The Nervous Mechanism of Plants

The following experiment shows that mere friction is sufficient to initiate the impulse. Experiment 4. — ’The petiole of Mimosa is held within clamping jaws to prevent any mechanical shaking. A suitable contrivance permits quick unclamping for observation of the responsive fall of the leaf. The petiole is subjected to the stimulus of friction by rubbing it with emery paper. If the clamp be now quickly opened the leaf will be at first found to retain its normal position, and it is only after a short interval that it exhibits the excitatory fall. The delay is due to the time taken by the impulse to travel through the intervening distance.

Even more convincing is the result already de.scribed of the effect of electric stimulation of moderate intensity on the intact plant (Experiment 2). As already pointed out, there is in such a case not the remotest possibility of exudation of sap or of injury to the wood. Both the theories of hydro-mechanical disturbance and transpiration-current are based on the supposition that a deep wound, causing an escape of sap or irritation to the wood, is essential to the transmission of impulse. I have just shown that transmission takes place, even in the complete

absence of any wound, under stimulation by superficial friction or by electric shock. The essential structure on which the two theories are erected thus falls to the ground. The theory of the transpiration-current involves three other assumptions ; that transmission takes place across a water-gap ; that the transpiration-current carries a hypothetical stimulant excreted by the stimulated wood ; and that the velocity of the sap-movement is the same as that of the transmission of impulse. The following experiments will be found to disprove all three assumptions.

I may begin by mentioning that Koketsu 1 cut across the petiole of Mimosa and connected the two portions by a water-tight tube filled with water ; no evidence was ever obtained that impulse generated by stimulation of the distal half was conducted across the water-gap to the other. My own experiments with the stem of Mimosa to test the supposed transmission across a water-gap were carried out with more than fifty specimens of Mimosa pudica, grown in vigorous condition in the grounds of my Institute. The experiments included diverse modes of intense stimulation ; yet the results in all cases were in perfect agreement. They were in some cases made with cut stems ; I have shown elsewhere ^ that the normal excitability of the cut specimen becomes fully restored in the course of a few hours, and that the response of the cut specimen is then very similar to that of the intact plant.

Experiment 5.— A divided stem had its two cut ends enclosed in a water-tube, and the two ends were brought in contact with each other. The sensitiveness of the upper and lower portions of the stem was such that separate ^ Koketsu, R. — Journal of the Department of A gricuUure, Kyashu Imperial University, vol. i, No. i, p. 55 (1923) : also Bose — Physiological and Anatomical Investigation on Mimosa pttdica — Proc. Roy. Soc., B. 98, 1925, p. 290. 2 Physiological Investigations with Petiole-Pulvinus Preparations of Mimosa pudica — Proc^ Roy. Soc., vol. 89, 1916.

application of induction-shock of minimal intensity, lasting for one second, caused the fall of the leaves of both portions. After recovery, stimulus was applied to the lower piece, its intensity being increased to the maximal; the duration of application was also increased from one to twenty seconds. In spite of this very intense stimulation there was no evidence of conduction across the water-gap. Experiment 6.— Stimulation was electro-thermal. A thin piece of platinum wire was wound round the lower piece of stem ; the passage of a strong electric current made the wire red-hot. Though the lo%ver piece was scorched by this intense stimulation, the upper piece remained unexcited, showing that no impulse was transmitted across the water-gap. This and the previous experiments were repeated with more than fifty different specimens ; in not a single instance was there any evidence of transmission across the gap.

The supposed transmission across a water-gap, in the case of the stems and petioles of Mimosa SpegazziuH, led Ricca to formulate the transpiration-current theory. Snow, in his recent paper, ^ states that ‘ in the leaf of M. Spcgazzinii excitation is regularly conducted by some mechanism that has nothing to do with the water-current.’ In opposition to the very far-fetched theorj? of pressure-variation put forward by Dixon, Snow asserts that ‘ changes of pressure in the tube-cells play no part in conducting excitation, even in the leaf.’ Snow is nevertheless of opinion that transmission in the stem is normally brought about by movement of water which carries the hypothetical stimulant. It will be presently shown that this supposition is without any foundation.

The transpiration-current theory involves the assumption of the excretion of a stimulant substance as the result of stimulation. I have failed, as the following experiment shows, to obtain evidence that any such substance is excreted. Experiment 7. Application of extract at cut end. — An extract prepared from internodes of Mimosa was applied at cut end of the stem. This caused no stimulation, though the experiment was repeated with twenty different specimens. It is conceivable that certain vegetable extracts might act as stimulants ; vegetable alkaloids of a poisonous nature, for example, produce excitation when applied in minute doses to the cut end of the stem. It would, however, be quite unreasonable to conclude froin this result that an alkaloid is excreted by the plant under the action of a minimally effective stimulus.

But even assuming the presence of a stimulating substance, the question of the velocity of its translocation still remains ; for, if the transpiration-current theory be true, evidently the velocity of the transpiration-current which conveys the stimulant must be identical with that of the transmission of the impulse. Snow finds that the highest rate of ascent of sap in Mimosa is 18*5 cm. per minute, or 3 mm. per second. I find that the highest velocity of transmission of excitation in thin petioles of Mimosa is of a very different order, being as high as 400 mm. per second. Again, under normal conditions, the ascent of sap is upwards, but excitation travels both upwards and downwards simultaneously.

Instead of comparing the velocity of the movement of sap and of excitation in different specimens, the results would be more conclusive if their respective rates were determined in an identical specimen. It is moreover possible to arrange the experiment in such a manner that there is practically no movement of sap in the direction of transmission of excitation;^ Simultaneous Determination of Movement of Sap AND Transmission of Impulse The rate of translocation of a stimulant can easily be ascertained (i) by the use of a staining agent which is also a stimulant ; or (2) by the use of a colourless stimulating fluid, the extent of translocation of which by the movement of sap can be detected by the employment of a suitable ‘ developer.’ I describe three typical e.xperiments carried out with three different species of plants.

Experiment 8.— Methylene blue, in a moderately strong solution, is* a stimulant. The thin and long flower-stalk of Biophytim sensitivim is a very efficient conductor of excitation. When the stalk is stimulated in any way by a mechanical or electrical stimulus or by a chemical irritant, the resulting impulse travels downwards, against the normal direction of ascent of sap, and reaches the rosette of leaves, causing the closure of the sensitive leaflets from the centre outwards. To hasten absorption of the stimulating stain, the top of the flower-stalk was cut across and a piece of moist cloth placed on the cut end. After recovery of the leaflets from the excitator^j- impulse caused by the cut. Methylene blue was applied at the cut end. The impulse initiated by the stimulant was found to rt;ach the sensitive leaflets and cause their closure. The time required for transmission was 30 seconds, the intervening length of the flower-stalk and stem being 60 mm. The extent of translocation of the staining solution downwards vas ascertained by examining the longitudinal section of the flower-stalk under the microscope. The translocation in 30 seconds was found to be quite negligible, certainly not more than through 0-3 mm. This was due rather to slow diffusion than to the movement of sap. hlven on the assumption that the transpiration-current can carry the sap downwards, the rate of transmission of excitation was of a very different order to that of .sap-movement, being

at least 200 times quicker. I obtained similar results with Eosm stain. specimen in this case was Mimosa pudtca the stimulant employed being a drop of dilute hydrochloric acid The extent of translocation was tested by application of silver nitrate solution, which produces a white precipitate. The acid was applied at the tip o he upprmost leaf. To promote absorption of the solulon It IS advisable to wash the tip with dilute ether Half a dozen experiments gave very similar results, of which

d stimulant travelled downwards and caused the successive fall of seven lower leaves in the course of 40 seconds, the distance of transmission being 120 mm. Examination of the section of the plant with silver nitrate solution showed that the acid had not been translocated downwards but remained practically localised at the point of appHcation! Experiment 10.— For this experiment I took a different species the sensitive M. Spegazzinir of tree-like habit. The potted plant was young ; the petiole of each leaf bore rom five to seven pairs of sub-petioles, each of which in their turn bore about thirty pairs of relatively large-sized leaflets. The passage of impulse is easily followed by the excitatory fall of the sub-petioles and leaflets in regular sequence. The pulvinules of the leaflets are highly sensitive ; the secondary pulvinus of the sub-petiole is also fairly sensitive; but the main pulvinus of the leaf is practically insensitive.

A drop of hydrochloric acid was applied to the extreme end of the uppermost sub-petiole to the right. The im- p^se traveUed inwards and reached the main petiole; afterwards it travelled in a centripetal direction towards the stem, the successive pairs of sub-petioles ahd their leaflets exhibitmg excitatory fafl (fig. 2). The length through which the impulse traveUed in the uppermLt sub-petiole was 64 mm., the time of transmission being 10 seconds. The impulse reached four out . of the five pairs

of sub-petioles and was transmitted through a total distance of 135 mm. in the course of 55 seconds. Chemical examination with silver nitrate solution showed that the acid applied at the tip of the sub-petiole had not been translocated, but had remained localised at the point of application. The results of the experiments described above prove conclusively that the transmission of impulse is in no way connected with the movement of sap. The theory of the

Fig. 2. Centripetal Impulse in Petiole of A/. Spegiusinii, under application of chemical stimulus at S. transpiration-current as the means of the conduction of excitation is therefore completely discredited. Normal transmission is, then, not hydro-mechanical, nor is it due to movement of the sap. There tlius remains but one alternative, that it is a propagation of protoplasmic excitation as in the nerve of the animal. My investigations on the subject,^ carried out for more than twenty years, afford conclusive proof of the nervous character of the transmission of impulse in plants. I propose to give in the following chapters a connected account of my

researches on the subject. I will first describe experiments m proof of the excitatory character of the transmitted impulse in Mimosa, and then deal with the characteristics of the conducting tissue. FinaUy I shall give an account of my recent discovery of a class of phenomena in Mimosa which mdicate a high degree of differentiation in its nervous system. It has been contended, on insufficient grounds, that the wood is the tissue which conducts impulse. Haberlandt’s experiments prove, on the other hand, that it is the phloem and not the xylem, which is the conductor. . '

^ The hydro-mechanical theory is based on the observation of Pfeffer that stimulation is only effected when the knife has penetrated the vascular bundles and allowed the escape of a drop of water. The excitatory fall of the leaf at a distance is thereforesupposed to be due to a transmitted mechanical blow or traction. It is here shown that stimulation can be readily effected without a wound and escape -of sap. The transmission is therefore not hydro-mechanical.

The theory of transmission by the transpiration-current IS based on the supposition that a hypothetical stimulant excreted as the result of irritation of the wood, is translocated by the transpiration-current with the same speed as the impulse which causes the successive fall of the leaves. Experiments have been made for the simultaneous determination of the velocity of the impulse and the rate of transport of the chemical stimulant which initiates it. The results show that while the chemical stimulant remained practically localised at the point of application, the impulse generated was transmitted to a considerable dikance. The transmission of impulse is therefore in no way connected with the movement of sap.

The results of the tests described in the last chapter show that transmission of impulse in Mimosa is due neither to a hydro-mechanical disturbance nor to the transpirationcurrent. I now offer evidence that transmission in the plant is physiological and is fundamentally of the same character as that of the nervous impulse of the animal. Satisfactory evidence is obtainable only by the employment of a mode of stimulation which is purely ph3'siological, and which causes no physical disturbance. Transmission of impulse must then be due solety to the propagation of protoplasmic excitation. Further confirmation would be afforded by effecting a physiological block which arrests an excitatory impulse but has no such effect on a hydromechanical disturbance or on the transpiration-current. The following are the results of my investigations on (i) the excitation induced by the discriminati\-e polar action of a constant current, and (2) on the electrotonic arrest of transmission of impulse.

It is well known that an electrical current causes .specilic excitatory reactions in animal tissues. Excitation is characteristically produced at the sudden starting or ‘ make ' of the current ; it is also produced at the stoppage or ‘ break ’ of the current. Thus when a feeble electric current is passed by means of two electrodes through a muscle, an excitatory contraction is initiated on the sudden starting of the current at the kathode where the current leaves the tissue, no excitation being produced at the anode, that is at the point of entrance of the current. At the break of the current, excitation is produced neither at the kathode nor at the anode. When the intensity of the electric current is increased, the excitation due to the make still takes place only at the kathode; whereas at the break of the current excitation now takes place at the anode.

These characteristic effects are produced not only by the direct application of the current, but also by its indirect application. Thus a nervous impulse, initiated at the kathodic point of a nerve by the make of the current, is conducted along the nerve, causing excitatory contraction of the terminal muscle. Similarly the break of a stronger current initiates an impulse at the anode. The characteristic polar effects of current on Protozoa were found by different observers to be, generally speaking, opposite to those in animal tissues. Hence it has been supposed that the laws of polar reaction in unfibrillated protoplasm must be different from those in highly differentiated animal tissues. My experiments on the polar action of electrical current on plants prove, on the other hand, that the reactions of the undifferentiated protoplasm of the plant-body are identical with those of the animal tissues. This is demonstrated by the following experiments with the primary pulvinus of Mimosa pudica.

Monopolar method. — One electrode from a battery is applied on the pulvinus and the second electrode attached to a distant indifferent point. Four cycles of operation can be performed with the commutator by which the current in either direction can be made or broken. Figure 3 illustrates the condition after sudden make of the current by tilting the commutator to the left, the pulvinus being made the kathode. By proper manipulation of the commutator, the pulvinus may be subjected (i) to kathodemake, (2) to kathode-break, (3) to anode-make, and (4) to anode-break.

Experiment ii. Effect of feeble current. — I used a leaf of Mimosa which was in a moderately sensitive condition. The electrical resistance between the two points of contact was found to be a million ohms. An E.M.F. of b volts was Electric connections are made with the pulvinus and a second in-* different point on the stem. Tilting the commutator to left makes the pulvinus kathode. Slight movement to right causes break of kathodic current. Tilting to t!ie right produces make-anodic current at pulvinus. Slight tilting back produces anode-break.

found to be effective in inducing the e.xcitatory fail of the leaf, the pulvinus being made the kathode. Excitation is induced by a sudden variation of the current and n(.)t during the continuance of the current. Hence, if after tlu; excitatory fall, the current is continued, the leaf re-erects itself. The current is now broken ; this induces no excitation. The commutator is next tilted to the right, the pulvinus being made the anode ; this again induces no excitation, nor is there any excitation on break at the anode.

Experiment 12. Effect of moderate current. — The E.M.F. of the acting current was next increased to 12 volts. In carrying the experiment through the usual cycle of operations, it was found that the kathode excited at make and not at break. The effect of the anode, on the other hand, was different ; it excited at break and not at make. The automatic record by the plant is given in Fig, 4. The normal signal-line below indicates no current; the up-line, kathode-make ; return to horizontal, kathodebreak ; down-line, anode-make ; and return to the horizontal represents anode-break. It will be noted that under 8 volts, excitation takes place only at kathode-make. Under 12 volts there is a stronger response at kathode - make and none at kathode break ; no excitation was produced at anode-make, but it occurred at anodebreak.

polar excitations is Fig- 4* tinder 8 volts excitation at kathode- j , ,, T, make Km; under 12 volts excitation at of experiments which I carried out with the sensitive organs of various other plants, such as the leaflets of Mimosa, of Biophytum, of Neptunia oleracm, of Averrhoa Carambola and of Averrhoa Bilimbi, The effective intensity of the current depends on the excitability of the particular specimen, a vigorous specimen being excited by a feebler current. It also depends on the relative excitability of different motor organs, the pulvinules of the leaflets being, in general, more sensitive than the main pulvinus. As an example of the response of the leaflets, I describe an experiment with Biophytum sensitivum, employing the Bipolar method. The two electrodes were applied at two points on the petiole which carries numerous pairs of motile leaflets.

Experiment 13. Effect of feeble current. — This caused excitation only at kathode-make and not at break. No excitation was produced at anode on either make or break. The formula for excitatory reaction to feeble current is thus Km. Experiment 14. Action of moderate CM/mii.— Excitation took place only at kathode-make and at anode-break. I give a sketch showing these effects (fig. 5) ; the figure to Fig. 5. Illustration to the left shows excitation induced by kathode-make, that to the right the elfect of anode-break {Biophytmn sensUivmn)^

the left shows the excitation initiated at the kathodic point at make, which did not remain localised but was conducted in both directions. After the recovery of the leaflets, the circuit was broken, and excitation was initiated at tlic point of anode-break. The formula for moderate intensity of current is KmAb. Numerous experiments were carried out with differiait plants which gave similar results. In the two tables on p. 27 are given the effects of feeble and of moderate current. The value of the current given is what was found to be minimally efiective for highly excitable specimens.

The specific reactions of plants to electric current are thus in every way similar to those of animal tissues. table L-Effecx op Feeble Cxxrrekx on various Sensitive Planxs Excitation only at kathode-make and at anode-break Formula KmAb. 7 he discnminahve excitatory transmission on kathode-make and on anode-hreak proves that it is due neither to hydromechanical disturbance nor to the transpiration-current, but is a propagation of protoplasmic excitation. '