Plant Physiol. (1993) 101: 1107-1111

Rapid Communication

Electrotropism of Maize (Zea mays 1.) Roots'

Facts and Artifacts

Hans-Cerhard Stenz and Manfred H. Weisenseel*

Botanisches lnstitut I, Universitat Karlsruhe (TH), Kaiserstrasse 12, 7500 Karlsruhe, Germany

lntact and decapped primary roots of maize (Zea mays 1.) were exposed to DC electric fields of 0.5 to 8.0 V/cm in low-salinity media to resolve conflicting results about the direction of electro- tropism. In DC fields of 0.5 V/cm or 1.0 V/cm, intact roots always curved toward the cathode. In a field of 8.0 V/cm, intact roots curved toward the anode and stopped growth. Decapped roots also curved toward the anode both in weak and strong fields. The results indicate that growth toward the cathode is the true response of healthy roots.

As a consequence of their ion transport activities, plant roots generate long-lasting electric fields in the apoplast and rhizosphere (Weisenseel et al., 1992). These electric fields can polarize cells and tissues and can affect growth of the root. For instance, electric fields may generate a lateral asymmetry of ions and hormones in the elongating zone. Application of DC electric fields can mimic endogenous fields and provide a basis for understanding the complexities of root differential growth and signal transduction.

Over the last few years, severa1 papers have been published that report conflicting results on the effect of DC electric fields in plant roots. For instance, growth toward the anode was reported of intact and decapped roots of maize (Zea mays L.) and some agravitropic mutants of Z. mays by Fondren and Moore (1986), Ishikawa and Evans (1990a, 1990b), and Moore et al. (1987). Curvature toward the anode was also observed in normal and nongravitropic roots of Pisum sativum (Ishikawa and Evans, 1990b), in roots of Tristerix aphyllus with a defect in cap development (Moore and Montenegro, 1991). On the other hand, growth toward the cathode was observed in roots of Lepidium sativum and Z. mays (Stenz and Weisenseel, 1991). In the latter investigation, it was shown that field strength, pH of the medium, and pH gradients in the medium are crucial parameters for the direction of cur- vature and the well-being of roots exposed to electric fields.

We believe that the conflicting results now existing in the literature warrant a thorough reinvestigation to answer the following questions. What is the true response of roots to DC

This investigation was supported by the Deutsche Agentur fiir Raumfahrtangelegenheiten (German Space Agency) DARA, grant number 50 QV 9108.

* Corresponding author; fax 49-721-608-4290.

electric fields? What is the physiological range of field strength and current density? What role does the root cap play in root electrotropism? As experimental material, we chose the main root of Z. mays seedlings because most studies of electrotropism to date have been conducted with this type of root.

MATERIALS A N D METHODS

Plant Material and Media

A11 experiments were conducted with 2-d-old seedlings of maize (Zea mays L. cv Bonny, LUFA, Karlsruhe, Germany). The maize caryopses were soaked in running tap water for 5 h and then stuck between two Plexiglas plates covered with moist filter paper. The two plates, held together by rubber bands, were placed vertically in a glass cylinder whose bot- tom was covered with tap water. After 48 h at room temper- ature, with light from the room and a nearby window, most caryopses developed a straight, downward-growing main root with a length of 15 to 30 mm. (The Bonny variety is a light-dependent cultivar with regard to gravitropism and was therefore grown in the light to yield straight roots.) For experiments with decapped roots, the caps were removed with a small scalpel squeezed gently into the cap/root junc- tion on the stage of a dissecting microscope.

Ten of the 17 experiments were carried out in medium A, containing the following salts: 1.0 m~ NaC1,O.l XYIM KCI, 0.1 m~ CaC12, 1.0 mM Mes, adjusted to pH 6.0 with Tris. Seven experiments were conducted in medium B, consisting of distilled water and 1.0 mM Mes, adjusted to pH 5.0 or 7.0 with Tris (cf. Ishikawa and Evans, 1990b). The conductivity of medium A was 185 pS/cm, the conductivity of medium B was 23 pS/cm at pH 5.0 and 74 pS/cm at pH 7. Conductivities were measured at 22OC with a Konduktometer (Knick, Berlin, Germany). Prior to a11 experiments, air was bubbled through the media for 1 d to saturate them with air. One hour before field application, the roots were transferred to the experimen- tal container to adapt them to the medium. In a11 experiments conducted with medium B, adaptation was made in distilled water as described by Ishikawa and Evans (1990b).

Abbreviation: pS, microsiemens 1107

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1108 Stenz and Weisenseel Plant Physiol. Vol. 101, 1993

Application of DC Electric Fields

Seven to eight seedlings were placed into the boreholes of the cover plate of the experimental container, with the roots oriented vertically downward. The seedlings were fastened in the bores with a small amount of inert putty attached to the side of the grain. The container, made from Plexiglas, was then filled with 70 mL of the appropriate medium, and after 1 h a DC electric field was applied at a right angle to the longitudinal axis of the roots. In the experiments with medium A, two electrodes, each consisting of five layers of platinum wire gauze (platinum/rhodium 90/10, mesh of 1024/cm’, wire strength 76 pm, Degussa, Hanau, Germany), were inserted 9 cm apart at both ends of the container. The electrodes were connected to a constant current source (Di- gistand 6426, Burster, Gemsbach, Germany) and supplied with an appropriate current. To establish desired fields, the necessary current was calculated according to: J = I/A = aE, where J is the current density, I is the current, A is the cross- section of the container, u is the conductivity of the medium, and E is the electric field strength. The stability of the current and the electric field was monitored by a multimeter (Nor- matest Digital, Gossen, Erlangen, Germany).

During the experiment, the medium was stirred continu- ously by two small magnetic bars installed undemeath the electrodes to prevent ionic and pH gradients (Stenz and Weisenseel, 1991). In the seven experiments with medium B, the following conditions identical to those described by Ish- ikawa and Evans (1990b) were established. The electrodes consisted of stainless steel plates of 2.0 cm X 1.8 cm area and, instead of stimng, air was bubbled through the medium close to each electrode. A11 experiments were camed out at a room temperature of 22 & 1OC. A temperature rise in the medium due to Joule heating was found to be insignificant, i.e. <O.l0C/3 h for the highest current density applied.

Registration of Root Crowth

Registration of growth and curvature of roots was con- ducted by taking shadow pictures with a Nikon F-801 camera (Nikon, Tokyo, Japan) on black and white film (Kodak TMX- 100). The camera was equipped with a macro lens (Micro- Nikkor 23/55 mm) and a Multi Control Back MF-21 for automatic photo sequences. The illumination for recording was provided by a glow box behind the experimental cham- ber and a 10-cm thick layer of water to prevent heating of the experimental medium.

RESULTS

At the beginning of our investigation, we repeated the experiment of Ishikawa and Evans (1990b) using experimen- tal conditions as described by these authors. At first, a field of 8 V/cm (corresponding to a current density of 184 JLA/ cm’) was applied to maize roots submerged in medium B, pH 5.0. The result was consistent with the observation of Ishi- kawa and Evans, i.e. the response developed quite rapidly and some roots showed 30° of curvature toward the anode within one-half hour (Fig. la). After 3 h of field application, a11 root tips pointed toward the anode (Figs. l a and 2a). When the field was disconnected and the roots were observed

further, some dramatic changes were noticed. These changes indicated that the roots had been injured by the electric field. First, the roots developed a brownish color, formed little hooks, and then stopped growth (Fig. la). Second, the seed- lings sprouted additional roots at the grain much sooner than healthy control seedlings did, probably as a response to the death of the primary root.

To avoid such harmful effects in further experiments, the electric field was reduced to 0.5 V/cm (corresponding to a current density of 12 pA/cm2). Maize roots growing in me- dium B, pH 5.0, first curved slightly toward the anode but then reversed growth direction, and by 3 h the majority of root tips pointed toward the cathode (Fig. 2a). During the next 24 h, the roots showed no signs of injury and continued to grow vigorously. Roots in which the inital curvature to- ward the anode was prominent maintained this direction of growth, suggesting that intact roots curving beyond a critica1 angle are unable to reverse growth direction. Previous exper- iments with cress roots have shown that the pH of the medium is a crucial factor in electrotropic responses of roots (Stenz and Weisenseel, 1991), and we conducted another two experiments in medium B buffered to pH 7.0 by adding a small amount of Tris. A pH of 7.0 was used in these experi- ments to intensify the expected response to the difference in H‘ cdncentration. Because of the addition of Tris, the con- ductivity of the medium increased, and the applied current density had to be raised three times over its value at pH 5.0. When maize roots were exposed to 0.5 V/cm in medium B, pH 7.0, about half of the roots first curved slightly toward the anode (Fig. lb). However, the direction of growth then reversed, and by 3 h of field application most roots started pointing toward the cathode (Figs. l b and 2a). Curvature and growth toward the cathode continued after termination of the field.

After these measurements in medium B, we conducted two further series of experiments, one with intact roots and one with decapped roots, using medium A, pH 6.0, platinum electrodes, and stirring of the medium to prevent the build- up of pH gradients during field application. When an electric field of 1 V/cm (corresponding to a current density of 185 pA/cm’) was applied, almost a11 roots curved toward the cathode (Fig. lc). This response started in some roots with a slight asymmetry of the root tip at about 1 h after onset of the field (Fig. l c and inset). After 3 h, the average curvature obtained by the root tips was approximately 45O. Curvature and growth of the roots continued after disconnection of the field and reached 90° in about half of the roots within the next 3 h. Finally, most roots curled before reverting to straight growth.

A typical result obtained with decapped roots exposed to the same field strength as intact roots, i.e. 1 V/cm (current density 185 pA/cm’), is shown in Figures l d and 2, a and b. As the figures illustrate, practically a11 decapped roots curved toward the anode. Decapped roots responded faster than intact roots, and already 1 h after onset of the field severa1 roots attained a curvature of 45O (Fig. ld). After 3 h in the field, the degree of curvature was between 50° and 80°. In contrast with intact roots, decapped roots started to reverse direction of growth within 3 h after termination of the field. They never curled during further growth.

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Electrotropism of Roots 1109

Aoodc pH 7.0 0.5 V/cm 12 "".I

I 1 ) 1 ) ) / : : E I II/ pQ!2

Figure 1. Typical response of maize roots to application of a DC electric field for 3 h and after disconnection of the field for 19 to 21 h (E = DC electric field). a, Experimental conditions: intact roots, stainless steel electrodes, air bubbled through the medium, medium 6 , pH 5.0, electric field 8 V/cm, current density 184 rA/cmZ. b, Experimental conditions: intact roots, stainless steel electrodes, air bubbled through the medium, medium B, pH 7.0, electric field 0.5 V/cm, current density 12 pA/cmZ. Open arrowhead points at the typical smooth anodal curvature; closed arrowhead. points at the sharp bending typical of cathodal curvature. c, Experimental con- ditions: intact roots, platinum electrodes, medium continuously stirred, medium A, pH 6.0, electric field 1 .O V/cm, current density 185 pA/cmZ. Inset, Enlargement of the three roots marked by asterisks at time 1 h showing bulging of the anodal flank of the tip at the beginning of curvature. d, Experimental conditions: decapped roots, platinum electrodes, medium continuously stirred, medium A, pH 6.0, electric field 1 .O V/cm, current density 185 pA/cmZ.

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1110 Stenz and Weisenseel Plant Physiol. Vol. 101, 1993

Electric Field(Medium , + /-root cap)

8.0 V/cm(Medium B, pH5,+cap)0.5 V/cm

(Medium B,pH 5, +cap)

0.5 V/cm(Medium B, pH 7,-t-cap)

1.0 V/cm(Medium A, pH 6, +cap)1.0 V/cm

(Medium A, pH 6, - cap)

Number of Roots growing

Anode

23

5

-

2

43

toward theVertical Cathode

.

2 9

4 12

2 28

4

Number ofExperiments

3

2

2

4

6

Ffgure 2. Final direction of growth of maize roots exposed to DCelectric fields and longitudinal, median section of an intact and adecapped root tip. a, Direction of growth of intact (+root cap) ordecapped (—root cap) maize roots after 3 h of exposure to differentDC electric fields and media, b, Image of a typical intact (left) anddecapped (right) maize root. The root cap was removed by applyinggentle pressure to the root cap without harming the adjacent tissues.Bar = 200 ̂ m

DISCUSSION

Three criteria must be satisfied with investigations of theeffect of electric fields on living cells and organisms to avoidartificial responses: (a) Toxic products formed at the elec-trodes during current flow must be prevented from reachingthe cells and organisms, (b) Chemical gradients in the bathingmedium, for instance pH gradients caused by electrophoresis,must be dissipated, (c) Harmful field strength or currentdensities must not be applied.

Most investigators have dealt successfully with criterion aduring their measurements. Criterion b has been neglectedin many older and some recent investigations. In particular,unnoticed pH gradients that develop very fast in unstirredmedia can have dramatic effects on the response (Stenz and

Weisenseel, 1991). We feel that a pH gradient was responsiblefor the anodal response of maize roots observed by Marcumand Moore (1990), as well as by Fondren and Moore (1986),because the medium adjacent to the roots was neither wellbuffered nor stirred during application of the field.

The method employed by Ishikawa and Evans (1990b) waswell suited to meet criteria a and b. However, criterion c wasprobably handled inadequately. The authors found that max-imum curvature of maize roots toward the anode occurred ina field of 8 V/cm. When we repeated the experiment usingthe same experimental conditions, we also observed curva-ture of the roots toward the anode, although bending of 90°was never obtained within 3 h. However, continued obser-vation of these roots showed without doubt that the rootshad been harmed seriously. When the field strength wasreduced to 0.5 V/cm, no sign of damage to the roots wasnoticed, and the number of roots growing toward the cathodeincreased with time and the pH of the medium. These resultsclearly indicate that growth toward the cathode is the trueresponse to DC electric fields of healthy, intact roots in low-salinity medium. On the other hand, harmful field strengthsand/or unphysiological pH values may cause artificial re-sponses, i.e. curvature toward the anode. However, damageto the roots may not become visible immediately and thethreshold for damage probably varies with the root species,because Lepidium roots, for instance, are not harmed by 3 V/cm, but maize roots are (Stenz and Weisenseel, 1991).

We conclude that the true response of healthy, intact rootsis growth toward the cathode, which is further supported bythe results with decapped roots and by the morphology ofcurvature. Decapped roots always grow toward the anode,and the site of maximum curvature lies at 4 to 8 mm behindthe tip, i.e. in the basal elongation zone. This is also true forintact roots that curve toward the anode. On the other hand,curvature toward the cathode always shows a maximum siteof bending at 2 to 4 mm behind the tip, i.e. in the distalelongation zone. The response toward the cathode also pro-ceeds with a smaller arc than the response toward the anode.And, in several cases, the response toward the cathode startswith bulging of the anodal side of the root tip. These mor-phological features of roots growing toward the cathode arevery similar to those occurring with gravistimulated roots(Iversen, 1973).

To establish a field of 1 V/cm in medium A required thesame current density as a field of 8 V/cm in medium B.Because the direction of root curvature was just the oppositein these experiments, field strength rather than current den-sity must be the dominating parameter in electrotropism.However, the amount of H+ and Ca2+ in the medium andthe apoplast of the root also may play an important role inelectrotropism. For instance, Gd3+, which is known to blockmany stretch-activated channels, putative gravity transducerchannels, and some calcium channels (Pickard and Ding,1992), abolishes curvature completely (H.-G. Stenz, unpub-lished results). This suggests the involvement of Ca2+ influxin electrotropism of maize roots. Moreover, a pH gradientmay develop in the unstirred root apoplast and stimulate cellenlargement at the site of higher H+ concentration, which isthe side facing the anode (Good, 1988). In fields that causeanodal responses, the H+ concentration on the anodal flank

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Electrotropism of Roots 1111

may reach harmful levels and may inhibit cell expansion. Because maize roots are very sensitive to H+ ions (Cormack, 1949), fields of more than 1 V/cm may cause such an H+ concentration on the anodal side of the root. However, a pH gradient across the root may not be sufficient for electrotropic curvature. Signals from the root cap seem to be involved, as well, because intact and decapped roots respond differently.

From our present results we expect that nongravitropic mutants curve toward the anode, responding similarly to decapped roots. We are aware that an anodal response has been observed by Moore et al. (1987), but this study was conducted without strict satisfaction of a11 criteria and, there- fore, warrants repetition.

Received August 26, 1992; accepted December 10, 1992. Copyright Clearance Center: 0032-0889/93/101/1107/05.

LITERATURE CITED

Cormack RGH (1949) The development of root hairs in Angio- sperms. Bot Rev 1 5 583-612

Fondren WM, Moore R (1986) Collection of gravitropic effectors from mucilage of electrotropically-stimulated roots of Zea mays L. Ann Bot 5 9 657-659

Good NE (1988) Active transport, ion movements, and pH changes. I. The chemistry of pH changes. Photosynth Res 1 9 225-236

Ishikawa H, Evans ML (1990a) Electrotropism of maize roots: rela- tionship to gravitropism (abstract No. 458). Plant Physiol93 S-79

Ishikawa H, Evans ML (1990b) Electrotropism of maize roots. Role of the root cap and relationship to gravitropism. Plant Physiol94 9 13-918

Iversen T-H (1973) Geotropic curvatures in roots of cress (Lepidium sativum). Physiol Plant 28: 332-340

Marcum H, Moore R (1990) Influence of electrical fields and asym- metric application of mucilage on curvature of primary roots of Zea mays. Am J Bot 77: 446-452

Moore R, Fondren WM, Marcum H (1987) Characterization of root agravitropism induced by genetic, chemical, and developmental constraints. Am J Bot 74: 329-336

Moore R, Montenegro G (1991) The structure and graviresponsive- ness of roots of Tristerix aphyllus (Loranthaceae) (abstract No. 180). Plant Physiol96 S-32

Pickard BG, Ping Ding I (1992) Gravity sensing by higher plants. Adv Comp Environ Physiol 10 81-110

Stenz H-G, Weisenseel MH (1991) DC-elechic fields affect the growth direction and statocyte polarity of root tips (Lepidium sativum). J Plant Physioll38: 335-344

Weisenseel MH, Becker HF, Ehlgotz JG (1992) Growth, gravitrop- ism and endogenous ion currents of aess roots (Lepidium sativum L.). Measurements using a nove1 three-dimensional recording probe. Plant Physiol 100: 16-25

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