Cellular responses to endogenous electrochemical gradients in morphological development

Adv. Space Res. Vol. 17. No. 6/7, Pp. (6/7)27-(6/7)33. 1996 Copyright 0 1995 COSPAR Printedin Great Britain. All rights reserved. 0273-l 177/96 $9.50 + 0.00 0273-l 177(95)00609-5

CELLULAR RESPONSES TO ENDOGENOUS ELECTROCHEMICAL GRADIENTS IN MORPHOLOGICAL DEVELOPMENT M. F. Desrosiers DFD Enterprises, 320 Clarendon Road, East Lansing, MI 48823, U.S.A.

ABSTRACT Endogenous electric fields give vectorial direction to morphological development in Zeu nurys (sweet corn) in response to gravity. Endogenous electrical fields are important because of their ability to influence: 1) intercellular organization and development through their effects on the membrane potential, 2) diiect effects such as electrophoresis of membrane components, and 3) both intracellular and extracelhllar transport of charged compounds. Their primary influence is in providing a vectorial dimension to the progression of one physiological state to another. Gravity perception and transduction in the mesocotyl of vascular plants is a complex interplay of electrical and chemical gradients which ultimately provide the driving force for the resulting growth curvature called gravitropism. Among the earliest events in gravitropism are changes in impedance, voltage, and conductance between the vascular stele and the growth tissues, the cortex, in the mesocotyl of corn shoots. In response to gravistimulation: 1) a potential develops which is vectorial and of sufficient magnitude to be a driving force for transport between the vascular stele and cortex, 2) the ionic conductance changes within seconds showing altered transport between the tissues, and 3) the impedance shows a transient biphasic response which indicates that the mobility of charges is altered following gravistimulation and is possibly the triggering event for the cascade of actions which leads to growth curvature. INTRODUCTION The electrical asymmetry which arises from gravistimulation, historically called the geoelectric effect, has been studied in detail for many years /l, 2/. Among the assumptions in the history of the geoelectric effect is that the externally measured potential is the product of an internal linear electrical gradient. I performed a series of experiments to determine if gravistimulation affected the bioelectric parameters of voltage, ionic conductance, and charge mobility in 5 day-old corn seedlings. Corn seedlings were chosen as the test sample because the mesocotyl of corn seedlings have a simple root-like anatomy with a central vascular stele surrounded by a cylindrical cortex and epidermis. The questions about gravistimulation of corn shoots I sought to answer were: 1) Does the internal bioelectric gradient mirror the external electric field? 2) Is there a correlation between the gravistimulated growth curvature and changes in the bioelectric parameters internally? and 3) Does the timing suggest that the measurable bioelectric events play a role in perception or transduction in gravistimulation? The external bioelectric gradient observed in the system by Tanada /3/ suggests that a lateral electrical field of 17 mV (Iower side positive) will develop within 5 mimnes of gravistimulation. Also this gravistimulated lateral gradient was confined to the areas of the shoot which will later show growth curvature. I found that there was indeed a lateral electrical asymmetry, but it included an internal bilateral symmetry also outward from the stele tissue. METHODS AND MATERIALS The plant material was 5 dayold Zea mays, (L. var. Silver Queen; Burpee Seed Co. Warminster Pa.), seedlings. They were grown in the dark at 27oC in wet rolled paper towels, handled under dii green light, and maintained in a vertical position during handling prior to the experiment. The mesocotyl of corn seedlings has a root-like anatomy with a central vascular stele surrounded by a

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M. F. Desrosiers

cylindrical cortex and epidermis. Two-mm-long samples were taken from each of the 3 regions of the mesocotyl: 1) the division xone where the cells are small and actively dividing, 4 mm below the mesocotyl node, 2) the extension zone where the cells are elongating adding length to the shoot, 12 mm below the meaocotyl node, and 3) the mature region, 35 mm below the mesoco~l node. The electrodes for the measurement of potential and impedance were 100 micrometer diameter platimrm wire. Relatively large electrodes were used so the electrodes would be embedded in the apoplast. They were inserted 1 mm into the tissue with a separation distance of 1.7 mm with one electrode centered in the stele. For the DC conductance m~~ernen~, glass AgCl/KCl electrodea of 100 micrometer internal diameter were used to prevent polarixation. ENDOGENOUS VOLTAGE BETWEEN VASCULAR STELE AND CORTEX It has been long observed that ~av~tim~ation induces an external lateral electrical gradient across a horizontal shoot /4, 51 and can induce membrane depolarization /6/. The hypotheses I examined is whether the potential difference between the vascular stele and cortex tissues is sensitive to gravistimulation and if the tram-tissue potential difference is a reflection of the externally measured potential differences seen by other inv~tigators. The potential difference between probes inserted into the stele (low) and cortex (high) was measured by an electrometer, (Reithley, model 614). The electrical parameters of the dry probe and leads were 1 pF capacitance and 5~10’~ohms leakage resistance. To measure electrical noise produced by the equipment during handling of the apparatus, a sample of tissue was boiled for two minutes and the voltage was monitored while the sample was moved between vertical and horizontal positions. There was no correlation between the orientation of the boiled sample and observed voltage. Reversal of leads also had no effect upon the observed voltage. The maximum magnitude of the electrical signals observed with the boiled sample due to handling was 10 mV.

Dktance below lvleeocotyl node 12mm ---em.........- a6lmn

20

Fig. 1. Representative time course of the voltage between steie and cortex for samplea taken from the division, extension and mature regions of the mesocotyl as the sample’s orientation is shifted. The cartoons show the position of the sample with electrodes. $Jravitv-InducedPotential Channes Alone the Shoot Figure 1 shows the voltage between the stele and cortex for sample8 drawn from the three regions of the meaocotyl as the sample underwent positional changea at 15 minute intervals. There was a large rapid change in the voltage caused by shifting the plant from a vertical to a horixontal position. In

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Electrochemical Gradients

particular, notice that the sample from the division region (upper 4 mm below the mesocotyl node) showed the opposite direction in its response to a positional shift compared to that of samples from the other two regions even though there is only 6 mm between the division xone sample and the extension xone sample. f

Potentnil chanpes Between Stele and Cortex To determiue if the gravity-induced voltage chauges between stele and cortex showed lateral asymmetric correlations, we measured voltages when the electrodes were placed between the upper, middle, or lower cortex and stele. The measured change in voltage after the orientation shift was called the eon volw and is defined as the difference in voltage from the beginning of the positional change to the voltage 10 mhmtes later. Figure 2 shows the average transition voltages at the three junctions for samples from the three physiological regions. All of the transition voltages are statistically different from zero for both orientation changes showing that there is a significant perturbation in voltage between the stele and cortex whenever the shoot is moved. There is a bilateral asymmetry in the vertical to horizontal transition between the lower and upper side transition voltages. This bilateral asymmetry arises in the area of the shoot which will later show growth curvat&e. The return from a h&ontal to a vertical position for the shoot showed no bilateral poetry. VeltkNl ->

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Fig. 2. The mean of the maximum change in voltage (within 10 mimrtes) between the stele and upper, middle, or lower cortex for samples from the three regions of the mesocotyl after gravistimulation, Plot A, or on return to a vertical position, Plot B. An asterisk marks cases where the observed bilateral transition voltage is statistically significantly different between the upper and lower tissue. Error bars are standard errors. ‘Ibe data show that the ~vi~-~u~ tram-tissue potentials are not simply a linear gradient of the external electrical potential. If only the cortex voltages are included in the analysis then it would appear that the internal voltages mirror the external surface voltages seen by other investigators /4/. With inclusion of the stele data, the internal electrical gradients are more complex than a linear gradient across the shoot and shows a bilateral asymmetry. The vertical stele is at approximately 170 mV relative to the cortex verticaliy and can change to -245 mV with gravistimuhtion. Thii magnitude of voltage (245 mV) over 1.7 mm electrode separation distance gives a field strength of 1.44 V/cm. Using an example of a 10s m diameter cell, this is a potential drop of 1.4 mV per cell. There are 2 major conclusions to be drawn from these data. First, internal electrical gradients can be considerably more complex than inferred from external surface measurements. Secondly,

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M. F. Dcmsiers

~~~~~~io~ can induce large electrical gradients in both lateral and lo~~d~ d~~i~~. In the case of gravistimulated corn shoots, the significant bilateral asymmetries occurred in the regions of the shoot where growth curvature occurs. Gus-~UCED DC CG~UCTA~CE MESGCGTYL STELE AND CORTEX

CHANGES BETWEEN THE

To determine if the ~av~t~~ transition voltages were caused by alteration in the transport of ions between the tissues, the DC resistance was calculated from the induced current in response to an applied 25 mV potential for a period of one second. Prior to each experiment the electrode cell constant was determiued using 0.1000 N KCI. To make the DC! resistance values independent of the specific electrode used, the DC re&tauce values were converted into specific conductance values using the electrode cell constant determined prior to each experiment. Figure 3 shows the time course of tbe specific ~~u~~ for position changes. In all casea there was a si~fic~t decrease in conductance with a vertical to horizontal shift. There was an observed bilateral symmetry of the transition conductance.

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Fig. 3. Time course and magnitude of gravity-induced DC conductance. Graph A shows the time course of the specific conductance as a function the shoot’s orientation. GRAVlTY-INDUCEDAC RESISTANCE CHANGES BETWE9JNTHE ME?SGCOTYLSTELE AND CORTEX If sn early event in gravity detection by plants is due to the motion of charged particles in the tissue then alternating current measurements of the mobility of their charged particles should show changes in those critic for the duration of the event. AC resistance measures the mobility of charges that are not free to migrate to an electrode but are able to oscillate over a smaller distance dependmg on the applied frequency. The mobility of charges as reported by the resistance parameter (mobility is inversely p~~~o~ to resistance) undergoes a transient response following ~avistim~~io~. The impedance at 0.1, 1, 10 and 100 KHZ was recorded with time following gravistimulation utilixing a Stanford Instruments Model SR720 LCR me&x with a custom interface box to adapt the BNC adsptor to the electrode leads and prevent enviro~emal noise aud high humidity levels from interfering with the integrity of the signals. The driving signal from the LCR meter was 0.1 V,,,,,. To eliminate possible artifacts due to the LCR meter’s 0.1 V, driving signal producing a temperature rise in the sample during the course of the experiment, the heat generated during the entire experiment was computed to be 2.3~10~ calories. If 2.3~10~ calories were applied in one second instead

Electmchemical Gradients

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of over the normal 45 minutes duration of the experhnent it would raise the temperature of the sample by 3xlW “c, a negligible amount. Care was taken to avoid cross talk between sensing leads. Teflon was used for insulation of all exposed wiring and terminals on the electrodes to avoid leakage between high and low leads due the high levels of humidity present. The dry electrode had an isolation resistance greater than 200 GGhms and a capacitance of 0.2 pF. Data were collected from the LCR meter by computer. To remove the effect of electrode polarization, the impedance values of a known conductivity standard that was similar in ionic content to the sample (O.~~M KCl) were subtracted from the sample values /7/. The data are therefore reported as the difference in resistance between the sample and 0. 1OOM KCl. A control sample consisting of 4% agar + 0.05 M KC1 was examined to detect possible equipment and handling artifacts. No change in the resistance was observed to correlate with changes between vertical and horizontal positions.

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Fig. 4. Time course of the normalized resistance at 100 KHZ between stele and cortex for a sample taken at 4 mm below the mesocotyl node showing the orientation transitions. The orientation was vertical, horixontal, vertical on a 15 minute interval. Plot A shows the effect of a vertical to horizontal transition after 15 minutes vertical (vertical dashed line). Plot B shows the effect of returning to a vertical position from a horixontal position (vertical dashed line). The sample had been vertical for 15 minutes first and then horizontal for the next 15 minutes. The major result was a transient resistance deviation lasting approximately 80 seconds following changes in orientation from vertical to horizontal and from horixontal to vertical. Figure 4 shows the normalized resistance following two changes in ori~~on for a sample taken at 4 mm below the mesocotyl node and at the upper cortex-stele junction when horizontal. There is a transient change of approximately 80 seconds duration in the resistance of the sample following the vertical to horixontal position change at 15 minutes and the return to a vertical position at 30 minutes. Gther brief resistance transients of shorter duration are seen in the data particularly in the first 10 minutes when the sample was vertical, but only the resistance changes after orientation changes were reproducible and consistent from ssmple to sample. The resistance transients which correlated with positional changes occurred in approximately 70% of the samples. The AC induced mobility of the charges present in tissue samples changed immediately following changes in orientation. This is significant because 1) the mobility can change due entirely to physical mechanisms and hence be prior to any biochemical responses, and 2) the fact that the gravity-induced resistance change is of limited duration emphasizes the short-lived nature of this early, possibly sigJASR 1r:W-o

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M. F. Desrosiers

nailing event. Any gravity-induced charge r~is~bution such as sedimentation of charged particles should return to equilibrium quicldy. Does the charge r~is~butio~ hrduce the biochemical cascade? If the gravity-induced resistance change is due to physical mechanisms, then it can occur prior to the biochemical response and can serve as the trigger for the biochemical cascade which results in and maintains the bioelectric potentials observed following gravistimulation. DEiCUSSION From the preceding data, we may answer the questions proposti previously. Does the internal bioelectric gradient mirror the external electric field? No, the internal bi~l~i~ gradient is a bilateral one, not a simple linear gradient as suggested by surface m~uremen~. Is there a correlation between the gravistimulated growth curvature and changes in the bioelectric parameters? Yes, there is a locational rel~io~hip* Ibe bilateral asymmetry occurs in the area where asymmetric growth occurs. Does the timing suggest that the induced bioelectric events play a role in perception or transduction in gravistimulation? Yes, the bioelectrical events occur prior to the gravistimulated growth curvature, Figure 5. From Figure 5 it appears that the DC conductance decreases faster than the change in potential. This leads to several interesting points concerning control mechanisms. One hypothesis is that the potential between the stele and cortex is maintained at a high level and the voltage is regulated by selective leakage through low resistance pathways which loads and hence lowers the voltage when the shoot is vertical. This explains the behavior at 4 mm below the mesocotyl node but not at 12 and 35 mm below the mesocotyl node where both the tuition voltage and DC conductance decrease together. Another hypothesis is that the decrease in DC endure is a m~h~ism whereby a plant can localize a response such as growth curvature. The evidence for this behavior is that the gravity-induced longi~di~ voltage at 4 and 12 mm below the mesocetyl node changes from 54 mV/7 mm to a maximum of 171 mVf7 mm on the lower side of the shoot when hor~n~. Also sup~~g this hypothesis is the bilateral asymmetry at 4 mm below the meaocotyl node where the difference in voltage is 62 mV13.4 mm. Figure 6 shows the vertical and horizontal endogenous voltages.

Fig. 5. A summary of curvature, potential, DC conductance, and AC resistance changes in response to gravistimulation, This figure shows the rapidity of the bioelectric parameters which occur in less than a minute after the beginning of the gravitropic growth response of the mesocotyl, which requires nearly 10 minutes for detection.

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The changea in internal bioelectric properties of a corn shoot can be correlated with the shoot’s orientation with respect to the gravity vector. In particular the bilateral asymmetric voltage occurs in the region of bilateral asymmetric growth which results in curvature leading to the general plant response, ~a~~~isrn. ‘The internal bioelectric parameters of voltage, DC conductance, and AC impedance are dynamic and show the ability to localize a morphological response to gravity.

117

Extm

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Fig. 6. The mean Potential in mil’iivoltsfor a vertical and horizontal positioned corn shoot with the stele at zero potential.

This research was supported by a grant from the Life Sciences Section of the Space Biology Program at NASA and the author gives special appreciation to Robert Bandurski for his support and advice. REFERENCES 1.

E.J. Lund, Bioelectric Fields and Growth,The University of Texas Press, Austin, 1947

2.

II. Ginsburg, AnaIysis of Plant Root El~o~tenti~s, (1972).

3.

T. Tanada and C. ~mten-Joh~en, hypocotyls, Ptanf, cell ~~n~o~~

4.

L. Grahm and C.H. Hertz, M~uremen~ of the geoelectric effect in coleoptiles by a new technique, PhysiotogiaPtaniamm, 15,% (1962).

5.

A.R. Schrank, Analysis of the effects of gravity on the electric correlation field in the coleoptile of Avena sativa, in: Bioe&wtricFietds and Grow&, ed. E.J. Lund, The University of Texas Press, Austin 1947, p.75.

6.

H.M. B&ens, D. Grad and A. Sievers, Mernbr~~~i~ responses fo~low~g gravistimulation in roots of Lepidium sativum L., Ptanra 163, 463 (1985).

7.

H.P. Schwan, Deviation

bob

of ~0~~~

Bison

37,380

Gravity induces fast electrical field change in soybean 3, 127 (1980).

of Biological Impedances, in: P~sic~ Tec~iq~s in Bioto~c~

Research WB, ed. W.L. Nastuk, New York, 1%3, p.323.