to 230C, as shown by a dashed line.
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3. 4.
5.
6. 7.
8. (h)
Figure 8. The electric potential and the elongation per 10 min measured in one root. The elongation is indicated by a bar chart. The temperature in the solution bathing the root,denoted by a dashed line, was changed at 90 min (20-22.50C) and at 210 min (22.5270C). At 140 min the new period of oscillations appeared even at a constant temperature.
9. 10. 11.
12.
fore, we cannot suggest a definite relation between the electric oscillations and elongation in short-term measurements. However, a clear connection appears in long-term measurements.
Regarding a relation between membrane potential and elongation, an acid growth theory has been proposed (3, 5, 15). The electric current loops formed around the organ of a stem and a root may play a key role in accumulation of protons (21, 27). The resultant acidification of the cell wall leads to wall loosening and acceleration of growth. Since the oscillations arise from the membrane at the xylem/parenchyma interface (22), direct acidification ofthe epidermal cell wall may not be expected. It may be one of the reasons why a real-time correlation did not exist between the elongation and the electric oscillations. Whereas the electric oscillation can be considered to reflect or play an important role on processes of elongation (e.g. wall loosening), there is also a possibility that oscillation and growth are independent in the cause/effect sense. Roots show the largest elongation around 28 to 30°C (14, 17). The present result implies that the frequency of electric oscillations increases on the whole with the larger elongation speed at higher temperatures (Fig. 4). Figure 8, however, shows that when the temperature was increased from 22.5 to 27°C, there was only a transient increase in the elongation followed by decrease. This result may be suggestive of a transient increase in H+ efflux found in excised roots (10). The present study showed that the roots exhibiting electric oscillations had the larger elongation speed. Explication of underlying biochemical steps is a future task.
13. 14.
15.
16. 17.
18. 19. 20. 21.
22. 23. 24. 25. 26.
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LITERATURE CITED Behrens HM, Weisenseel MH, Sievers A (1982) Rapid changes in the pattern of electric current around the root tip of Lepi-
27.
Plant Physiol. Vol. 93,1990
dium sativum L. following gravistimulation. Plant Physiol 70: 1079-1083 Bowling DJF (1972) Measurements of profiles of potassium activity and electrical potential in the intact root. Planta 108: 147-15 1 Brummer B, Felle H, Parish RW (1984) Evidence that acid solutions induce plant cell elongation by acidifying the cytosol and stimulating the proton pump. FEBS Lett 174: 223-227 Ezaki S, Toko K, Yamafuji K, Irie F (1988) Electric potential patterns around a root of the higher plant. Trans IEICE E71: 965-967 Hager A, Menzel H, Krauss A (1971) Versuche und Hypothese zur Primarwirkung des Auxins keim Streckumgswachstum. Planta 100: 47-75 Heathcote DG (1966) A new type of rhythmic plant movement: micronutation. J Exp Bot 17: 690-695 Jenkinson IS, Scott BIH (1961) Bioelectric oscillations of bean roots: further evidence for a feedback oscillator. I. Aust J Biol Sci 14: 231-247 Jenkinson IS (1962) Bioelectric oscillations of bean roots: further evidence for a feedback oscillator. II. Aust J Biol Sci 15: 101114 Kamiya N (1953) The motive force responsible for protoplasmic streaming in the myxomycete plasmodium. Annu Rep Fac Sci Osaka Univ 1: 53-83 Kennedy CD, Gonsalves FAN (1988) H+ efflux and trans-root potential measured while increasing the temperature of solutions bathing excised roots of Zea mays. J Exp Bot 39: 37-49 Kristie DN, Jolliffe PA (1986) High-resolution studies of growth oscillations during stem elongation. Can J Bot 64: 2399-2405 Ogata K, Kishimoto U (1976) Rhythmic change of membrane potential and cyclosis of Nitella internode. Plant Cell Physiol 17: 201-207 Oata K, Toko K, Fujiyoshi T, Yamafuji K (1987) Electric inhomogeneity in membranes of Characean internode influenced by light/dark transition, 02, N2, C02-free air and extracellular pH. Biophys Chem 26: 71-81 Pahlavanian AM, Silk WK (1988) Effect of temperature on spatial and temporal aspects of growth in the primary maize root. Plant Physiol 87: 529-532 Rayle DL, Cleland RE (1970) Enhancement of wall loosening and elongation by acid solutions. Plant Physiol 46: 250-253 Scott BIH (1957) Electric oscillations generated by plant roots and a possible feedback mechanism responsible for them. Aust J Biol Sci 10: 164-179 Sutcliffe J (1977) Plants and Temperature, Chap 4. Edward Arnold Publishers Ltd, London Sweeney BM (1987) Rhythmic Phenomena in Plants, Ed 2. Academic Press, San Diego, pp 61-62 Toko K, Hayashi K, Yamafuji K (1986) Spatio-temporal organization of electricity in biological growth. Trans IECE E69: 485-487 Toko K, Iiyama S, Tanaka C, Hayashi K, Yamafuji KA, Yamafuji KE (1987) Relation of growth process to spatial patterns of electric potential and enzyme activity in bean roots. Biophys Chem 27: 39-58 Toko K, Fujiyoshi T, Tanaka C, Iiyama S, Yoshida T, Hayashi K, Yamafuji K (1989) Growth and electric current loops in plants. Biophys Chem 33: 161-176 Toko K, Souda M, Matsuno T, Yamafuji K (1990) Oscillation of electric potential along a root of higher plants. Biophys J 57: 269-279 Ulrych TJ, Bishop TN (1975) Maximum entropy spectral analysis and autoregressive decomposition. Rev Geophys Space Phys 13: 183-200 Usa M, Kobayashi M, Scott RQ, Maeda T, Hiratsuka R, Inaba H (1989) Simultaneous measurement of biophoton emission and biosurface electric potential from germinating soybean. Protoplasma 149: 64-66 Weisenseel MH, Dorn A, Jaffe LF (1979) Natural H+ currents traverse growing roots and root hairs of barley. Plant Physiol 64: 512-518 White A, Handler P, Smith EL (1968) Principles of Biochemistry, Ed 4, Chap 11. McGraw-Hill, New York Yoshida T, Hayashi K, Toko K, Yamafuji K (1988) Effect of anoxia on the spatial pattern of electric potential formed along the root. Ann Bot 62: 497-507
