The Distribution of Electromotive Forces in the Neighbourhood of Apical Meristems

The experiments to be described form part of the early stages of a research whose general object is to investigate the organization of plant material as it develops from a meristematic mass to differentiated tissue. Two major classes of control can be recognized in the organization of any living material, one, characteristic of and located in the cells of the developing organs and the other characteristic of the surroundings of the changing cells. This second controlling factor has been named the biological field, and much is known of its properties in the case of animal embryology.

It is a striking fact that wherever a biological field can be recognized and the electrical condition of the region tested, an electrical field is found to be present. An investigation of the behaviour of the electrical field while organization of plant tissues is in progress appears therefore to offer a promising lead to knowledge of the biological field.

Early experiments made in this laboratory and a study of the literature showed a surprisingly erratic behaviour of the electric potentials measured in plant tissues. For this reason attention was given to the question of explaining this and finding a satisfactorily reproducible background for the description of the normal electrical condition of a growing plant.

An examination of the literature reveals that most investigators are in agreement in finding the regions adjacent to apical meristems electrically positive to older parts of the organs, on the surface of the plants. Such a polarity has been found by Thomas (1939), using bean (Vicia Faba) roots, Ramshorn (1934), using sunflower hypocotyls and asparagus shoots, and Lund (1947), working with onion roots. However, some of the results of Clark (1937) are contrary to these findings. He states that the apices of Pisum, Vicia and Impatiens seedlings are negative to their bases. As the plants mentioned have apical meristems, it is seen that Clark’s findings are contrary to those of the aforementioned authors, though his results on the external polarity of Avena and Zea coleoptiles agree with those of Lund. The results of Lundegardh (1940) are on the whole not inconsistent with the results of the first three authors, if attention is limited to the first 8-10 mm. from the apex.

The following experiments were carried out to determine the distribution of e.m.f.’s both inside and outside the plant tissue in the neighbourhood of the apical meristems in turnip {Brassica Rapa) seedling hypocotyls and in maize (Zea Mays) seedling roots. The main results have been checked by experiments with other plants.

Particular attention was paid in this work to obtain unambiguous experimental results under conditions of specified control so that a sound background might be developed for further work.

It is shown that the measured potential is due to electromotive forces produced by the plant and not to static charges on its surface. The plants studied supplied power, through external conductors, of the order of 10⁻⁹ W. from approximately 2 mm.³ of tissue without change in the e.m.f. generated by the plants.

The e.m.f.’s in the neighbourhood of apical meristems were obtained by slightly different techniques each designed to check the other. In all of these the measurements gave (i) potential differences on the surface of the plants between the meristematic region and locations away from it and (ii) potentials between the inside and surface of the plants in the region of the meristem and the corresponding p.d.’s a few millimetres away from the meristem. The results in all cases showed a greater p.d. in the meristematic region than in the region remote from it, and it is thought that a firm foundation is laid for the theory that the meristematic mass is negative to the differentiating tissue that derives from it.

It is recognized that the individual radial p.d.’s measured are functions of external factors such as concentration of ions in the contracting medium, but it is considered that the experiments with their varied techniques estabfish the reality of the difference between the radial p.d.’s in different regions. It is important to recognize, however, that when other meristems are near an apex, as, for example, when secondary roots develop from a primary root in bean, the essential nature of the potential pattern may be obscured.

The voltage measurements were made with a high input resistance, three-valve electrometer using a type 954 pentode connected to give a grid current of the order of 10^-14 amp. (Nielsen, 1947) and followed by two stages of direct-coupled amplification. The plant voltage was applied between the isolated, highly insulated grid and an earth connexion, and the output read on a 0·50µ A. meter. The maximum sensitivity was 2·5µ A./mV. A voltage-regulated power supply was used, and zero drift reduced to that due to discharging the filament accumulators.

Electrical contact with the plant to be measured was made with two non-polarizable Ag/AgCl electrodes which consisted of glass tubes containing agar jelly saturated with AgCl into which dipped an AgCl-coated Ag wire. A copper wire soldered to this was connected to the electrometer. The electrodes were made in a photographic dark-room and painted black to reduce the rate of reduction of the AgCl.

The electrodes were placed in the ends of glass tubes with pointed tips containing agar jelly and 0·3 % Knop’s solution. In making measurements on the surface of the plants small agar-Knop’s solution blobs were placed on the appropriate spots on the plant and the glass tips rested on these. The glass tips were of the order of 20µ diameter, which is less than the length of most of the plant cells encountered in the experiments. The small p.d. existing between parts of the electrode system alone was subtracted from each reading.

Turnip hypocotyls and maize seedling roots were used as the main experimental material, a few of the experiments being repeated with onion roots, turnip secondary roots, oat roots, and bean seedling hypocotyls. The seeds were germinated on wet filter-paper in Petri dishes in an incubator at a temperature of about 30 ° C. The hypocotyl of the 2-4-day turnip seedling ranges in length from approximately 3 mm. in a 2-day plant to approximately 20 mm. in a 4-day plant. The meristematic region is at the top of the hypocotyl immediately below the cotyledons. The vascular tissue is like that in the root and is confined to the centre of the hypocotyl. Cells at the top of the hypocotyl are packed closely together and are about 40µ in length. At the base of the hypocotyl the cells range between 100 and 200µ in length. The average diameter of the hypocotyl at the meristem is 0·3 mm. and nearer the base is 0·7 mm.

The maize roots used were from 20 to 100 mm. long with a diameter of 1-1·5 mm. The diameter of the stele alone is rather less than half of this. The meristematic region is in the first 1 or 2 mm. of the root tip.

The seedlings were transferred from the Petri dishes to a paraffin-wax insulated glass dish, and the hypocotyl or root rested on paraffin wax during subsequent measurement. The seedlings were disturbed as little as possible during the transfer and were allowed to assume a relatively stable potential before measurement. This usually took 3-5 min. While under experiment they were grown in a moist atmosphere with the root supplied with water from wet cotton-wool. The cell containing the plant was placed on a stage and observed with a dissecting microscope. The illumination was from a 24 W. spot-lamp. The contact tips described above were moved by means of Fonbrune pneumatic micromanipulators.

If the plant is an ohmic system, it can be represented by the circuit of Fig. 1 in which E is the equivalent electromotive force and R_s the plant’s equivalent resistance. Experiments such as the one to be described have shown that this is so, and that there is no polarization of the e.m.f., that is, E remains constant with currents of the order of 10^-8 amp. flowing in the circuit. The circuit is completed from two spots on the surface of the plant through different resistances, the potential drop across these being measured with an electrometer of practically infinite resistance.

It is concluded that no polarization effects are set up in the plant for the range of currents considered. That is, E remains constant in value even though the plant is nearly short-circuited externally and is supplying current of the order of 10⁻⁸ amp. (see Fig. 2). This maximum current averaged 3 × 10⁻⁸ amp. for the turnip and 6 × 10⁻⁸ amp. for couch grass. This corresponds to an average power generation of 10⁻⁹ and 2 5 × 10⁻⁹ W. for turnip and couch grass respectively.

The threshold value of the current required to polarize the plant e.m.f. is not yet known but is suspected to be of the order of 10⁻⁷ amp. (Compare Lund and Berry’s (1947) value of about 4 × 10⁻⁸ amp. for the onion root.)

Numerous experiments have shown that there exists a potential gradient on the surface of 3-5-day-old maize roots and turnip hypocotyls, the region near the meristem being nearly always positive to the older parts of the organ. In older plants, activity developing farther back in roots may cause these regions to become more positive than the primary apex. The potential decreases approximately linearly with length over the first 6-8 mm. from the meristem.

It will be shown in the next section that this surface p.d. can be thought of as the sum of two radial e.m.f.’s and a small longitudinal p.d.

When measurements are attempted to obtain p.d.’s inside plant organs, considerable trouble is experienced in getting constant results. This variability is due, at any rate in part, to permanent injury and temporary stimulation. The effects of damage by exploring tips is more marked in turnip hypocotyls than in maize roots. In all cases, however, when appropriate precautions were taken, the results could be represented by the diagram shown in Fig. 4A, which should be examined together with Fig. 4B, a diagrammatic representation of the plant showing where measurements were made. In this diagram the symbols have the following meaning. As in § V, a and b are positions on the surface of the plant organ with a near the meristematic region and b some 5-10 mm. away from it. a ′ and b ′ are positions near the axis of the organ beneath a and b respectively.

An examination of Fig. 4 A shows that the average state of affairs in maize roots and turnip hypocotyls is as follows:

• (i) The inside of the organ is negative with respect to the outside at any plane between a and b.

• (ii) The magnitude of V_aa. is greater than that of V_bb. That is, the section of the hypocotyl or root at aa′ (the plane of the apical meristem) has the larger p.d. between the surface and axis.

• (iii) V_a._b. is small; b′ is usually slightly positive with respect to a′.

Thus, the distribution of potential is consistent with a conducting path following the course of the central tissue of the plant. It is inferred that there is a current flowing inside the plant organs around the circuit abb′a′a due to the larger e.m.f. generated between a′ and a, against the smaller e.m.f. between b′ and b. Resistance measurements have substantiated this and indicate that this current is probably largely dependent on the moisture condition of the outside of the plant.

Two contact tips were employed, these being placed in agar blobs on the surface of the root at a and b. The root was immersed in paraffin oil to conserve constant external conditions during measurement. A reading with the tips in this condition gave V_ab. The tip at a was then pushed into a′ and time allowed to elapse until the voltage between the tips had assumed a relatively stable value. In certain cases this value was close to the initial reading when the tip was first pushed in. It is regarded as likely in these cases in which the initial reading was the stable one that damage and subsequent injury current flow was at a minimum. Also, in these cases only was the surface p.d. V_ab very nearly the same after the experiment as before. In other cases it took a considerably longer time to obtain a stable reading, and this was very different from the initial reading. A large difference in before and after the experiment correlated with this; when such conditions were encountered the experiment was discarded.

The difference between V_ab and the stable value of V_ab gave V_aa.; similarly, V_bb. was obtained as the difference between V_a._b and V_a._b, the latter being the stable reading when the tip at b was pushed to b’.

The experiments in which V_ab (after) was within ± 4 mV. of V_ab (before) are recorded in Table 1 and are seen to form a consistent pattern, i.e. that of Fig. 4A.

In Exp. ii uncertainties due to the time rate of change of the potential between the tips when one or both were inside the root were partly eliminated by recording V_aa. and V_bb. as the initial difference between the voltages on the surface and inside the root.

Contact tips were placed initially at points a, b and c on the surface of the plant, that at c being a fixed reference tip and not penetrating the plant, while the exploring tips at a and b were subsequently pushed in to the centre one after the other. The positions a, b and c were in order proceeding from a at the surface of the meristematic region towards the base of the root. The lengths between a and b, and b and c were approximately equal. Small agar blobs were placed at these spots.

Referring to Fig. 4 A, V_ab, the surface p.d. was obtained as the difference between V_ab and V_aa. Next, V_aa. was obtained by pushing the exploring tip at a rapidly into the centre of the root to a′. The initial difference between V_aa and V_aa was taken to be the p.d. which existed between the surface of the root and the axis (V_aa) prior to disturbing it, that is, it is assumed that the potential inside the plant is not abruptly changed when the latter is entered. If time is allowed to elapse between entering the plant and reading the p.d., in general the result will be different due to diffusion of the contents of damaged cells. This method of experimenting is justified by the consistent nature of the results, both internally and compared with those of Exp. i. Similarly V_bb. was obtained by subsequently pushing the tip at b to b’ on the axis of the root below b. The readings of V_ab, the potential drop along the axis of the root, were the differences between the readings V_ab and V_aa.. Table 2 gives the results of experiments such as these, and they are summarized in Fig. 4A.

When the tip at b penetrated the plant, the point a in most cases was observed to become more positive with respect to c. The experiments of Table 2 are those in which the change was less than 2 mV. It is, of course, possible that the process, whatever it is, may have affected both a and c nearly equally. For this reason the value of V_ab. cannot be regarded as very significant.

In this case a much more fragile organ was used, the diameter being about one-third that of the maize roots.

The experiment was similar to Exp. ii described above. Two exploring tips were used, one after the other being pushed rapidly into the centre of the hypocotyl and the initial changes in potential being read. The ‘settled down ‘voltages did not form a consistent structure, due, as pointed out earlier, to injury effects.

The justification for choosing one set of results rather than the other to represent the static potential distribution inside turnip hypocotyls is found (i) in the consistency of the results (Tables 3 A and B), (ii) in the fact that these results suggest a pattern similar to that given by maize with both types of experiments, and (iii) the likelihood of damage to such fragile material.

It was realized that when one tip is pushed into b′, for example, the potential of a′ relative to b′ may be changed by a transient impulse. In addition, the tip now at b′ does not serve as a fixed reference point for the other tip when it is pushed into a′ (compare Exp. ii with maize) because of the injury currents flowing near the point V. The two sets of experiments recorded in Tables 3 A and B, were designed to eliminate these effects as far as possible by altering the order in which the tips are pushed into the interior of the plant. The significant differences between the two sets of figures are explicable by these causes, but it will be noted that the principal features of the observations are the same in each case.

In one set of twenty experiments on the hypocotyls of 3-day-old turnip seedlings, of which ten are given here, the tip at a entered before that at b and in the other, vice versa. The averages are different, and in particular the value of the p.d. V_ab can only be an indication of the state of affairs in the unstimulated plant.

The above work was planned as a step towards the understanding of the role of electric fields in living matter during its growth and organization. An examination of literature and early work in the laboratory showed that research was necessary into the distribution of e.m.f.’s in the region of apical meristems. Even in this simple case the results published and observed were surprisingly erratic. Several slightly different types of experiment are described, designed to allow for or eliminate the many uncertainties encountered in attempting to measure the potential of inside regions of plants relative to their outsides.

The absolute values of the radial p.d.’s measured as described are dependent on the concentration and nature of ions in the contacting fluid and in the contact tips, on the extent of short-circuiting between outside and inside and on the difference in diffusion potential between contact tip and fluid when outside and between tip and cell sap inside the plants. However, there is enough consistency in the results which show a larger p.d. at the meristematic region than elsewhere to give a basis for the theory, mentioned in the introduction, that meristematic tissue is electrically negative to differentiated tissue surrounding it, in a simple apex.

Details of the distribution of potential between the surface of the organs and their axes has still to be investigated, a large part of the radial e.m.f.’s being thought to be at the interface between exodermis (or epidermis) and contact fluid.

This work has been done at the Biophysics Laboratory of the University by members of the research staff and of the staff of the Commonwealth Scientific and Industrial Research Organization. We wish to thank C.S.I.R.O. for its support of this work.