Plant Response as a Means of Physiological Investigation

IN the last two chapters the effects of various agencies on the power of conduction were demonstrated qualitatively. It is important, however, to obtain, if possible, the quantitative values of this conductivity and its variations. ' The absolute value of conduction can be obtained from the determination of the velocity of transmission of excitation through the tissue. This determination of velocity may be made roughly, by observing the time taken for the application of a stimulus, say by cut or hot wire contact at a given point, to produce motile effects on a leaflet at a known distance.

A result thus obtained, however, would, for reasons to be given presently, prove very indefinite, and no two such results in succession could be trusted to agree. In order to ascertain the exact quantitative effects of various agencies on conductivity, we must first be completely assured that our determinations of velocity under normal conditions are trustworthy. Difficulties in exact determination of velocity of transmission of excitation.— In the course of the investigation carried out on this subject, I have found that the discrepancies between the velocities, determined in the way described, are largely to be accounted for, first, by indefinite changes of excitability at the point of application, due to the injury caused by excessive stimulation ; and, second, to the changes of conductivity, caused by fatigue, in the rest of the tissue. I have also found that the velocity of transmission is only a determinate quantity when the intensity of stimulus is constant It undergoes variation, with changes in the stimulation-intensity.

These difficulties are met by using a stimulus which does not cause injury, and which can be repeated at uniform intensity. Such a stimulus is given by means of the condenser discharge. As regards the changes of conductivity due to fatigue, I have found that fatigue is removed, and conductivity fully restored, after a definite period of rest, which, in the case of Biophytum, is about four to five minutes. The next difficulty to be overcome is concerned with the question of recording the exact moment of application of stimulus, and that of the initiation of response at a distant leaflet. A further source of uncertainty in the last respect, lies in the existence of an unknown latent period of the leaflet, which may delay the visible response, even after the effect of stimulus has reached the point at the base of the leaflet.

It is evident that the times of application of such rude modes of stimulation as cut, or contact of hot wire, cannot be accurately determined, and the exact moment of the beginning of the responsive movement of the motile leaflet is equally difficult to ascertain by the unaided eye. These difficulties are, however, removed, if we use the discharge from a condenser as our mode of stimulation, and the magnified movement of the spot of light from the Optic Lever, as the indicator of the commencement of response. The observer, following the spot of light from the Optic Lever, makes two

marks on the revolving drum — one when the discharge-key is pressed, at the moment of application of stimulus, and another when the spot begins to move, that is to say, at the commencement of response. It is then easy, knowing the rate of movement of the drum and the distance between the two marks, to determine the exact time-interval between the two. There then remains only the question of allowing for the loss of time due to the latent period of the responding organ. This is accomplished by means of a separate experiment, in which the stimulus is directly applied at the base of the motile organ. The latent period thus ascertained is subtracted from the time-interval already determined, and we have thus the true time of transmission of excitation through the given distance ; from this the velocity, or rate of transmission per second, may be deduced.

Exact determination of velocity.-— I now give an account of an actual experiment for the determination of the velocity of transmission of excitation in the petiole of Biophytum. The two points A and B are connected in the circuit of a condenser through the usual non-polarisable electrodes (fig. 14). An indicating leaflet, L, is attached to the Optic Lever, by which the exact moment of its response may be recorded on the revolving drum. If now we make B kathode, during the charge of condenser, an excitatory wave will start from B and travel inwards towards L, in this particular case in a centripetal direction, i.e. towards the main stem. A mark is made, as already explained, on the revolving drum, at the exact moment when the tapping-key excites the plant. In this case, the capacity of the condenser was *oi microfarad, and E.M.F. twelve volts. The time of the stimulus reaching L, as indicated by the movement of the spot of light, is also marked, as explained, on the revolving drum. As has been said before, the time-interval is accurately determined from the speed of the revolving drum. As an additional precaution, the same time-interval is taken by means of a stop-watch.

From a separate experiment, by direct stimulation of the base of the petiolule, it was found that the latent period of the leaflet was so small a fraction of a second, as, for our present purpose, to be negligible. In this way, in my first experiment, I found the time taken by the excitation to travel the distance of 27 mm. between B and L to be 14-3 seconds. I allowed the plant a period of rest of three minutes, and again performed the experiment under similar conditions. The time taken was found to be 14*5 seconds, which is practically the same, within experimental error, as the result first obtained. The slight difference was due to the residual effect of fatigue. In any case, the extreme difference between the two results amounts to less than 1-4 per cent. — or 7 per cent, from the mean value of 14'4. From this we find that in the particular plant under experiment the velocity of transmission in a centripetal direction was 1*88 mm. per second. In order to show how consistent successive results are, I give successive time-intervals taken by stimulus in two different cases, to travel the intervening distances.

In all these cases, the second experiment was undertaken after an interval of rest of three minutes. The slight retardation uniformly observed is due, as already explained, to residual fatigue. It is, however, so small as to be negligible. At any rate, making allowance for all possible sources of uncertainty, the variation of these determinations will be less than 2 per cent. We have to remember that, owing to the slow velocity of transmission of impulses in plants, and also to the comparatively great length of tissue, that can, when necessary, be brought under examination, the total interval of time that has to be observed may be made as large as twenty to forty seconds. In such periods, a mean error of even '2 second

would hardly produce an inaccuracy of 1 per cent, in the result. This would compare favourably with the determinations that have been made of the velocity of transmission of nervous impulses in animals. In a frog's nerve, for example, owing to the high velocity and comparatively short length of nerve available for experiment, the total interval of time which has to be observed is of the order of some thousandths of a second. To obtain an accuracy within i per cent here, would mean the recording and measuring of an interval of something like the 2 0 * 0 0- part of a second.

The velocity of transmission in a given plant is found, under normal conditions, to be constant. It varies in different species, and even in the same species the value changes with the season of the year and the physiological condition of the specimen. A velocity determined in winter under less favourable physiological conditions, is very much lower than the velocity of transmission in the same plant in summer. The exact determination of the velocity of nervous impulses in animals has, therefore, been a matter of some

uncertainty. For example, Helmholtz found this velocity in man to be about thirty-three metres per second. Some recent determinations, again, give a value twice as great. Owing, moreover, to the difficulty in exactly discriminating the rising part of the curve, the same record may be interpreted to give results which differ from each other by as much as 20 per cent.1 Preferential conductivity. — I shall next pass to the consideration of the very curious and interesting pheno-

Fig. 104. Diagrammatic Representation of Electrical Connections for Determination of Velocities of Centrifugal and Centripetal Transmissions menon of preferential conductivity, by which it is seen that the state of excitation travels through a tissue with greater facility in one direction than in the opposite. For the purpose of this demonstration, I took a fresh leaf of Biophytum. The two condenser connections (capacity *oi microfarad charged to twelve volts) were made at A and B (fig. 104). The indicating leaflet L was situated somewhere between. On charge, B became the kathode, and an excitatory wave was started in a centripetal direction. On the abatement of this wave, the condenser was discharged ; A now became the kathode, and an excitatory wave was transmitted in a centrifugal direction. From these two successive experiments, we are now able to determine the two velocities in opposite directions in the same leaf.

The following table exhibits the results obtained in this manner with two different specimens : Excitatory discharge preferentially directed. — These two observations show that the velocity is greater in the centrifugal direction. In some instances I have found the centrifugal velocity to be nearly twice as great as the centripetal. These experiments seem to indicate that under certain conditions, excitatory discharges will take place preferentially in one direction only. We may imagine any intermediate point in the midrib of the leaf, to be acted on locally by a gradually increasing or accumulating stimulus from

external sources. Immediately on this reaching the threshold of response, it will give rise to an excitatory discharge. And it is clear that this excitation will be transmitted preferentially along the line of least resistance, that is to say, in the direction of the greatest conductivity, or outwards. I have been able to obtain further experimental verification of this conclusion, by applying a gradually increasing stimulus of condenser discharge to an intermediate point on a petiole of Biophytum, When this stimulus had reached a certain value, it was found that while excitation, as indicated by the fall of the leaflets, travelled through a great distance forwards, or outwards, its transmission backwards was extremely limited. Could we have adjusted the stimulus, so as to have been slightly above the threshold of response, there would have been no transmission backwards. When stimulus, on the other hand, is excessive, the entire excitatory effect cannot be carried forward ; there is an overflow backwards ; and under these conditions excitatory movements take place in both directions.

Effect of fatigue on velocity of transmission. - We shall next deal with the modification of the velocity of transmission by fatigue. Specimens of Biophytum were used for the purposes of this investigation, and experiments were performed by ascertaining the times taken for the transmission of a repeated uniform stimulus, through the same distance, under shortening periods of rest. A stimulus was given to a leaf of Biophytum, and the record of time taken— the transmission being in a centripetal direction. The plant was now given an interval of rest of three minutes. Stimulus was again applied, and the timerecords obtained in the usual manner. The next stimulus was applied after a resting-interval of two minutes, the following after one, and the last after half a minute, the time of transmission and the records of response being taken throughout. From the results given below, it will be seen how regular is the decrease in velocity with the increase of fatigue. The distance to be traversed, 27 mm., was kept the same in all cases. The time taken at the beginning, when the plant was

fresh, to traverse this distance was 14*3 seconds. In the next experiment, when the stimulus was applied after three minutes, there was a slight residual fatigue, and this prolonged the time to 14*5 seconds. On the third occasion, a still shorter time, namely two minutes, was allowed for rest, and the rate of transmission became slower, the time being now 157 seconds. The next interval of rest was still further shortened, to one minute, and the time of transmission was correspondingly increased to 16*4 seconds. And lastly, when the stimulus was given after an interval of only half a minute, the velocity was still further retarded, the time now taken being 175 seconds.

The following table gives the different velocities under increasing fatigue, and the heights of the corresponding responses. It has already been said that the distance through which the stimulus was transmitted was in all cases the same, namely 27 mm. Table showing Variations of Velocity of Transmission and of Amplitude of Response in Biophytum, with Increasing Fatigue It will be seen from this table that, while the variation of velocity due to the difference of conductivity between three minutes' rest and an indefinitely longer period is slight, there is a considerable diminution of this velocity when the restingperiods are still further shortened. It will be noted, moreover, that increasing fatigue is shown not only by a regular decrement in the speed of transmission, but also in an independent and still more striking manner by a steady diminution in the heights of the responses themselves.

Fig. 105. Curve showing Decline in Heights of Responses, with Diminishing Periods of Rest Abscissa represents resting-periods, and ordinate heights of response motile excitability under shortened periods of rest. This curve when produced will cut the abscissa. Such a point would mark the time-interval between two successive stimuli, at which the response would be zero, that is to say, the motile excitability would be abolished. In other words, the leaf would, when the resting-interval was shortened to this period, prove refractory to stimulus. In Chapter XXI I the existence of this theoretical refractory period will be demonstrated by experiment.

It is thus seen that owing to imperfect protoplasmic recovery, or, in other words, to residual molecular strain, not only is the conductivity of the tissue gradually diminished, but the excitability also. Thus we obtain some idea of the processes by which fatigue is brought about. Effect of intensity of stimulus on velocity. — We have next to study the variation in the velocity of transmission. of the excitatory condition, with increasing strength of stimulus. In the case of animal nerve, it has been ascertained by different observers (Helmholtz, Vintschgau, and Fick) that the velocity of transmission of the nervous impulse is not independent of the strength of stimulus, but increases with increasing intensity. The experimental verification of this with conducting animal tissue is, however, extremely difficult, owing principally to the shortness of time involved.

I have carried out an investigation on this subject with vegetable tissues, which shows in an unmistakable manner that velocity does undergo an increase, with increasing stimulus. These experiments were carried out with leaves of Biophytum. I first demonstrated this in a qualitative manner, by using thermal stimulation. It was thus found that the excitation caused by a strong, would travel with a greater velocity than that due to feeble stimulus, the one being sometimes double the other. I next tried to obtain quantitative results, by using a form of stimulus which was measurable, and could be increased in a graduated manner. For this I employed the method of stimulation by condenser discharge, the stimulus being increased by increasing the E.M.F. that charged the condenser (*oi microfarad).

In one series, with a stimulus of eight-volt charge, the velocity found was r8 mm. per second. When the stimulus was increased by charging the condenser to twelve volts, there was an increase of velocity to 1*9 mm. per second. And finally, with a sixteen-volt charge, the velocity was found to be 2"i mrn. per second. These velocities referred to centripetal transmission. In the next series, the experiments were carried out on a much more excitable leaf, and the velocity was determined in a centrifugal direction ; with a charge of eight volts, the velocity was 3*27 mm. per second ; with sixteen volts, it rose to 376 mm.; and with thirty-two volts, it became 3-83 mm. per second. The two following tables exhibit these results of increasing velocity with increasing stimulus :

Tables showing Increase of Velocity with Increasing Stimulus Specimen I. — Centripetal transmission. The distance traversed by stimulus was 27 mm. Specimen II. — Centrifugal transmission. Distance traversed by stimulus was 38 mm. The stimulus, it is to be remembered, is increased by increasing the voltage. But, on an undue increase of this charging voltage, to about forty volts or upwards, I have often found that the velocity undergoes an actual diminution. This is to be ascribed to the fact, which was demonstrated in the chapter on Polar Effects of Currents, that the excitatory value of the kathode reaches a limit, with a certain E.M.F., and that if the E.M.F. be carried far beyond this, the excitatory effect is reversed, that is to say, it is now the anode that excites (p. 206). We have then the curious case of a negative direction, as it were, of transmission. For whereas, with moderate voltage, the excitatory disturbance travels in the interpolar region from kathode to anode, it now, with excessive voltage, travels in the opposite direction, from the anode towards the kathode.

We can now see with what great accuracy it is possible to measure these changes of velocity, from which we can deduce the variations of conductivity, not merely qualitatively, but also quantitatively. This opens out to us the further possibility of studying the quantitative effects of various external agencies in modifying conductivity. I shall here relate a simple experiment which affords an example of the method to be followed in such an investigation.

Effect of lowering of temperature on velocity of transmission.— In order to study the effect of lowered temperature on conductivity, I applied ice-cold water over an area of 10 mm. of the conducting petiole in Biophytum. The length of the conducting tissue experimented upon was 38 mm., and the time taken for the stimulus of a condenser-discharge (ten volts and "Oi microfarad) under normal conditions, i.e. before the application of ice-cold water, to traverse this length, was icri seconds, giving a velocity of 376 mm. per second. But after the application of ice-cold water, the conductivity was so diminished that the transmitted excitation did not produce any response of the motile leaflet ; on allowing the temperature of the cold water applied, however, to rise a few degrees, the stimulus was found to be effective ;

but the velocity of transmission was now found to be much reduced. Instead of icri seconds being necessary for transmission through the entire length, it was now found to take 148 seconds. The difference of 47 seconds here, represents the additional time taken for transmission through the 10 mm. length of cooled tissue. In other words, whereas transmission through 10 mm. of normal tissue had taken about 2'6 seconds, it now took about 2'6 4- 47, or 7*3 seconds ; that is to say, the conductivity was reduced by cooling to nearly one-third.

Effect of rise of temperature on velocity. — It has been proved that conductivity is reduced by lowering of temperature. We should therefore expect that a rise of temperature would produce the opposite, namely, an increase of conductivity. That this is the case was shown by an experiment on a leaf of BiopJiytinn. Care was taken that the leaf should not be too young, since, as will be shown later, the effect of a rise of temperature on a young leaf is to initiate automatic response. I found that in this specimen excitation travelled a distance of 41 mm. in a centrifugal direction in 11 seconds, the temperature being 300 C. The velocity at this temperature was therefore 37 mm. per second. On now raising the temperature to 350 C. the time taken for transmission was reduced to half, i.e. 5-5 seconds. The temperature was next raised to 370 C, and the time was now found to be further reduced to 4*5 seconds, the velocity being thus 9/1 mm. per second, or nearly three times as great as at 300 C.

Table showing Effect of Rise of Temperature otf Velocity of Transmission in Biophytum Channels for conduction of effect of stimulus. Before concluding this chapter, it is important to consider the channels through which stimulus is conducted with the greatest facility. Since the conduction of stimulus is due to the transmission of protoplasmic change, it is clear, as already said in a previous chapter (p. 60), that such changes will be conducted most easily along those paths in which there is least interruption of protoplasmic continuity. It is evident, therefore, that certain elements in the fibro-vascular bundles will furnish the best conducting medium for the transmission of stimulus. It also follows that in the fibro-vascular tissue itself, the conduction along the length would be more rapid and complete than across.

On the other hand, the cells of indifferent tissue, such as the parenchyma of the leaf, are divided from each other by more or less complete septa, the fine filaments by which neighbouring cells may be protoplasmically connected being so minute that the conduction of stimulus through such imperfect channels must be exceedingly feeble. These theoretical conclusions I have been able to verify by direct measurement of conductivity in different kinds of tissue. In this investigation, as motile tests of the state of excitability were not available, I devised an electrical method — to be referred to briefly in the next chapter, and described more fully elsewhere— by which to attack the problem.

Using this method of investigation, I found that plantorgans which contained fibro-vascular elements, such as the stem, peduncle, and petiole, were the best conductors of the state of excitation, and that conduction in such organs is much greater along the length than across it ; in the peduncle of Musa, for example, the conductivity lengthwise is three times as great as that crosswise ; and finally I found that though indifferent tissues like the parenchyma are directly excitable, yet there is practically no transmission of that state of excitation through such tissue.

From anatomical and other considerations, Dutrochet and Haberlandt came to the conclusion, that it was* certain elements in the fibro-vascular bundle which were concerned in transmitting the disturbance in Mimosa. This transmission was, however, regarded rather as a hydro-mechanical than as a true excitatory propagation. Such a conclusion, as we have already seen, appeared at one time to be probable in the light of the experiment on the transmission of excitation through a narcotised area. I have, however, already shown on p. 229, that abolition of motile excitability need not always imply the abolition of conductivity. Haberlandt describes an experiment according to which the excitation in Mimosa is said to have been propagated over dead tracts of the petiole, these portions having been destroyed by scalding. But it is extremely difficult to ensure the death of interior tissue by such means as superficial scalding. I have found that a portion of a plant-tissue when subjected locally to the action of boiling water, afterwards exhibited signs of true excitatory electric response. It is only by prolonged immersion in boiling water that one can be quite sure that the interior tissue is really killed by scalding, and unless this is done thoroughly it is easy to see that the inner cells may conduct the stimulus.