Planta

Planta 150, 431434 (1980)

9 by Springer-Verlag 1980

The Influence of Small Direct Electric Currents on the Transport of Auxin in Intact Plants D.A. Morris Department of Biology, Building 44, The University, Southampton, S09 5NH, U.K.

Abstract. When a d.c. potential of 9.0 V was applied

to the stem of intact pea seedlings (Pisum sativum L. cv. Meteor and cv. Alderman) via 10 m M KC1soaked filter paper electrodes placed ca. 50 m m apart the stem passed a steady current of 15-20 ~tA (resistance ca. 100 kf~ cm *). The basipetal transport of [1-1~C]IAA applied to the apical bud was completely inhibited over the portion of the stem through which current flowed and 14C-labelled compounds accumulated in the vicinity of the upper electrode. The inhibition of transport was independent of the polarity of the applied potential. The basipetal transport of I A A in the stern above the electrode was not affected. Labelled auxin accumulated at the upper electrode both as unchanged I A A and as a compound tentatively identified as indol-3yl-acetyl aspartic acid (IAAsp). These compounds were only slowly remobilised when the current was interrupted. However, the ability of the transport system to move freshly-applied I A A was rapidly and fully restored when the potential was removed. No injury to the plant was detected after maintaining a current flow for up to 72 h. No leakage of a4C-labelled compounds into the KC1 solution bathing the electrodes was detected. Key words: Auxin transport

Electric c u r r e n t - P i s u m -

Potential gradients.

to electric fields frequently have had inhibitory effects on plant growth, a few workers have reported significant increases in growth rate and ion accumulation by plants following the application of small d.c. currents (e.g. Black et al. 1971). The mechanisms by which applied currents m a y influence plant growth are poorly understood. In m a n y cases inhibitory effects on growth can be attributed directly to cellular injury; however, growth stimulation by electric currents has been attributed variously to stimulation of membrane-associated ion pumps, to effects on water movement in the plant and to effects on the distribution and/or metabolism of plant hormones (Black et al. 1971; Bratton and Henry 1977). Although there is some evidence to indicate that lateral redistribution of indol-3yl-acetic acid (IAA) m a y occur under the influence of applied potentials (Schrank 1953), little is known about the effects of longitudinally-applied potentials on the longdistance polar transport of hormones in intact plants. This report presents observations which indicate that the passage of a small d.c. current through the stem of the intact pea plant can reversibly inhibit the basipetal transport of apically-applied I A A under conditions which appear to cause no permanent injury to the plant. Material and Methods

Introduction Plant Material. Seedlings of P. sativum L. cv. Meteor (dwarf) or

Modifications to plant growth and development have been reported following the exposure of plants to electric fields or the passage through them of small direct (d.c.) or alternating (a.c.) currents (see reviews by Lund 1947; Schrank 1953; and Ellis and Turner 1978). Although electrical stimulation and exposure

cv. Alderman (tall) were grown singly in 90 mm diameter plastic pots of vermiculite at 21~ C+ I~ under illumination from 'daylight' fluorescent lamps (16 h photoperiod; ca. 12 W m 2). The seedlings were watered as required with Hoagland's mineral nutrient solution. Plants were usually used in experiments when 14 to 16 d old and the transport experiments themselveswere carried out in diffuse daylight in the laboratory at 20 22~ C. Electrical Circuits. Direct current was applied to the plants via

Abbreviations: IAA=indol-3yl-acetic acid; IAAsp=indol-3yl-ace-

tyl aspartic acid

an electrode attached around the upper part of the stem, usually on internode 5, and the return was provided either by a stout

0032-0935/80/0150/0431/$01.00

432 copper rod (100 mm long- 1.75 mm diam.) inserted in the vermiculite rooting medium or by a second electrode attached to the base of the stem on internode 2. Each stem electrode consisted of a strip of Whatman No. 1 filter paper (4 mm wide. 15 mm long) through which was threaded a length of fine copper wire (0.25 mm diameter). The electrode was pinched lightly around the stem in such a way that only the filter paper made contact with the stem. The electrodes were kept continually wet with 10 mM KC1 solution either by frequent applications from a syringe or, in long-term experiments, by means of a cotton wick one end of which dipped into a glass vial containing KC1 solution. The potential difference was provided by 1.5 V dry cells connected in series. In each experiment several groups of plants, each consisting of 4 individual plants wired in parallel, were connected to the source through a switched moving-coil meter (0-100 gA; internal resistance < 1 kf~) which allowed the current in each circuit to be measured in turn. The internal resistance of the meter was less than 0.3% of the combined resistances of the plants in each circuit9 In all the experiments reported here a potential of 9.0 V was applied. The direction of current flow was reversed where required by means of a switch which allowed the connections to the d.c. source to be rapidly changed. Control plants were fitted with KCl-soaked electrodes in the normal way but no potential was applied.

Application, Extraction and Measurement of 14 C - I A A . [1-'4C]IAA (specific activity 2.13 GBq retool; Radiochemical Centre, Amersham) was reduced to dryness under nitrogen from the toluene/ acetone solution supplied and was redissolved in 0.01% Tween 20 in distilled water at a radioactive concentration of 551 kBq ml a (equivalent to 46 gg IAA ml-~). In the transport experiments the compound was applied to the plants as a 4.0 gl droplet between the stipules of a small, unexpanded leaf still enclosed in the apical bud (usually leaf 6 or 7 in 14-d old plants) using a calibrated micrometer syringe. After the required transport period the apical buds were quickly removed and the stems were cut into 5 mm long segments which were individually extracted overnight in 1.0 ml cold ethanol in polyethylene counting vials. 14C was determined by liquid scintillation counting after the addition of 10 ml Bray's fluid to each vial, and the results were corrected for quenching by automatic external standardization. Transport profiles were constructed for each plant individually by plotting loglo radioactivity per segment against distance from the point of application of labelled IAA (measured to the centre of each segment), After each experiment the filter paper electrodes were dropped into counting vials containing 10 ml scintillation fluid and checked for leakage of t4C from the tissues. In some experiments the form of the transported 14C-labelled compounds was checked by ascending paper chromatography in isopropanol :ammonia :water (10:1 : 1, v/v) and comparison of Rf values of radioactive spots with the Rf of samples of authentic IAA and indol-3yl-acetyl aspartic acid (IAAsp).

Results

Electrical Characteristics of the Plant. U n d e r the a p plied p o t e n t i a l o f 9.0 V each p l a n t initially p a s s e d a c u r r e n t o f 25-30 ~tA when the e l e c t r o d e s were separ a t e d by a d i s t a n c e o f ca. 50 ram. This c u r r e n t decreased d u r i n g the first 70-90 rain to reach a s t e a d y value o f 15 20 g A w h i c h was m a i n t a i n e d for u p to 48 h w i t h o u t change. The m a g n i t u d e o f this s t e a d y c u r r e n t was n o t affected by the p o l a r i t y o f the a p p l i e d potential. Reversal o f the p o l a r i t y once a s t e a d y cur-

D.A. Morris: Electric Potentials and Auxin Transport rent h a d been a t t a i n e d resulted in an i m m e d i a t e increase in the c u r r e n t p a s s e d f o l l o w e d b y a g r a d u a l fall until s t e a d y c u r r e n t c o n d i t i o n s were regained. This o b s e r v a t i o n suggests t h a t the initial fall in the c u r r e n t p a s s e d m a y have resulted f r o m the electrop h o r e t i c a t t r a c t i o n o f m o b i l e ions t h r o u g h the stem t o w a r d s the electrodes. U n d e r s t e a d y c u r r e n t c o n d i t i o n s the electrical resistance o f the stems o f b o t h A l d e r m a n a n d M e t e o r was a p p r o x i m a t e l y 100 kf~ c m -1 length. T h e r e was no i n d i c a t i o n t h a t the t r e a t m e n t s in a n y way affected water m o v e m e n t in the stem a n d p l a n t s were n o t visibly d a m a g e d b y the p a s s a g e o f the c u r r e n t s for u p to 72 h. Over 50 m m the a p p l i e d p o t e n t i a l o f 9.0 V was e q u i v a l e n t to a v o l t a g e d r o p o f the o r d e r o f 18 m V per cell for files o f cells 100 ~tm long.

Infhtence of the Applied Potentials on the Transport oflAA. In Fig. 1 typical t r a n s p o r t profiles are s h o w n for i n d i v i d u a l p l a n t s o f M e t e o r d w a r f p e a in an experi m e n t in which a 9.0 volt p o t e n t i a l was a p p l i e d to the stem t h r o u g h o u t t r a n s p o r t . [ 1 - I 4 C ] I A A was a p plied to the apical b u d 80 min after the c u r r e n t h a d been switched on (i.e. w h e n s t e a d y c u r r e n t c o n d i t i o n s h a d been a t t a i n e d ) a n d t r a n s p o r t was a l l o w e d to p r o ceed for 2.5 h o r 5.7 h. In c o n t r o l p l a n t s (no current) the l o g a r i t h m i c profiles of t4C d e v e l o p e d in the stems were typical o f those o b t a i n e d in earlier t r a n s p o r t experiments. T h e y possessed well-defined c u r v i l i n e a r fronts w h i c h m i g r a t e d b a s i p e t a l l y at a s t e a d y velocity o f 12.4_+0.86mm h -1 leaving a ' p l a t e a u ' o f r a d i o active m a t e r i a l in the stem as they a d v a n c e d (see Eliezer a n d M o r r i s 1979). T h e p a s s a g e o f the c u r r e n t p r e v e n t e d the m o v e m e n t o f l a C - l a b e l l e d I A A b e y o n d the u p p e r e l e c t r o d e on the stem regardless o f the p o l a r i t y o f the a p p l i e d p o t e n t i a l . I n e x p e r i m e n t s in which the u p p e r e l e c t r o d e was p o s i t i o n e d s o m e distance b e l o w the apical b u d I A A c o n t i n u e d to m o v e t h r o u g h the stem a b o v e the e l e c t r o d e a n d a c c u m u l a t ed in the vicinity o f the e l e c t r o d e itself, i n d i c a t i n g t h a t the i n h i b i t o r y effect o f the c u r r e n t on t r a n s p o r t was confined to the region o f the s h o o t t h r o u g h which the c u r r e n t passed. This is e v i d e n t f r o m the results p r e s e n t e d in Fig. 2 for an e x p e r i m e n t i n v o l v i n g the A l d e r m a n v a r i e t y in w h i c h the u p p e r e l e c t r o d e was sited 70 to 100 m m b e l o w the a p i c a l b u d . T r a n s p o r t times in this e x p e r i m e n t were 4.0 h a n d 23.0 h. L a b e l l e d I A A which a c c u m u l a t e d in the vicinity o f the e l e c t r o d e was n o t i m m e d i a t e l y released for t r a n s p o r t when the c u r r e n t was switched off a n d only after a p r o l o n g e d r e c o v e r y p e r i o d (16 h) was there a n y i n d i c a t i o n that s o m e r e m o b i l i z a t i o n h a d o c c u r r e d (Fig. 3). O n the o t h e r h a n d when [1-14C]IAA was

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Fig. 3a and b. Behaviour of transport profile fronts in 15-d old Meteor pea seedlings during the passage of a small direct current (a; 12.5 gA; 9.0 V; shoot negative to ground), and at varying time intervals after the current was switched off (b). In b IAA transport was allowed to proceed for 7.25 h with current flowing and the plants were harvested 0-16 h after the current was interrupted

Fig. l a and b. Inhibition of IAA transport in 14-d old Meteor pea seedlings caused by the passage of a small direct current (19.5 gA ; 9.0 V) through the stem. Transport profiles are for typical individual plants and were constructed 2.5 h (a) and 5,7 h (b) after the application of [I-~4C]IAA to the apical bud. Horizontal b a r = position of upper electrode ; broken line = background radioactivity

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Fig. 2a and b. Inhibition of IAA transport in 16-d old Alderman pea seedlings caused by the passage of a small direct current (11.3 gA ; 9.0 V). Transport profiles are for typical individual plants and were constructed 4,0 h (a) and 23.0 h (b) after the application of [IJ4C]IAA to the apical bud

Fig. 4a and b. Recovery of the IAA transport system following the interruption of a small direct current passed through the stem. Transport profiles are for typical individual plants t r a n s p o r t i n g under the following conditions: a Current passed throughout the transport period (19,5 laA ; 9.0 V; shoot negative to ground; transport period=3.75 h); b the same current was passed for 4.0 h and [I-I*C]IAA was applied to the apical buds 0 4 h after the current was switched off (transport period= 3.75 h

applied to the apical bud of dwarf pea plants immediately after a current which had been flowing for 4.0 h was switched off, normal transport was observed (Fig. 4). This indicates that the transport system itself was not injured by the current flow and that its ability to transport IAA was very rapidly restored when the potential was removed. Chromatographic analysis of ethanol extracts of l~C-labelled material which accumulated in dwarf pea stems in the vicinity of the

upper electrode when current was flowing indicated that in addition to IAA itself a substantial proportion of the radioactivity was associated with a compound which had the same Rf value as authentic IAAsp in the solvent system used. It has been shown elsewhere that this compound is a major immobile metabolite of IAA in the dwarf pea (Morris et al. 1969). In none of the experiments was any 14C detected in the KC1 solution which bathed the electrodes.

434 Discussion

The results presented here show that the slow, longdistance basipetal transport of I A A applied to the apical bud of the intact pea plant is completely and reversibly inhibited in the region of the stem through which a small direct current is made to flow under the influence of an applied potential difference. The passage of the current for up to 72 h appears not to injure the plant and the ability of the stem to transport freshly-applied I A A normally when the current is switched off indicates that the functions of the specific auxin transport system are not permanently impared. W o r k reviewed by Schrank (1953) has shown that the application of small d.c. currents to one side of intact A r e n a coleoptiles and to coleoptile segments may induce auxin-dependent growth curvatures towards the stimulated side and can counteract growth curvatures induced by unilateral illumination. Webster and Schrank (1953) found evidence for a lateral redistribution of auxin under the influence of transverse currents in the Arena coleoptile, auxin moving towards the side of the coleoptile in contact with the negative electrode. This, clearly, cannot be due to the electrophoretic movement of I A A anions. Little or ~othing seems to be known about the effects of applied currents on the long-distance basipetal transport of auxin, Black et al. (1971) found that direct currents of similar values to those employed in the present experiments significantly increased growth and ion accumulation by intact t o m a t o plants when the stem was made negative to ground. Reversing the polarity reduced growth. Calculation of the total charge applied indicated that the observed ion accumulation was not simply a passive movement under the influence of the applied potential, and it was suggested that small currents might have stimulated the activity of ion pumps or might have influenced ion distribution indirectly by altering the distribution of endogenous growth regulating compounds. More recently, Bratton and Henry (1977) have reported that direct currents of 3 to 7 gA (plant negative to ground) applied to intact t o m a t o plants increased IAA levels in the leaves and petioles compared with untreated plants, but reduced the levels in the root system, The observations reported here suggest that these changes in the distribution of I A A may be explained by an inhibition of longitudinal transport. The information available to date does not permit a ready explanation of the mechanism by which the applied currents caused the inhibition of long-distance auxin transport. It was probably not the result of physical damage to the transporting cells - the effect was rapidly reversible, and no indication of tissue

D.A. Morris: Electric Potentials and Auxin Transport damage was observed after passage of the current for prolonged intervals (continuously for up to 72 h in the present work and intermittently for up to 20 days in that of Black et al. 1971). The observed independence of the inhibition on the polarity of the applied potential suggests that inhibition could not have been caused by an electrophoretic attraction of IAA anions or of charged IAA-carrier complexes towards the upper electrode. It is likely that the application to the stem of a d.c. potential of either polarity would result in a change in extra-cellular p H (by removing protons towards the cathode), and possibly result in m e m b r a n e depolarization. Such changes would be expected to influence the degree of dissociation of I A A molecules and to disrupt the protonand emf-dependent polar transport of I A A postulated by the ' c h e m i o s m o t i c ' model (Rubery and Sheldrake 1974; Raven 1975). Whatever the mechanism involved in the inhibition of long-distance polar auxin transport by the passage of small electric currents through the stem, the apparent absence of injury to the plant and the rapid restoration of transport when the current is interrupted suggest that use may be made of the effect to reversibly inhibit endogenous auxin transport. If so, the effect could provide a valuable technique with which to investigate the role o f auxin transport in the regulation of plant growth and development. I am grateful to Mrs. R P Bell for able technical assistance. References Black, J.D., Forsyth, F.R., Fensom, D.S., Ross, R.B. (1971) Electrical stimulation and its effect on growth and ion accumulation in tomato plants. Can. J. Bot. 49, 1809 1815 Bratton, B.O., Henry, E.W. (1977) Electrical stimulation and its effects o n i~doleacetic acid and peroxidase ]evels in tomaW plants (Lycopersicon esculentum). J. Exp. Bot. 28, 338-344 Ellis, H.W., Turner, E.R. (1978) The effect of electricity on plant growth. Sci. Prog. (Oxford) 65, 395407 Eliezier, J., Morris, D.A. (1979) Effects of temperature and sink activity on the transport of lr indol-3yl-aceticacid in the intact pea plant (Pisum sativum L.). Planta 147, 216-224 Lund, E.J. (1947) Bioelectricfields and growth. University of Texas Press, Austin Morris, D.A., Briant, R.E, Thomson, P.G. (1969) The transport and metabolism of 14C-labelled indoleacetic acid in intact pea seedlings. Planta 89, 178-197 Raven, LA. (I975) Transport of indoleacetic acid in pIant celIs in relation to pH and electrical potential gradients, and its significance for polar IAA transport. New Phytol. 74, 163-172 Rubery, P.H., Sheldrake, A.R. (1974) Carrier-mediated auxin transport. Planta 118, 101 12I Schrank, A.R. (1953) Electronasty and electrotropism. In : Encyclopedia of plant physiology, vol. XVII, pp. 148-163, Ruhland, W., ed. Springer, Berlin G6ttingen Heidelberg Webster, W.W., Schrank, A.R. (1953) Electrical induction of lateral transport of 3-indoleacetic acid in the Arena coleoptile Arch. Biochem. Biophys, 47, 107 118

Received 25 July; accepted 25 September i980