Plant Response as a Means of Physiological Investigation

Chemical reagents are found to have the same effect on the response of growth as on ordinary response, or as on the autonomous response of Desmodium gyrans. Carbonic acid gas induces a preliminary acceleration, followed by retardation and arrest of growth. On readmission of fresh air growth is revived. Ether, when applied internally, causes a preliminary acceleration, followed by retardation and arrest of growth. Minor differences of effect may be observed if the application be made externally.

A very dilute solution of sodium carbonate is an excitant, and accelerates growth ; but stronger solutions cause retardation. Solution of sugar also stimulates growth, if the strength of solution be not excessive. Alcohol causes spasmodic alternations of growth. Too strong a solution arrests growth. As in the case of autonomous response of Desmodiumy so in the response of growth also, acids and alkalis are found to have antagonistic effects. Acid causes relaxation and ultimate arrest of growth ; alkali causes arrest of growth in the contracted position. The arrest brought about by one of these agents may be counteracted by the other.

The reaction of a specimen to a chemical reagent is determined by the strength of the dose, and by the duration of application. A stimulating agent if given in too strong a dose causes depression. A poisonous reagent, again, if given in a sufficiently small dose, acts as an excitant. A clear insight into the nature of ' constitution,' so called, as a factor in determining the reaction of a specimen to a given drug, is afforded by the induction of definite artificial constitutions. This may be carried out by subjecting the plant to four different typical temperature conditions—the ordinary, the optimum, post-optimum, and maximum.

It is then found that a plant which has been made to acquire excess of internal energy can struggle against, or even overcome, the influence of such adverse circumstances as the action of poison, whereas under ordinary circumstances it quickly succumbs. The nature of the response is thus seen to be determined in a definite manner by the constitution, or tonic condition, of the plant, this factor being in its turn dependent on the sum total of the energy which is latent in the organism.

Statement of the problem of apogeotropic response— Mode in which stimulation is brought about : radial-pressure theory, and theory of statoliths — Mechanics of responsive movement — Experiment demonstrating responsive curvature as brought about by unilateral pressure of particles— Record of curvature induced by gravitation — Record of different rates of curvature when specimen is held at angles of 450 and 1350 to the vertical— Determination of the true character of apogeotropic response — Responsive curvature of acellular organs— Curvature of grass haulm under gravity — Growth of grass haulm on a klinostat.

It is well known that growing organs exhibit certain directive movements under the influence of gravitation. Horizontally laid shoots, for example, bend upwards, or against the direction of gravity ; while roots react in precisely the opposite way — that is to say, they bend so as to lie in the direction of the force of gravity. In the case of the various forms of stimulation hitherto studied, the action of the plant is well defined and intelligible, consisting of concavity of the excited side, in response to a stimulus which is clearly understood. But in the case of geotropism the mode in which stimulation is brought about is not quite evident, and we find, moreover, two directly opposed effects brought about by apparently the same force of gravity : in the root, as already said, a positive movement, that is to say a movement in the direction of gravity ; and in the shoot a negative movement, away from the direction of gravity.

The seeming impossibility of explaining effects so divergent as due to a single common cause, has led to the modern idea that these responsive movements are ' executed at the suggestion of changes in the environment, not as the direct and necessary result of such changes ; ' or in other words that ' light and gravitation could be classed together as external agencies acting, not directly, but in some unknown indirect manner.' ! There are two distinct points to be borne in mind in connection with the effect of gravity : first, the question as to how gravity exercises stimulation ; and secondly, that of how, in answer to this stimulus, a definite responsive curvature is induced.

Mode in which stimulation is brought about. — Now it is clear that, as regards the former of these points, the only conceivable way in which gravity could produce stimulation is by the effect of weight ; not, that is to say, by the weight of the plant as a whole, but by the differential effect of weight in the cells. Two important theories have been advanced which offer very rational explanations of the means by which gravi-perception may be induced. According to these, the necessary differential weight-effect may be due to the weight of the cell-contents, whether of the sap itself, or of those heavy particles like starch-grains which are contained in it. When, therefore, the cell is laid horizontally, it is the lower tangential wall which has to support the relatively greater weight (fig. 203). This theory of hydrostatic or radial pressure was suggested by Pfeffer, and supported by Czapek. The other theory, of statoliths — advocated by Noll, Haberlandt, and Nemec— substitutes for the weight of the

1 A luminous resume of our present state of knowledge on this subject, with all its difficulties and obscurities, is contained in the addresses of Francis Darwin delivered before the British Association in 1891 and 1904. From these I have made the quotations which appear in the text. Figs. 203, 204, and 209 appear as illustrations of the latter address in Nature of September 8, 1904, Fig. 203. Diagrammatic Representation showing Differential Effect of Weight on Lateral Walls of Cells

In the figure to the right the cell is laid horizontally, and the lateral wall, D, has to bear greater weight than c (after Francis Darwin). Fig. 204. Diagrammatic Representation of a Multicellular Organ On the upper side the statoliths act on the inner, and on the lower side on the outer, tangential wall (after Francis Darwin). water-column the weight of certain relatively heavy bodies, such as starch-grains, differentially exercised upon the lower tangential walls. In the case of multicellular plants, laid horizontally (fig. 204), EE and E'E' may be regarded as regions in which stimulation is caused by the weight of the particles. The effects produced on the upper and lower halves are evidently antagonistic, and in spite of this we obtain in the case of shoots a resultant curvature upwards. This shows that the stimulation of one half must be greater than that of the other. The inequality must be due to this difference, that the statoliths in EE rest on the inner, and in E'E' on the outer, tangential wall. It would thus appear that one of these must be less excitable than the other.

Mechanics of responsive movement. — From these hydrostatic or statolithic differences of weight, bearing on the ectoplasm of the cell, it is understood that graviperception arises, that is to say that the plant perceives the direction of gravity. But there is no explanation as to how stimulation is produced ; nor is there any satisfactory explanation as to the mechanics of the responsive curvature. It is generally supposed, as has been said, that this curvature is not the direct and necessary result of some environmental change, but that it is an instance ' of a plant reading a signal and directing its growth' accordingly. It is supposed further that in an apogeotropic cell the curvature takes place by the development ' of relatively accelerated growth on the side on which the pressure is greatest.'

This view, that the curvature induced in some unknown way in a geotropic organ, by gravitation, depends upon the accelerated growth of the convex side, is apparently supported by Elfving's experiments on grass haulms in which growth had been at standstill. ' He found that the pulvini of grass haulms placed on the klinostat increase in length. This experiment shows incidentally that the klinostat does not remove but merely distributes equally the geotropic stimulus ; also that geotropic stimulus leads to increased, not to diminished, growth. The same thing is proved by the simple fact that a grass haulm shows no growth in its pulvinus while it is vertical, so that when curvature begins (on its being placed horizontally) it must be due to acceleration on the convex, since there is no growth on the concave side in which retardation could occur.' l

In the cases of response to stimulation, hitherto studied, however, it will be remembered that the fundamental effect was always a contraction and concomitant retardation of growth, the expansion and acceleration of growth being always a secondary and indirect effect ; but in the case of the response to stimulus of gravity, the interpretation of results which has just been quoted would make it appear that the responsive action brought about by gravity was essentially distinct in its character from the responsive reaction with which we have hitherto been familiar.

Experiment demonstrating responsive curvature as brought about by unilateral pressure of particles.— My own object in the course of the present chapter is, however, to demonstrate the fact that the curvature of an apogeotropic plant-organ in response to gravitation is the result of the direct effect of stimulus, its reactive peculiarities being in no way different from those other instances of the response of the plant to stimulation, which we have already studied And in order to simplify my explanation, I shall here

1 B.A. Report, 1 891, p. 671. It ought to be mentioned here that in other plants when placed on the klinostat this increased rectilinear growth was not observed, leading to the supposition that in such cases a simultaneous increase and decrease of growth-rate on opposite sides of the rotating plant is produced. Ibid. : Nature, describe a typical experiment showing the excitatory effect of pressure in inducing responsive curvature. That unilateral pressure or contact does induce curvature is known in the case of tendrils ; but in the present experiment I shall show that this responsive reaction is not peculiar to tendrils alone, but is exhibited by all organs alike. I was also desirous of making the action on my experimental specimens in every way parallel to that of gravitationally excited tissue in which the statolithic particles exert their weight on a particular side of the responsive organ.

I took a specimen of Crinum Lily and mounted it vertically. I next took a thin strip of india-rubber, the inner surface of which was studded with iron particles, adhering by means of shellac varnish. This was adjusted laterally, on one side of its zone of growth, so as almost, but not quite, to touch the specimen. On the opposite side was placed an electro-magnet, which when excited attracted I the strip to which the iron particles adhered, and thus

produced a unilateral pressure on the specimen, the magnetic particles functioning as so many statoliths (fig. 205). A recording microscope, which will be fully described in a later chapter, was now focussed on the index, I, attached to the specimen. Before the application of pressure, the quiescent condition of the specimen had been ascertained by noting the stationary position of the index in the field of view of the microscope. On now applying unilateral pressure by exciting the magnet, we might expect to obtain two different effects. The first of

Fig. 205. - Diagrammatic Representation of Experiment showing Curvature Induced by Unilateral Pressure Exerted by Particles F, flower-bud of Crinum ; s, indiarubber strip studded with iron particles attracted by electromagnet, M, causing unilateral pressure on growing region ; 1, index attached to flower. these would be the sudden magnetic pull which would cause a movement of the flower away from the pressed side. This effect would be instantaneous, and quickly come to a stop. The second would be the excitatory effect on the specimen of the unilateral irritation caused by the pressure of the iron particles. If this response, like response in general, were to take the form of a contraction, a curvature concave to the source of stimulus would be produced, by which the flower should be seen to bend towards the stimulated side. This stimulatory effect, unlike the mechanical disturbance, would go on increasing with time. In this way it is easy to demonstrate that unilateral irritation of pressure of particles induces contraction, which in growing organs retards the

normal rate of growth, the side acted upon thus becoming concave (fig. 206). It should also be borne in mind that in consequence of Ftc. 206. Record of R^ponsfve Curvature this responsive coninduced in Criniim under Experimental traction some of the Conditions shown in fig. 205. MagnificapvnpiiPf4 watpr finf4c its way to the opposite side, thus increasing its turgidity, with consequent acceleration of rate of growth and convexity of that side. The experimental proof of this latter phenomenon will be given in the next chapter. Moreover, in consequence of the curvature induced by this contraction of the excited side, the further side will be stretched, and tension is, as we know, an agent tending to increase the rate of growth. Thus we see that in this case the contraction of the excited side is the active factor, and the convexity of the further side is the secondary or subsidiary effect in the growth-curvature.

If then such stimulation by unilateral weight-effect, whether statolithic or hydrostatic, be the cause of the apogeotropic curvature induced by gravitation, it follows that the active factor in the process lies in the contraction of the upper side, and that the expansion and convexity of the lower must be merely the subsidiary effect. But it is usually supposed, as we have seen, that the predominatingly active factor in these gravitation-curvatures is the accelerated growth of the convex side. Hence the crucial experiment by which the correctness of one hypothesis or the other may be determined will evidently consist in demonstrating which of the two, contraction or expansion, is actually the essential element in the responsive growth-curvature. And if, further, we should succeed in proving that contraction was the essential element, we should then have established a unity, as between the phenomena of response to gravitation and those which are the results of other forms of stimulation. But before I describe this particular investigation I must explain the method of continuous record, which I employ for observing gravitational curvatures.

Record of curvature induced by gravitation. — We have in the Optical Lever a means by which the responsive effect of gravitation-curvature and its variations may be recorded quickly and continuously, and with as great a magnification as is desirable. The horizontally laid specimen, say the scape of Uriclis Lily, has its terminal upper end attached to one arm of the Lever, the other arm being weighted with a slight counterpoise. A continuous record is then taken, on a revolving drum, from which we obtain the responsive curvature and its time-relations. It will be seen from the record that the scape first bent down during a period of forty minutes, after which the effect of gravitation was seen by the reversal of the curve, which indicated the curving up proper to gravitation (fig. 207).

It is sometimes thought that this preliminary lapse of time before the appearance of the gravitation-effect, sometimes known as the presentation time, is the interval necessary for the statoliths to fall ; but I shall presently describe some experiments which will show that the time taken for variation in response to the varying action of gravitation is very much shorter, probably less than a minute. In the present case we must remember that the movement in response to gravitation has to overcome opposite mechanical movements before it can be made perceptible at all ; for we have, firstly, the weight of the organ, which tends to make it bend down ; and secondly, on account of this bending, we have greater tension on the upper surface, which, as we have seen, increases the

Fig. 207. Record of Apogeotropic Response in Scape of Uriclis Lily The up curvature due to apogeotropic action proper commenced forty minutes after the specimen was laid horizontally. rate of growth, tending to make that side convex. The differential effect due to gravity has to overcome all this, and becomes visible only when it has done so. After this stage has been reached, the rate of movement upwards goes on increasing until a fairly constant rate is attained.

Record of different rates when specimen is held at angles of 45°and 1350.— Czapek has found that the effective stimulus of gravitation is greater when the organ is held at 1 350 to the vertical than when held at 45 °. This difference of effect can be obtained quantitatively with great accuracy by taking successive records with the specimen in the two positions ; and in order to eliminate the effect of any chance disturbance, or of spontaneous variation, I took four alternate records, first with a specimen at 45 °, then at 1350, then back once more at 450, and then again at 1350, each position being maintained just long enough for the attainment of the permanent effect. The change from one position to the other was made quickly, in the course of less than a minute. The experiment was carried out with the unopened flower of Crinum Lily, in which we have already found that the rate of growth is very regular and considerable.

The first record was taken after the curvature movement, due to gravitation at an angle of 45 °, had attained a constant value. The tip of the flower was then found to be moving at a rate of four divisions per minute. On changing the angle to 1350, the rate of movement showed an immediate increase, and attained in the course of five minutes a permanent rate of 15*5 divisions per minute. It has been said that the experimental adjustment of change of position took only about a minute to effect, yet on renewing the record we find that the effective increase thus established in the gravitationstimulus was immediately perceived and responded to by the organ, in an accelerated rate of curvature. The permanent increased rate at 1350 is thus found to be nearly four times that at 45 °. The organ was now returned to its position at 450, and the rate once more fell till it became nearly equal to what it had originally been at 450, being only very slightly greater. The organ was once more placed at 13 5°, and the rate again rose, till it reached slightly beyond its former value at that angle. The ratio of the second rates determined at 450 and 13 50 was also found to be almost as 1 is to 4 (fig. 208).

With reference to the cause of this difference of gravitational effect, Haberlandt suggested that it may lie in the fact that the weight of the statoliths in 450 position is on the basal half, while at 1350 it is on the apical half (fig. 209) The next point is, to account for the greater reaction of the Fig. 208. Response Records showing Differences in Rate of Curvature according as Specimen is held at Angles of 450 and 135° FlG. 209. Diagrammatic Representation of Different Positions of a Single Cell, according as the Specimen is held at an Angle of 45° or 1 350, showing Consequent Redistribution of Statoliths (after F. Darwin)

apical half. Some light may, perhaps, be thrown on this subject from the results of my experiments on Biophytum. I found that on subjecting this plant to the favourable tonic condition of rise of temperature, the younger leaflets began to show spontaneous excitatory response much earlier than the older leaflets. This shows that in an excitable tissue the younger portions are, generally speaking, the more sensitive. This will probably account for the difference of geotropic reaction in the case under consideration, for the apical halves of the cells are relatively younger than the basal halves.

Determination of the true character of apogeotropic response. — I shall now proceed with the crucial determination of the true nature of gravitational response. In the diagrammatic representation of the multicellular organ, we have the upper and lower responding layers of cells represented by E and E' (fig. 204). Each of these may be one or more layers in thickness. Since in apogeotropic organs the curvature is upwards, this response may be due (1) to the relative expansion or acceleration of growth of the lower side, or (2) to the relative contraction or retardation of growth of the upper side. As regards the first hypothesis, the curvature induced in grass haulms has been assumed, as stated before, to have proved that the gravitational response is one of accelerated growth. On the other hand, the curvature may be due to the active contraction of the upper side, the response then being of the same nature as was seen demonstrated by the irritating pressure of the magnetic particles. The crucial experiment in deciding between the two alter-

native hypotheses will lie in determining which factor is actively concerned in the production of responsive curvature. If the lower should prove to be the side actively concerned, then the response will be one of expansion ; the activity of the upper, on the other hand, would demonstrate the contractile nature of the response. The principle of the mode of investigation which I adopted in order to decide this question will be understood, if we remember that motile response can be abolished temporarily by application of cold. Thus, if we cool the pulvinus of Mimosa, it ceases to exhibit any responsive contraction under stimulation. Now, in a horizontally placed organ, if the continued responsive curvature be due to the excitatory contraction of the upper side, then local application of cold on that side ought to arrest it. The application of cold on the lower side, however, should produce little effect. But if, on the other hand, the lower side should be actively concerned in the production of response, then the application of cold on that side would have the effect of arresting the curvature, while its application on the upper would have little or no effect. In connection with this, it should be remembered that a twitch may sometimes be produced by the transient excitation due to sudden variation of temperature, but the effect will be short-lived. The permanent arrest of hitherto continuous responsive movement due to gravitational stimulus will only take place when the active side has its power of response abolished by cold.

An experiment carried out in this manner would thus decide the question as to whether it is the upper or the lower side that is actively concerned, and also the question as to whether the gravitational response, like all other forms of response to direct external stimulus, is, or is not, fundamentally one of contraction. In carrying out experiments on the principle described above, I first took a record of the responsive curvature of a Crinum Lily, which was lying horizontally. When a uniform rate of upward movement had been attained, the tip of the