Plant Response as a Means of Physiological Investigation

Fig. 82. Determination of Deathpoint in Allium Tube by Ob- servation of Volumetric Contraction, causing sudden Expulsion of Water A, record given by older specimen ; death-point 630 C. b, record of younger specimen ; deathpoint 590 C. preliminary rise of the Mimosa leaf or curling of the Passiflora tendril. But at the death-point this movement was at first arrested, and then reversed with accelerated speed. The condition of this particular specimen was afterwards tested by the electrical method, when it was found that, while a portion of the peduncle previously cut off gave the normal response of living tissue, the specimen which had been subjected to the death-temperature gave no response.

The death-point of a plant, under heat-rigor, is concomitant to the disappearance of the true excitatory response of galvanometric negativity. After this point is passed, hydrostatic disturbance generally gives rise to the reverse positive response. When an anisotropic organ like the pulvinus of Mimosa is gradually raised in temperature, then, at a certain critical temperature, a spasmodic movement is produced which proves to be the death-response of the organ. This is a true excitatory response. The critical death-points of similar specimens are very definite and practically identical.

The death-point is modified by the physiological condition of the tissue. Other things being equal, death occurs earlier with a young than with a mature tissue. The death-response of an anisotropic organ is composite. In Mimosa it consists of a down, followed by an up, movement. This is due to the death-contraction being followed later by the post-mortem relaxation of the organ. In the spiral tendril of Passiflora, the death-response is given at the critical point by a sudden uncurling.

In the case of the hollow peduncle of Allium, the deathresponse is exhibited by volumetric contraction, producing sudden expulsion of contained water. Death-spasm in anisotropic organ due to differential longitudinal contraction — In radial organ the death-contraction is purely longitudinal — Death-point determined from point of inversion of a thermo-mechanical curve — The complete record thus constitutes a curve of life-and-death, the two being separated by the death-point — Characteristic thermo-mechanical curve as resultant of variation of temperature and variation of length — The necessity of specifying the rate of rise of temperature — The thermo-mechanical curve characterised by sharp and definite inversion at point of death — No inversion of thermomechanical curve after death of plant— Death-contraction under heat-rigor in plant analogous to similar phenomenon in animal — The Morograph, a perfected form of apparatus for determining critical point of death — Remarkable identity of thermo-mechanical curves obtained with two similar specimens — Death-point almost as definite as a physical constant — Vanishing of point of inversion with age— Determination of death-point under cold-rigor — Constancy of death-point.

We have seen how the death-point can be determined in anisotropic organs, by the occurrence of a spasmodic lateral movement. We have also seen that this deathmovement is an excitatory response, at once initiated and terminated at the point of death. It has been shown further that the anisotropic is simply an instance of differential longitudinal response. It follows that if the death-spasm in the anisotropic organ be indeed caused by true excitation, then from a radial organ, at the moment of death, we should obtain a purely longitudinal contraction. We may look Upon this phenomenon, again, from a purely molecular standpoint. We can then see that if death be brought about by a sudden molecular change, such an event might be expected to exhibit itself in a correspondingly sudden change of form. Let us

then take a radial tissue, and subject it to a gradual rise of temperature, taking a continuous record of its variation in length. From what has already been said it will be understood that, the variation being gradual, no responsive contractile effect will be induced during the process, but, on the contrary, some relaxation. At the death-point, however, a sudden inversion of the curve, due to death-contraction, may be expected to appear, and thus the whole record will constitute a curve of life-anddeath, this point of inversion separating the two. Should the inversion prove to be very abrupt, the turning-point will afford us a means of determining the temperature at which death occurs, with very great accuracy.

In order to prove, further, that this specific response is definitive, we may, after passing the death-point, bring the tissue back to its original temperature once more, and repeat the process. The record ought now to show no inversion characteristic of a transition from life to death. Means of obtaining thermo-mechanical record. — I shall now proceed to describe the manner in which I have obtained this thermo-mechanical record. I took a specimen of a radial organ, in this case the long style of Datura, and fixed it to a small glass rod which in its turn was fastened to a weight, the whole being placed in a vessel of water. The free end of the style was attached to one arm of the Optic Lever. The bath was now warmed gradually, a thermometer indicating the rise of temperature. Variations of length corresponding to the rise of temperature were progressively recorded, from the movement of the spot of light. For this purpose the mode of procedure was as follows : The vertical movement of the spot of light — occasioned by the variation of length of the specimen — was converted into lateral, by reflection from a second mirror. The paper wrapped about the recording drum was divided into millimetres. It was required that the abscissa of the thermo-mechanical curve should represent temperature, and the ordinate the corresponding length. The position of the spot of light, at any given temperature, was marked on the drum. At each rise of temperature of

1° C. the drum was rotated, say, through a distance of 2 mm., and the position of the spot recorded. In this way, by connecting the recorded points, a curve was obtained, in which the length corresponding to each temperature was known. In this curve an abrupt inversion, due to sudden deathcontraction, was found to occur at about 590 C. The curve thus obtained, however, though the successive points recorded were very near each other, is the result of intermittent observations. Again, two observers were required, one to read the temperature, and the other to take the record. It was therefore subject to error of thermometric reading.

Means of obtaining automatic record. — For this reason I was desirous of obtaining a curve which should be continuous and practically automatic, the plant itself being made to record its own variations of length, and its own death-point. The problem resolves itself into that of making the reflected spot of light partake of two motions simultaneously, namely, a horizontal movement proportional to the change of temperature, and a vertical movement proportional to change of length. The horizontal, or thermometric, component of the movement I secured as follows : I constructed a thermo-electric element of iron and nickel, one junction of which was placed in melting ice, and the other junction in the vessel of water containing the specimen whose temperature was being subjected to change. This element was placed in circuit with a resistance box and a sensitive reflecting galvanometer. The amount of the movement of the galvanometer spot of light could now, by interposing suitable resistance, be brought to any appropriate value. In my experiments, with a particular galvanometer, the movement of this spot of light, for each degree of rise of temperature, was 2*5 mm. — i.e. one-tenth of an inch — when the recording surface was at a distance of 125 cm. from the galvanometer. This extent of movement was quite sufficient for the purposes of the experiment, as it enabled estimates to be made with ease, correct to one fifth of a degree. By interposing smaller resistances, however, one-twentieth of a degree could easily be discriminated. The excursion of the spot of

light was now found to be strictly proportional to the rise of temperature. In order to combine this horizontal thermometric movement with that vertical movement occasioned by the variation in length of the specimen, the vertically moving spot of light from the Optic Lever was thrown on the galvanometermirror. The apparatus, it should be mentioned, was so arranged that the two mirrors were as close together as possible. The spot of light now, having been reflected from two mirrors, directly described a curve in which the abscissa gave temperaturevariation, and the ordinate, variation of length. When the source of light is a point, that is to say, a pinhole with electric light behind — the excursion of the reflected ray upon a photographic plate will produce an automatic record. Or the movement of the light may be followed continuously with a pen.

Conditions for securing accurate death-point. — Here I must point out certain conditions which must be kept in view if we are to obtain a very definite death-point. We know that if a plant be placed in an unfavourable environment, or in a temperature much above the optimum, for a prolonged period, death will ultimately ensue. But inasmuch as these temperatures would only cause the death of the plant by indirect and cumulative action through progressive derangement of the several functions, they cannot in themselves be said to constitute death-points. To be scientifically precise in such a determination it is necessary that we should discover a temperature which is of itself efficient to initiate sudden death. On the other hand, again, as the contraction of death is a phenomenon of response, we see that it must have a certain latent period. Some interval elapses, moreover, during which the tissue is attaining the temperature of the bath in which it is placed. Now if the rate of rise of temperature be too rapid, then, owing to the lag caused by these last two factors, by the time the death-response commences, the recorded temperature may have gone beyond the actual death-point.

We thus arrive at two conditions which must be regarded as mutually somewhat antagonistic. In the first place, in order to obtain the immediate point of death, it is essential that the plant undergo an exposure which is not too prolonged. In order, on the other hand, to make due allowance for the latent period and for attainment of the surrounding Imperature, the rate of rise must be gradual and definite. 1 the case of tissues which are not too thick, the latter of ese conditions is amply fulfilled by a rate of i° C. per inute and a half. We see, therefore, that in precise deterinations of the deathpoint, the rate of rise must always be »ecified. With thick stems, however, owing to relative want of ermal conductivity, the attainment of surrounding tem- >rature and occurrence of death throughout the whole of the organ is a very protracted process. The experiments which I shall describe were made with specimens which were not too thick, death at the fatal point being ensured, when the rate of rise of temperature was that prescribed, namely, i° C. per every minute and a half.

This definite rate of rise may be secured by using an electric heating apparatus, such as is commonly employed for boiling a tea-kettle. The current from the street-mains, which is 220 volts, heats water too rapidly. But the desired rate may be obtained by interposing an electrolytic rheostat of copper sulphate, the current being brought to a suitable value, by separation of the two electrodes through which the current enters and leaves the electrolyte.

In this case, when placing the specimen in the experimental bath, it is advisable to secure it to a bent glass rod, which rests outside. For if it is placed in the metallic vessel itself, the record will be subject to a certain disturbance, owing to the expansion of the supporting metal while heating. The expansion of the glass rod is so small as to be negligible. In this way, using for experiment a filament of the corona of Passiflora, I obtained a record, showing a very abrupt inversion, corresponding to the death-point, which was

between 590 and 6o° C. I shall presently have occasion to describe in detail the various characteristics of this curve. Having described the apparatus with which these curves were recorded, it is necessaVy to point out the difficulties which were encountered in working with it. It must be remembered that the excursion of the spot of light, in this case, represented a high magnification of the actual movements involved. The spot of light, moreover, was reflected from two separate instruments, and was liable to be disturbed by the slightest jar or tremor in either of them. Though the instruments were placed on a steady stone pedestal, even this precaution could not be made wholly effective, in the heavy traffic of a town. It was only, therefore, in intervals of quiet that approximately perfect results could be obtained. This difficulty led me to the devising of a much simpler and more perfect instrument, which I shall designate as the Morpgraph} This is a small and portable apparatus, self-contained, in which the necessity of a galvanometer is obviated. By its means, moreover, the record is unaffected by any earth-vibration.

The Morograph. — The thermometric record is produced by means of the curling and uncurling of a spiral compound strip, of two metals, having different coefficients of expansion. In order to give strength and steadiness, this helix, which is about 2*5 cm. in diameter, is made of somewhat thick strips of brass and tinned iron, soldered together. By increasing the number of turns in this spiral, the extent of movement per degree in the thermometric record may be increased at will. In my own Morograph, a helix of three circles was found to answer all requirements. The last half-circle of the lower end of the spiral is fixed to a heavy circular stand of brass, 3 cm. in diameter. The topmost half-circle, on the other hand, has had the tinned-iron strip cut off, and therefore consists of brass alone. It will thus be understood that a line drawn diametrically across this last half-circle

1 This word is derived from the Sanskrit root mrt, Latin mors, death. would rotate round a vertical axis passing through the centre of the spiral, under the influence of the differential expansion or contraction produced in the compound strip of metal by rise or fall of the temperature. When the outside of the circumference of the spiral consists of the more expansible metal, brass, then a rise of temperature will produce the movement of curling. The difficulty in the construction of this part of the apparatus lies in securing equal angular rotation of the diameter about the axis, with every equal rise of temperature. These indications were at first extremely irregular. I was able, however, to remove all traces of irregularity by careful and repeated annealing. In any case the thermometric indications of the compound helix may be previously calibrated.

The axis of the Optic Lever — one arm of which is attached to the plant-specimen, and which is to give the record of its variation in length with rise of temperature — is now supported on the diameter of this last half-circle of the helix and is thus rotated bodily, with rise of temperature (fig. 83). And it will thus be seen that the motion of the spot of light, reflected from the single mirror of the Optic Lever, is a resultant of two movements, which take place at right angles to each other — namely, the horizontal movement, due to thermometric variation and the corresponding vertical movement, due to the changes of length of the experimental plant-tissue. Owing to the fact that the spot of light in this apparatus is reflected only once, it is extremely bright.

I shall now proceed to describe the manner in which the plant is mounted, and other accessories of the apparatus. Fig. 83. The Thermometric Spiral and Optic Lever of the Morograph The circulaf brass stand on which the helix is mounted has at the centre a small tube, in which the lower part of the specimen is clamped. The plant-organ thus occupies the vertical axis of the spiral, its upper end being connected by a thread with the short arm of the Optic Lever. It may be pointed out here, as is better explained in the diagram, that in order to give room to the specimen, the axis of the Lever is made to rest upon T-pieces, which are erected at the two ends of the diameter of the helix. The plant-organ being thus placed at the centre, the inclosing spiral thermometer gives an accurate indication of the temperature to which it is exposed.

The circular stand, supporting both the specimen and the recording apparatus, is placed in what I shall describe as the inner thermal cylinder, within the circumference of which the base fits exactly, while the helix is free, to the extent of •25 cm. all round. This internal cylinder is made of copper, coated with silver. It is filled with water and placed inside an outer, or heating, cylinder of brass, which is also filled with water. Heat is applied, by means of a spirit-lamp, to the bottom of the outer cylinder ; thus the water in the inner vessel is subjected to equal heat, on all sides at the same time. Had the heat been applied directly to the inner cylinder, convection-currents would have caused great disturbance of the recording spot. With these precautions, however, there is no trace of such disturbance.

The whole apparatus is supported on a steady stand. Below it is the spirit-lamp, which may be raised or lowered till a distance is found which gives us the standard rate of rise of temperature, that is to say, i° C. per minute and a half. Above the apparatus and on a sliding holder is the electric lamp, with focussing lens ; the light from this falls veitically on the mirror of the Optic Lever, which is inclined at an angle of 45 ° to the horizon. The horizontally reflected light is then thrown on a screen, which carries either semitransparent recording paper or a photographic plate. In the former case the observer, standing behind the screen,

traces the movement of the spot of light with a pencil. The whole recording apparatus and the source of light being thus placed on the same stand, any ordinary disturbance will affect all equally, and cause no irregularity therefore in the movement of the recording spot (fig. 84). Record may be taken by following excursion of spot of light on screen to the left. For photographic record, plate-holder is substituted for screen. I have given a great deal of space to the description of these details, because on them depends the accuracy and perfection of the results. The record may now be made on any scale of magnification, without misgiving. In fact

I have obtained very perfect records even when the passing traffic was at its thickest. How true this is may be seen from the photographic record of a. thermo-mechanical curve, given in fig. 85. It will be noticed from the curve that, as the temperature rose, there was a continuous preliminary elongation, which was suddenly reversed by the excitatory contraction at the death-point, found in this case to be 59'6° C. If desired, the photographic curve itself may be made to indicate the different temperatures at different parts of the curve. This is secured by interrupting the light for a time at, say, every half degree of rise of temperature. As in the anisotropic death-responses, described in the last chapter, we have in this case also, though not shown in the present Fig. 85. Thermo-mechanical record, the post-mortem relaxation

graphically (Coronal Filasucceeding the contraction of rigor a sudden inversion of the point of inversion was indeed the curve, due to spasmodic irreVersible death-change, I took the curve once more, after allowing the specimen to return to its original temperature. The curve now obtained showed no reversal-point. Remarkable agreement between thermo-mechanical curves of similar specimens. — It was pointed out in the last chapter that the death-point is almost as definite as a physical constant. And not only is this true of the death-point, which I find in different phanerogamous specimens, under normal conditions, to be almost invariably close upon 6o° C, but it is also more or less true of the whole curve, those given by similar specimens being almost identical. In this way

the thermo-mechanical curve is, in a sense, characteristic of the plant in a given condition. This is well seen in the two records which I have obtained from the styles of two flowers — both on the point of opening — of a single plant of Datwa alba (fig. 86). These two curves are so extraordinarily similar in The possibility of securing such uniformity of results, enables us to attempt an investigation on the influence of various agencies. For any deviation from the standard characteristic curve will then form an indication of the action of such agents.

Standardisation of curves. — Different plants, again, will exhibit differences in their characteristic curves, and in order to render these strictly comparable with one another, we must know the absolute value of relaxation or contraction in each part of the curve. By absolute value, is here meant the amount of relaxation or contraction per unit-length of the specimen. This is rendered simpler if we adopt a uniform standard for all specimens ; that is to say, the horizontal distances representing temperature may in the standard curves be 1 inch (2-5 mm.) per degree. Vertical distances, again, of 1 inch maybe made to represent a relaxation or contraction of one part in a thousand. The standardisation is carried out in the following way : first, the recording surface is moved,

Fig. 86. Thermo-mechanical Curve of Two Different Specimens of Style of Datura alba, obtained from Flowers of the same Plant till one degree of rise of temperature produces a horizontal movement equal to i inch. After this, keeping the distance of the recording surface constant, the length of the short arm of the Lever, to which the plant is attached, is so adjusted that the vertical magnification is two hundred times. The length of specimen used, unless the contrary is stated, is always 5 cm. A movement of the light-spot through a vertical distance of one division ('i inch) will then represent an expansion or contraction of one part in two hundred of a specimen whose length is 5 cm., that is to say, one part in a thousand of a specimen whose length is one centimetre. In fig. 85, the original record has been reduced to onefourth. The distance between two horizontal lines represents a contraction or relaxation of 1 per cent.

In order to exhibit the differences in the characteristic curves of different specimens, or of the same specimen at different ages, I append three records taken under the same standard Fig. 87. Thermo-mechanical conditions : (D) that of the Style of Records or s, Young Speci- v ' * ~ menotspirogyra ; s' Older Datura alba ; (s) of a young specimen horizontal lines represents these three experiments, the rate of stances having been the same, it is instructive to compare the different parts of the different curves.

Taking first the curve of Datura, we find its death-point to occur at 6o° C. The relaxation undergone by the specimen during the rise of temperature from 350 C. to the deathpoint, was at the mean rate of 2*1 parts per thousand per degree for the unit-length. This, for convenience, we shall call the coefficient of relaxation. But after the death-point, the sign of response undergoes an abrupt change to the negative, that is, contraction, the coefficient of contraction being fifty per thousand, or nearly twenty-four times the coefficient of relaxation.

The next specimen whose curve (s) is given was young Spirogyra of light-greenish colour. From the slight differentiation of these simple algal forms, and from their lack at this young stage of any efficient protecting envelope, we should expect them to offer but feeble resistance to the effect of heat, and we find the death-point lowered to 47 °, that is to say, 1 30 below that of the phanerogam Datura. Along with this, we find also a difference in the coefficients of relaxation and contraction. The mean coefficient of relaxation was in this case 'OOI, and that of contraction '007.