Plant Response as a Means of Physiological Investigation

HAVING explained the means by which it is possible to apply a quantitative stimulus of uniform or increasing intensity, and also how the responsive effect and its timerelations are accurately recorded, we shall next turn to the study of the various characteristics of the response itself, as given for example by the plant Mimosa or Biophytum. In order that we may inspect different parts of the responsecurve in greater detail, it will be necessary to take the record on a fast-moving drum, so that the curve may be drawn out, and its several features more easily distinguished. As we wish, moreover, to study the excitatory effect on the motile organ, stimulus will be applied directly on the pulvinus.

Automatic method of record. — As it will be necessary in the course of the following investigation to measure the times of reaction accurately, to small fractions of a second, the record must be obtained automatically. It is to be remembered that, as said before, response in vegetable tissues is relatively more sluggish than in animal, and it is superfluous to arrange for measurements of time up to more than hundredths of a second. The experimental method that I am about to describe would, however, enable us, if necessary, to make determination of time-intervals of one-tenth this magnitude, the question being only one of using a recording drum with the requisite increased speed. The drum used for these experiments was one constructed by Verdin, and provided with a very perfect governor. Some little time elapses after starting the drum before it acquires uniform speed, which it afterwards maintains, however — at least during the short time required for the experiment — with great perfection. The record is not taken until this uniform condition is attained. The mirror of the Optic Lever throws a spot of light upon a sensitive photographic film, wrapped round the revolving drum. In order to produce records of the required rapidity, I employ sunlight, proceeding from a pinhole, which after reflection from the mirror of the Optic Lever falls on the drum, appropriately focussed by means of a condensing lens placed at the end of a focussing tube, as seen in the figure. It is understood that, the record to be obtained being photographic, this experiment is carried out in a dark room, the sunlight required for the record being directed upon the pinhole by a heliostat outside.

The stimulus consists of a single strong break-shock, from a RuhmkorfFs coil, one electrode of which, by means of non-polarisable connections, is attached to the pulvinus of a leaf, and the other to the main stem lower down, of a specimen of Mimosa. The shock is applied by the recording drum itself, at a particular moment in the course of its revolution ; and at the same instant the curve of response begins to record itself automatically. These two acts — of imparting stimulus, and of opening a shutter by which the recording ray of light is allowed to fall upon the moving film — are performed simultaneously ; and both alike are initiated by the stroke of a rod which is fixed to the axis of the drum underneath.

This rod, which I shall designate as the striker, at a certain period in the revolution of the drum, impinges upon a balanced electric key, K,, thus closing an electro-magnetic circuit, and so releasing the shutter S, which is immediately in front of the pinhole, by which sunlight is admitted. This drop of the shutter, simultaneously producing a break of the Ruhmkorrfs coil circuit, gives an excitatory electrical shock to the plant. When the speed of the drum has become uniform, K2 is closed, and the striker, in connection with the drum, closes the electro-magnetic shutter circuit. The dropping of shutter, S, interrupts by k:} the primary circuit of the induction coil, the secondary of which gives a shock to the plant. The fall of the leaf pulls down the Optical Lever, O, producing record on drum. k4, short-circuiting key of secondary.

The electro-magnetic circuit of the shutter is interrupted by a key, K2, and this is kept open till the speed of the drum has become uniform. On closing K„ the circuit is still incomplete ; but the striker, impinging against the balanced key, Kl5 completes the circuit, and actuates the shutter. A thread connects the shutter with one arm of a second balanced key, K3. This arm is so overweighted that when freed, it causes two prongs, at the opposite extremity of the lever, which complete the primary circuit of the Ruhmkorff's coil, to be lifted out of their cups of mercury, and thus the circuit is interrupted. But the thread is of such a length that when the shutter is set, so as to close the pinhole, the prongs, dipping into the cups of mercury, complete the primary circuit. The overweighted arm of K3 falls, with the drop of the shutter lifting up the prongs, and thus suddenly interrupts the primary current, giving rise to a break-shock in the secondary, which passes through the plant. During the course of the preliminary adjustment, when primary circuit of the coil is made, a make-shock is produced, but this is prevented from affecting the plant by a key, K4, which short-circuits the secondary. When the adjustment has been made, this short-circuiting keyis opened.

Briefly to recapitulate the procedure : The drum, carrying the sensitive film, is released, and begins to revolve. The key K2 of the shutter-circuit is kept open, until a uniform speed is attained. It is then closed. The striker connected with the drum now closes the balanced key, K, ; the shutter drops, and simultaneously interrupts at K3 the primary current of the induction coil, thus causing an excitatory shock to be given to the plant. The determination of the latent period. — It will be seen from the upper of the two photographs, given in fig. 1 10, that for a period of -^ of a second the record remains horizontal. This represents the latent period, after which the tissue begins to respond. For a further period of half a second the leaf is seen to fall with a considerable and approximately uniform speed. The rate of movement of the tip of the leaf is now 71 mm. per second. After this the leaf continues to fall, but with a diminishing speed, till the maximum contraction fall is reached. From records obtained on

slower-moving drums, I find that this is attained in different specimens, in a period of 1*5 to 2-5 seconds after the shock. This maximum contraction persists for a further period of about thirty seconds. The leaf now begins to erect itself, and full recovery is attained in the course of a further period of about six minutes. These statements refer to reaction in vigorous Mimosa, at a favourable season of the year, like summer. In an unfavourable season, like winter, however, the reaction becomes very sluggish, and recovery is not then

complete in less than eighteen minutes, or three times the normal period. Effect of cold on latent period. — I shall next refer to the sluggishness induced by cold, prolonging the latent period. The extreme instance of this is seen when iced water is applied to the pulvinus, and too great cooling being thereby effected, the response is abolished. With moderate cooling the latent period is found to be prolonged to several seconds. This effect cannot conveniently be shown, however, within the limits of a fast record. In order, therefore, to show the comparative effect of cold on the latent period, in the case of the same specimen whose record is seen in the upper of the two photographs in fig. 1 10, I was careful to cool the pulvinus very slightly. In the lower of these photographs it will be seen that the latent period has become prolonged, from the normal ^ to f3^- of a second.

Fig. 1 10. Photographic Record of Response of Mimosa, Exhibiting the Latent Period in its Variation The upper curve was taken under normal conditions, and the lower when the pulvinus was slightly cooled. Time-marks = tenths of a second. Original record reduced to half. [The duplication, which will be observed in each record, is due to the fact that the heliostatic mirror was silvered behind, thus producing two reflections, one from the surface of the glass and the other from that of the silver.]

The rate of responsive movement is also seen to have undergone considerable diminution. In the first, or normal, case, during half a second after the commencement of response, the rate of movement was, as said before, 71 mm. per second. In the second case, however, after slight cooling, it is seen to have been reduced to 26 mm. per second, or almost one-third of the original rate. Record by means of electric sparks : prolongation of latent period by fatigue. — In order to overcome the difficulty of the insensitiveness caused by keeping the plant in a photographic dark room, I have recently devised a method of

Fig. in. Electric Spark Record, Showing Increase of Latent Period by Fatigue, in Successive Responses of a Leaf of Mimosa Latent period in normal topmost record seen to be f-^ second ; this increased in next — taken 1 minute before full recovery — to ^ second ; latent period further increased in last case — taken 3 minutes before full recovery — to — second. Note also progressive change in slope of curve. record by means of a series of punctures produced by electric sparks on a recording paper surface. The sparks occur at the short gap between the end of the long arm of the recording aluminium lever, and the drum, these being connected respectively with the two electrodes of a Ruhmkorff's coil. The electrical disturbance does not affect the plant, as the leaf is attached to the lever by a long silk thread. One great advantage of this method lies in the 'fact that the timeintervals, which may be as short as desired, are indicated by the distance between successive punctures, which are determined by the frequency of the vibrating interrupter of the coil. In the case of which the record is given (fig. 1 1 1) the

interval between successive sparks was fe of a second. By this means, it was found that increasing fatigue induced a corresponding increase in the latent period of a leaf of Mimosa, from the normal ffo to jVtt °f a second. Response of Biophytum. —I shall now proceed to describe effects essentially similar to the last, seen in Biophytum. In order to be able to observe in detail the various responsive peculiarities of the curve, subsequent to stimulation, records were taken in this case on a much slower-moving drum. The record given in fig. 112 shows the mechanical response to stimulation produced by discharge of condenser (capacity -oi

Fig. 112; Response of Biophytum ; Electrical Stimulus having been Applied at the Pulvinus of the Motile Leaflet microfarad, charged to nine volts). The exact moment of stimulation is marked on the record. It will be seen that the leaflet begins to respond almost instantaneously. The maximum contraction in this case, being considerably more rapid than in that of Mimosa, is almost attained in the course of half a second. An interesting point to be noticed in the record is the flattening of the top of the curve (fig 112). That is to say, the maximum contraction persists for a considerable time before recovery begins. In the present case this lasted for ten seconds. This period varies in different specimens, from a maximum of ten to about two seconds. But in this particular specimen the period

in question remained at least approximately constant, in successive experiments. After this there was commencement of recovery, which was completed — as seen by the return of the spot of light to its exact original position — in the course of five minutes. In order to form an idea of the consistency of results which may be expected from a good specimen, I repeated this experiment six times in succession, commencing the record each time at the same point on the recording surface as before. The degree to which all these curves coincided with each other in detail is almost incredible. Their rising portions, their flat tops, and their gradual descent during recovery, were all so coincident that the six successive curves appeared as but one.

In specimens of plants which were not in good condition, fatigue was shown by the gradual diminution in height of successive responses. For accurate standard experiments it is therefore necessary to have specimens which are vigorous. We have already seen the prolongation of the latent period which is induced by cold, in the case of Mimosa. I have obtained similar results also in working with Biophytum. For example, in a certain experiment, moderate cooling induced a prolongation of two seconds in the latent period. When the plant was allowed to return to the surrounding temperature of the room, however, the increase of latent period disappeared.

Latent period diminished by increased intensity of stimulus. — It has been said before that there are innumerable gradations between the extreme cases of motile sensibility in plants. As regards motility, an extremely sensitive leaflet was that of Mimosa pudica. Somewhat less quickly reacting were those of Biophytum sensitivum, and we had in the leaflets of Philanthus urinaria an instance of extreme sluggishness. The latent period of the leaflet of Philanthus, under moderate stimulus, is as long as three minutes, and the maximum contraction is not attained under forty minutes ; but with a stronger stimulus the latent period is reduced to less

than one minute, and the maximum contraction takes place in a relatively short period of about fifteen minutes (fig. 30, b and c, p. 44). Response of Biophytum on the ' all or none ' principle. It is well known that in the case of a contractile skeletal muscle, there is a minimal intensity of stimulus which -is necessary in order to produce contraction. From this point onwards, as the stimulus is gradually increased, the response increases, till a maximum contraction is arrived at, beyond which still further increase of stimulus produces no increase in effect. In cardiac muscle, however, the range of stimulus between minimal and maximal is practically narrowed to a point, so that the minimally effective stimulation is also at once maximal. It is to be remembered, at the same time, that the differences between cardiac and skeletal response are a question of degree, rather than of kind.

Curiously, the response of Biophytum is, in this respect, somewhat similar to cardiac response. In an experiment with a particular specimen of Biophytum, the intensity of stimulus was increased by successive increments of the E.M.F. used for charging the condenser. With an E.M.F. of seven volts there was no response. With a charge of nine volts there was always a response, and this was maximal. A charge of eight volts was almost on the threshold of response. That is to say, when I started experimenting, the leaflet was in a somewhat sluggish condition, and an eight-volt charge was ineffective. But after obtaining response to a nine-volt charge, I could obtain response also at eight volts. This was due to the fact that molecular inertness had been removed by the preceding effective shock. Thus we have two determinate values of stimulation, giving respectively maximum response and absence of response, the charges, namely, of nine and seven volts. The effective stimulus is, of course, constant for a given individual, but differs with the excitability of different specimens. Here, then, we have an instance of the ' all or none ' effect. The leaflet either responds to the utmost, or not at all.

Refractory period. — We shall next consider the peculiarities of the refractory period which I have discovered in the case of plant-tissues ; and for the material of this investigation we shall use the plant Biophytum, taking, in fact, the very specimen whose successive responses have already displayed such remarkable consistency (fig. 112). Such uniformity in successive responses is only possible when we allow sufficient time of rest for complete protoplasmic recovery, by which the excitability is fully restored. Eut it has been shown in Chapter XX. that if sufficient time of rest be not allowed, the protoplasmic recovery is incomplete, and the excitability is diminished. Hence the extent of response, which is an outward indication of excitability, is diminished, and this effect is known as fatigue.

We also arrived, in the same chapter, at the theoretical conclusion that there is a minimum resting interval, t|ie diminution of which results in such a loss of excitability as to abolish response, and this period we know as the Refractory Period, because the leaf then apparently takes no account of stimulus, or is 'refractory' to it (fig. 105 J. We shall now enter into greater detail regarding the peculiarities of this refractory period. After taking the six curves in response to separate single stimuli which were so extraordinarily similar, I proceeded to take a curve of response to two equal stimuli of the same intensity as before — namely, nine volts, charging •01 microfarad — the two stimuli following each other at an interval of one second. The application of the second stimulus appeared to produce no effect, the extent and general character of response being the same as in the case of single stimulus, with only the difference of a slight elongation of the flattened top of the curve. I next tried the effect of two stimuli at an interval of five seconds. The leaflet was still refractory to the second stimulation, but when I applied it at an interval of ten seconds, the second stimulus became effective. It will thus be seen that Biophytum has rather a long refractory period, during which, as far as can be seen, it takes no account of the impact of a new stimulus. This refractory

period is a matter of several seconds, but varies somewhat with different specimens. I have, again, in some cases observed a very curious phenomenon of two refractory periods. We thus find several very interesting parallelisms between the response of Biophytum in plants and that of cardiac muscle in animals. We find in both that the minimal response is also the maximal, increasing stimulation producing no increase of response. In the response-curve itself

the flattened top is common to both ; both have a prolonged refractory period ; and we shall see later that in both there is a tendency to the production of multiple rhythmic responses. In all these respects the responses of Biophytum resemble the cardiac responses, rather than those of skeletal muscle. But they have one peculiarity in which they share the characteristics of the responses of skeletal muscle. In the responses of cardiac muscle, successive effects are not additive, perhaps because that muscle undergoes the maximum contraction possible. In Biophytum, however, while any effective stimulus — whether minimal or largely supermaximal — will produce response of the same extent, yet this response, though the greatest possible for a single stimulus, is not the utmost of which the leaflet is capable. Hence, if we superpose successive stimuli, taking care that they do not fall within refractory periods, we shall obtain an extremely interesting response, showing the separate additive effects. I give here two curves, exhibiting these

Fig. 113. Additive Effects seen in Re- sponses of Biophytum to Stimuli which Fall outside the Refractory Period The record to the left shows the effect of stimuli, applied at intervals of one, and that to the right of half a minute. effects of superposition of stimuli. In the right-hand record in fig. 113 the stimuli were applied at intervals of thirty seconds. The successive responses, except the last, show a regular decrease. In the left-hand record, successive stimuli were applied at intervals of a minute, and appear much more equal.

But even in these additive effects we find one peculiarity which is also characteristic of cardiac response. In the latter case, though on repetition of stimulus there is no summation of height of response, yet the apex-time of the second response is shorter than that of the first. We see this in the case of BiopJiytum, in the right-hand curve. The first response has a slightly rounded top, but this is reduced to an acute angle in the second. The record, having been reduced to one-eighth for reproduction, does not show this so plainly as does the original.

Though each single response of Biophytum is maximal, yet from fig. 113 we have seen that this maximal response does not represent the utmost movement of which the leaflet is capable. In the particular plant here used for experiment, the maximal response to individual stimulus was always about thirty-eight divisions, but four superpositions produced a total movement of ninety-four divisions. It must be remembered that such effects can only be possible when the second stimulus is timed to fall at the expiration of the preceding refractory period.

If the absolute value of each individual minimaHy effective stimulus be represented by S, and if the whole be added together, or, in other words, if a stimulus of 4S be given at once, we may regard such a stimulus as made up of one minimally effective, plus three others which fall within the refractory period, and are thereby rendered totally ineffective. In other words, we may regard a very strong stimulus as made up of so many minimally effective stimuli. It is as if the first effective fraction alone acted, the succeeding portions, which arrive within the refractory period, being inoperative.

In view of certain other phenomena not altogether disconnected, it seems unfortunate that the term ' refractory period ' should be used with its present significance. For this term might be held to imply that the tissue refuses to take any account of the superfluous energy that is impressed upon it. It is more likely, however, that by some peculiar mechanism, the superfluous stimulus — i.e. what is over and above the amount necessary for producing maximal response— is prevented from overflowing. This excess of energy may, then, at least in some cases, remain latent, to be manifested at a later period in the form of excitatory impulses. Such impulses, again, attuned by some regulating process, may give rise to periodic or rhythmic overflow. That this is actually the case will be demonstrated in the next chapter.

The latent period of response is protracted by cold. It is also protracted by fatigue. It is shorter under strong, than under moderate stimulation. In vigorous specimens of Biophytum leaflet, the minimally effective stimulus is also maximal ; under normal conditions, this minimally effective stimulus has a definite value. There is also a definite refractory period in the response of Biophytum. If a second stimulus fall within this refractory period, it appears to produce no effect.

The response of the leaflet of Biophytum resembles in many respects that of cardiac muscle. In both, response is on the 'all or none' principle, and both exhibit a relatively long refractory period. Multiple electromotive responses due to a single strong stimulus — Multiple Ielectrotactile responses— Multiple mechanical responses -in Biophytum— Cyclic variations in multiple responses — Multiple retinal excitations— Intermittent pulse in man and plant — Semi-automatism —Continuity of multiple and automatic response — Conversion of Biophytum into automatically responding plant ; conversion of Desmodium into ordinarily responding plant— Similar polar effects of current in Biophytum and in Desmodium leaflet, at standstill— Moderate stimulus in Biophytum and in Desmodium at standstill produces single response ; and strong stimulus, multiple response.

I HAVE already explained in Chapter III. that the excitatory wave initiated by a stimulus has a concomitant electromotive wave. If the plant experimented on is provided with motile leaves or leaflets, the excitation is evidenced by the simultaneous mechanical response of the motile organ, and the electrical response of galvanometric negativity. " Multiple electromotive responses due to single strong stimulus. — In my investigations on electrical response in plants, I was surprised to find that whereas a single moderate stimulus gave rise to a single electrical response, a very strong stimulus very often initiated a multiple series of responses. I have obtained such multiple responses to a single stimulus with all kinds of plants, ordinary and 'sensitive,' and under the action of various forms of stimulus — chemical, thermal, and mechanical.