Propagation in Vicia faba Stem of a Potential

Plant Cell Physiol. 26(7): 1273-1283 (1985) J S P P © 1985

Propagation in Vicia faba Stem of a Potential Variation Induced by Wounding Gabriel Roblin and Jean-Louis Bonnemain University of Poititrs, Station Biologique de Beau-Site, 25 Faubourg Saint-Cyprisn, 86000 Poitiers, France

Key words: Variations potential — Vicia faba — Wounding.

Long distance transmission of stimuli by means of bioelectrical impulses takes place in plants with different characteristics according to the type of stimulus, namely injurious or not injurious (Bose 1907, Ricca 1916, Houwink 1935, Sibaoka 1954, Umrath 1959). In particular, when the stimulus is injurious (cutting, wounding, burning, etc.), a so-called variation potential is triggered. It appears as a wave of negativity with a variable shape and amplitude (Sibaoka 1953, Roblin 1979) which can pass through dead tissues. Ricca (1916) showed that astimulating substance was released by tissue wounding and he supposed that this material was transported in the transpiration stream. This type of conduction presents a widespread occurence in plants; it was thus detected in Tradescantia (Pfirsch 1966), Lupinus (Paszewski and Zawadzki 1976), Lycopersicon, Xanlhium, Cucurbita, Gossypium (Van Sambeck and Pickard 1976a), Vicia (Roblin

1985). In this respect, this type of stimulus transmission may represent a basic mechanism of functional coordination between different parts of a plant (Pickard 1974). In a previous work (Roblin 1985), it was shown that the time course of the variation potential in Mimosa, Vicia and Lycopersicon, recorded either by platinum wires or nonpolarizable Ag/AgCl electrodes can be divided into two components respectively called, components "A" and "B". Component "A" looks like a negative spiky bioelectrical change lasting about 1 min with an amplitude of 20-50 mV spreading in front of the component "B" depicted by a more smooth and long-lasting wave of negativity. The present paper extends these observations on the variation potential by studying some modalities of the transport of the stimulating substance in Vicia faba stem. 1273

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Heating of leaf blade in Vicia faba triggered bioelectrical events ("variation potential") spreading throughout the shoot, preferentially in the acropetal direction. The spreading of a variation potential, previously described in Mimosa and other plants, is related to a concomitant transport of a stimulating substance in the transpiration stream. This conclusion is supported by the observation in Vicia faba stem that conduction in the basipetal direction is promoted when the transpiration stream is inverted. The conduction is promoted in the vascular bundles in direct relation with the burnt lobe of the leaf, but a lateral conduction may exist, as electrical variations are recorded in isolated bundles of the orthostichy opposite to the wounded leaf. A local cooling (1°C) of the stem has no influence on the conduction rate but it completely inhibiti the bioelectrical variations in the cooled zone. This last result reinforces earlier demonstrations that the substance is transported in the vessels, according to a physical process, but that the electrical events require metabolic integrality in other cellular members.

G. Roblin and J. L. Bonnemain

1274

Materials and Methods

experimental

proced ure

L4

B incision Fig. 1 Isolation of a vascular bundle in Viciafaba stem and relative position of the electrodes (1, 2, 3, 4) inserted in the four orthostichies. Procedure A: control experiment. Procedure B: partial isolation of the bundle. Procedure C: entire isolation of the bundle.


Plants—Viciafaba L. (cv. Aguadulcc) plants were grown in vermiculite on a cycle of 10 h of darkness at 25CC and 14 h light at 27°G. Light intensity at pot level was about 6.6 W m~2 and was provided by four 40 W fluorescent tubes (Phytor ACEG-Charleroi-Belgium). Relative humidity ranged from 55 to 65%. Plants were fertilized daily with Hoagland and Snyder's solution (Hoagland and Snyder 1933). Leaves of all plants were numbered starting with the first reduced leaf (LI) above the cotyledons; in most experiments, plants had four fully developed bifoliate leaves (L3, L4, L5, L6). Monitoring equipment—Each experimental plant was set with its pot in a petri dish of nutrient solution inside a Faraday cage in the same experimental conditions as described above. The details of the recording method for the bioelectrical events by extracellular electrodes can be seen elsewhere (Stoeckel and Pfirsch 1975). Wires (50,um diameter 10% IridiumPlatinum) were inserted in plant internodeSj reference electrode being inserted in the vermiculite of the potted plant. Electrodes were linked through an impedance adaptor (1013 Q input resistance) to potentiometric recorders (Kipp and Zonen BD 9, Paris). It has been shown in a previous work (Roblin 1985) that the bioelectrical variations recorded by platinum wires present the same general pattern as those recorded by nonpolarizable Ag-AgCl electrodes connected to the plant by means of an agar-coated cotton thread protruding from a glass pipette containing 0.1 M KC1 gelled with 1% agar (Van Sambeek and Pickard 1976a). The main advantage of the recording with platinum wires is that they permit long-lasting experiments with recordings always taken at the same points. This is a good procedure because it allows comparative studies on the conduction rate to be made. The conduction rate is measured by the time lapse between the arrival of the electrical variation under two electrodes which were placed a known distance apart. Stimulating procedure—Stimulation was brought about by heating a leaf blade with forceps that had tips provided with heated disks of a known size (from 3 to 400 mm2). The disks were heated by dipping them in boiling water for at least 10 min (so that, their temperature when applicated on the leaf blade was about 100°C). This procedure permits to obtain the same bioelectrical variations than those triggered by classical burning of the plant tissues; it allowed a similar wounding technique to be used in all experiments. It has been demonstrated in Mimosa pudica (Sibaoka 1953) that conduction rate depends on the intensity of stimulation. It is important to note the limitation of this method because the heated leaf part is damaged. Nevertheless, it is possible to make a statistical comparison of the results obtained on various plants stimulated in the same way. In order to study the conduction rate in the acropetal and in the basipetal directions, leaves in upper and lower position respectively were heated (in most experiments, namely L4 and L6). Isolation of a vascular bundle—The platinum wires were inserted into the main vascular

Spreading of variation potential in plants

1275

Results The bioelectrical characteristics of the variation potential in Vicia faba stem It has been deduced that the vascular system of the stem is the transmission pathway for excitations, as well as the main passage for nutrient transport (Ricca 1916, Houwink 1935). Support for the occurence of the bioelectrical variations recorded by electrodes inserted in the orthostichies in the main vascular bundles of the broadbean comes from the observation that the pith is resorbed in this part of the stem, giving way to a central cavity. Typical variation potential

Following heating of the leaf blade, bioelectrical events were evoked, time course of which is represented by typical curve in Fig. 2. The analysis of the variation potential thus triggered has been made more extensively in a previous work (Roblin 1985), and it was concluded that the time course can be split into two components: an "A" spiky component with an amplitude of about 20 mV comes before the rather smooth "B" component of which a quantitative study has been made in later experiments using three parameters: the time at the maximum of the

mV = Fig. 2 Time coune of the variation potential recorded in a vascular bundle of Vicia faba stem by wounding of a leaf Tl, latent time separating the wounding blade (30 mm1). from the bioelectrical variation; Tm, time at the maximum of the potential variation; Tr, time at which the potential regains its basal value. Negative is up on the ordinatc.

com po nen ts 'A' "B-

z

oOB--

a. S

o o x i-

(0

OATI heating

TIM


bundles of the broadbcan which can be located externally in the four stem generating lines (orthostichies) (Fritz 1973). The bundles of an orthostichy can be isolated by incision with a razor blade up to the central cavity (Fig. 1). This isolation is made on an internode portion (procedure B) or on the entire distance separating two leaves (in the experiment shown L4 and L5); in this last case, the incision has been extended on every side in order to ensure that the nodal connections were totally cut off (procedure C). Procedure A refers to plants in normal conditions before the operation. The stimulations were begun when the plants had recovered from this treatment. Local cooling of the stem—The device to induce local cooling consisted of two circuits cooled by the circulation of a water-alcohol mixture whose temperature is regulated by a cryostat (Everat-Bourbouloux 1981). This allows a stem part, 5 cm long, to be cooled. In our experiments, the leaf 5 was removed to allow the assembly of the jacket at the 4-5 internode level. The electrodes were teflon insulated except at the wire tip; four were inserted into the orthostichy of leaves 4-6, two were inserted inside the cooling part and two outside, one in upper position, one in the lower position. The reduction from environmental temperature (28°C) to experimental value (1°C) took two hours.

1276

G. Roblin and J. L. Bonnemain

potential variation (Tm), the amplitude of the bioelectric variation at this time (signal amplitude) and the time at which the potential regains its basal value (Tr). The recorded signals, in particular the "A" component, are not float-up artefacts as those noted by Van Sambeek and Pickard (1976a) characterized by a simultaneous ascent under all the electrodes. In our experiments, the "A" component rose under each electrode after a latency time (Tl) in relation with the distance separating them each other, so that a conduction rate can be calculated. This point was also verified by placing the ground in various positions along the broadbean stem: in all cases, there was no marked changes in the pattern of the bioelectrical changes provided that reference electrode and recording electrode were enough separated to avoid biphasic recording. Differences between acropetal and basipetal conductions

Effect of the stimulation

intensity

The stimulation intensity is quantified by wounding the leaf blade with heated disks of known areas. For this experiment two platinum wires separated by a known distance (5—7 cm) were inserted into the internode 5 and 6 of the orthostichy bearing the leaves 4 and 6. The basipetal

direction

B acropetal

direction

©basipetal conduc''

•23

®" basipetal

direction

®-l_

0

I

40 2 40 2 4 MEAN BATE m m *"'

Fig. 3A Time course of bioelectrical variations induced by heating of a leaf blade (30 mm 1 ) in acropetal or basipetal direction recorded under four electrodes at 1 hour interval. Negative is up on the ordinate. B Variations of the mean conduction rates of the variation potential measured between successive electrodes inserted in different parts of a Viciajaba stem in acropetal direction (following heating of L4) or in basipetal direction (following heating of L6). Histograms are the results of three successive experiments carried out at 1 hour intervals. Electrode positions arc given on the figure.


Sibaoka (1953) has shown in Mimosa pudica that conduction is promoted in the acropeta direction since higher values of the conduction rate are observed in this way. The histograms in Fig. 3B point out that at the same stimulation intensity, the conduction rates are higher in the acropetal direction in spite of the variability of the results from one experiment to another. The obvious fact is that the basipetal rate rapidly damps. On the other hand, the acropetal rate is relatively constant in spite of transitory decreases or increases of its value. Such a relation is also found in the amplitude of the bioelectric variations (Fig. 3A), since the electrical wave rapidly damped in basipetal direction. A complete statistical outline of the results can be seen in Table 1.

b

r-

2

-z C

-.-

8

.a

x 8

'9

Direction of the conduction

Wounded area No. of (mm2) determinations

3 20 9 12 10 8

15

17 19 10 11 10 8

11 6 6 6 4

11

11 11 6 6 6 4

Acropetal 3 (Followin the 7 heating ~ B L ~ ) 30 100 400 whole foliole ( = I , 500) Basipetal (Following the heating of Ls) 7 30 100 400 whole foliole (2:1,500)

Parameter ElectrodeNo.

Tr (min)

Amplitude of the "BJ' (mW

~component t e

Tm (min)

Mean

Amplitude of the component ~ ~ ~ - "A" ~ (mV1

27+9.5(VS) 46f12(S) 59+28(VS) 81+26(VS) 130f35(VS) 194k22

~

6 1.5(VS) 7.4f 1.6(NS) 7.2+1.6(VS) 11.6+2.0(NS) 12.7*4.5(VS) 22.6k 7.5

25+10(VS)

~

30+18(VS) 66+27(NS) 75+24(VS) 120+21(S) lllk35(NS) 105+-22

5.2+1.3(VS)

35+13(VS) 59f18 (VS) 86+29(VS) 114+28(VS) 172+36

~

16+9(S) 23+8(NS) 20f3(VS) 29k9(NS) 25,6(NS) 26rt5

35+35(S)

6.1+1.3(VS) 7.3k 1.8 (VS) 12.4f2.3(NS) 12.7+4.6(VS) 30.2+ 10.2

O

1.9+0.5(VS) 3.4k1.8(NS) 4.8+2.3(NS) 5.4+2.4(S) 8.5+3.8(NS) 7.6k2.5

10+7(VS)

61+43(NS) 80f35 (S) 119+36(NS) 1171-34(NS) 100+32

C

l.lC0.3(S)

18+9(NS) 1 9 k 6 (NS) 22+4(NS) 22+5(NS) 23+4

+

1.6+0.6(NS) 2.0k0.7 (S) 3.3+1.2(S) 5.3+2.4(NS) 6.5k1.4

35 (9) 16 (5)

1

24 (7) 23 (7)

2

18 (6) 29 (9)

3

13 (4) 33 (9)

4

T1 (sec)

36 (3) 33 (3)

33 (4) 16 (4)

22 (7) 33 (3)

32 (1) 29 (2)

Amplitude of the component "A" (mV) 1 2 3 4

159 (7) 153 (16)

105 (4) 31 (3)

66 (18) 132 (5)

147 (12) 110 (3)

Amplitude of the component "B" (mV) 1 2 3 4

Table 2 Values of parameters depicting the bioelectric variations induced by heating of a leaf blade, recorded under electrodes inserted in each orthostichy of the stem following partial or complete lateral isolation of a vascular bundle of an orthostichy

Statistical significations are calculated by Student-Fisher Test (VS: very significant P>99%; S: significant >95%; NS: not significant P