Comparative Electro-Physiology: A Physico-Physiological Study

But if, on the other. hand, it could be shown that these objections were not valid, and if, further, some crucial experi- ment were devised to demonstrate that excitatory action was attended by a concomitant responsive movement of water in the tissue, it might then be claimed that the physiological theory of the ascent of sap had been established on a firm basis. The attempt to do this will form the subject of the next chapter. The first question that falls within the scope of our investigation, then, is as to whether the reactions of the root are or are not similar to those of digestive organs in general. We have seen, in the case of the latter, that as there are two opposite activities, of secretion and absorption, so also there are two opposite responsive phases, negative and positive, the

former being the more characteristic of the fresh condition, and the latter of a specimen which has been previously sub- jected to continuous stimulation. Before giving any account, however, of these electrical responses, it will be interesting to demonstrate here the occurrence of secretion in young or fresh specimens, when subjected to excitation. . The fact that young rootlets secrete, on excitation by contact, has corrosion of marble, mentioned above. But I shall now describe a new experiment, in which this fact is even more convincingly demonstrated. I took a specimen of Colocasza, srowing in marshy soil. The plant was lifted bodily, with earth adhering, and placed in water, so as to expose the roots gradually, without causing injury. It was then kept overnight, with the roots in normal saline solution, which was slowly absorbed by the tissues. Next morning, again, it was carefully washed till there was no trace of salt ad- hering. One of the very young roots was now immersed in very dilute solution of silver nitrate. If the previous washing haa oeen effective, there ought now to be no white precipitate, or only the merest trace, formed in the silver solution. The two electrodes of a Ruhmkorff’s coil were next connected, one with the silver solution,*and the other with the stem of the plant. On now passing tetanising shocks, the immersed root became excited, and secreted its contained salt solution, this being seen in the silver nitrate as streams of white precipitate.

Turning next to the electrical mode of investigation, we have found that in the digestive organs, the galvanometric negativity, which is the characteristic response of a specimen in the fresh condition, becomes reversed to positivity under continuous stimulation. In the case of Mepenthe, very young pitchers exhibited this normal response of negativity, which was converted, under continuous stimulation, into diphasic, tending towards positivity. Older specimens, again, pre- viously stimulated by the presence of excitatory food-material,

were found to be in the positive phase, giving rise to response by galvanometric positivity. 3 | In the case of the root, it is interesting to find that there is a similar alternation of responsive phases. For this demonstration I again took the root of Colocasia, and recorded its responses to equi-alternating electric shocks. Among the mass of roots there are naturally some which are dead and decaying. One of these was selected for one electrical contact, while the other was made with a young and vigorous root. The responsive reaction, under these conditions, was found to take place by galvanometric negativity (fig. 214). Under long-continued stimulation, however, I have often found this normal response by galvanometric negativity to be reversed to its opposite, positivity. From this we may pass to the consideration of response in older roots, where the phasic reaction is typically positive. a ee ae Now we have seen in previous Record of Normal Nega- chapters, as will be remembered, that ns Response..of Young oot of Colocasia there are two different conditions under which the positive may be substituted for the normal negative response. The first is that of reversal under long- continued stimulation, which we ,have just seen. And the second occurs when the stimulus falls below the critical level which is necessary to the evoking of true excitation. In this latter case, as we saw further, the incident stimulus increases the internal energy, and causes expansion, positive turgidity-variation, and galvanometric positivity. It would thus appear that one identical stimulus may induce one effect, that of galvanometric negativity, in a highly excitable tissue, and the opposite, or galvanometric positivity, in a tissue that is less excitable. In connection with this question the ex- perimental results which I am about to describe are very significant.

We have seen that a young root of Colocasia, when fresh, gives the normal response of. galvanometric negativity. Taking next an older root of the same plant, and employ- ing the same intensity of stimulus as before, I found the responses to take place, generally speaking, by galvanometric positivity (fig. 215). This would appear to suggest a ten- dency towards specialisation of fufction, galvanometric nega- tivity being associated, as we have seen, with secretion, and | positivity, in all probability, with the opposite—namely, absorption. A similar specialisation of certain cells for secretion and others for absorption is manifested more unmistakably in the digestive organs of the higher animals. |

Thus in the young roots the pre- dominant reaction would seem to be secretion, reversed under continuous stimulation to absorption. In the older roots, on the other hand, the pre- dominant reaction must be supposed to be absorptive. Here, then, judging from the electrical indications, we Fic. 218. Photographic would seem to have proof of that ! giving rise to the so-called ‘root- pressure. We can also see how, by the summated activities of numerous roots, this ‘ root-pressure ’ is kept approximately constant for a certain length of time. Taking longer periods into account, further, we can see that this physiological activity is likely to undergo periodic change, a fact which is evidenced by the known periodic variation of root-pressure. The question, however, of the actual influence of excitation on the process of the ascent of sap will be dealt with in the next chapter.

One form of stimulus to whose action the roots must often be subjected is that of the chemical substances present in the soil, and I undertook to test the electrical variations induced by these. The results obtained, at least with the specimens which I have tried, are in general parallel to those obtained by the electrical form of stimulation. Thus, in young roots, in the majority of cases, when subjected to the action of so dilute a solution as ‘5 per cent. of sodium car- bonate, an electrical change of galvanometric negativity was induced. The continued action of this solution, however, tended to induce a reversal to galvanometric positivity. But the reactions of older roots were different—that is to say, in the greater number of the latter cases, a solution of °5 per cent. induced galvanometric positivity ; and it required a much stronger solution, of from 5 to 10 per cent., to bring about the reaction of galvanometric negativity.

We have thus seen that in the root, as in the digestive organ, there are alternating phases of secretion and absorp- tion, and that it is by means of the secreted fluid that solid inorganic substances are rendered soluble, for subsequent absorption as food. We have seen moreover that the elec- trical reactions in the two cases are similar; that in the young root, as in the young glandular organ of Wepenthe, the characteristic response is by galvanometric negativity ; and that long-continued stimulation induces diphasic variation, with a tendency towards the reversal to positive. We saw further that older roots, like the glands in the older pitchers of epenthe, have a phasic reaction which is predominantly positive. And now, having thus completed our first line of inquiry, we shall turn to the second---the question, namely, as to whether the ascent of sap is or is not essentially due to physiological reaction.

The possible explanations of the ascent of sap may be grouped broadly under two different heads, as either physical or physiological. Under the former of these must be named such theories as those of atmospheric pressure, capillarity, osmosis, and evaporation from leaves. Under the latter, the physiological, the movement of water is regarded as mainly due, in some hitherto undefined way, to excitatory actions by which the sap is propelled in a uni-directioned

manner, this primary movement being aided by accessory factors. Afnong the physical theories which have been pro- pounded for the explanation of the ascent of sap those of atmospheric pressure and of capillarity are admitted to be inadequate. But that of osmosis and transpiration, put forward by Dixon, Joly, and Askenasy, is of much greater transpiration from leaves. The fluid in the mesophyll cells of the leaves becomes concentrated by evaporation; thus osmotic attraction is set up by the leaves, and the suction thereby exerted is supposed to be transmitted backwards as far as the roots, through cohering columns of water. The difficulties in the way of this theory lie (1) in explaining how a slow osmotic action could produce so rapid a water-current ; and (2), in the absence of any conclusive proof that, under actual conditions within the plant, the water-column could have sufficient tensile strength. Even apart from these objections, however, the fact remains that energetic water- movements take place in the plant in the entire absence of

a tree which may exert a pressure as great as that of a column of liquid 13 metres in height. It is thus seen that there is an independent activity o: some kind which maintains the movement of water through the plant. That this activity, moreover, is not resident in the root merely is seen from the fact that exudation of water takes place from the tips of grass-blades when their cut stems have been placed in water. Criticising the theory of trans- piration, Strasburger rightly remarks that transpiration only makes a place for inflowing water, but cannot furnish the force necessary to convey a large volume of fluid rapidly for a considerable distance through wood. From the considera- tion of these and other facts, Pfeffer, in his summary, was led to the conclusion which he states as follows: ‘A satis- factory explanation of the means by which the transpiration- current is maintained has not yet been brought forward. If

no vital actions take part in it, then it is obvious that we have only an incomplete knowledge of the causes at work and of the relationship of the different factors concerned.’ * The inadequacy of these theories to explain the ascent of sap has, then, been freely admitted. With special refer- ence, further, to that of osmosis, I shall myself be able to show that the movement of water often takes place in the plant, in a direction contrary to what it would be if osmosis alone were involved. The fact that the absorption of water is not a merely passive process, but a phenomenon connected with irritability, will be further shown in the depression of the rate of water-movement by such conditions as depress irritability, whereas the opposite circumstance will be found to enhance it. :

We are thus driven to examine the possibility of a physiological explanation of the ascent of sap. On such a theory it must be supposed to be brought about by the action of stimulus, inducing reactions expressed in the re- sponsive movement of water. The objections made to the physiological explanation have already been recapitulated. They are (1) that the movement of water is known to take place rapidly, and by preference, through woody tissues which are supposed to be dead; and (2) that when the roots have been killed by hot water, or when poison is supplied, the transport of sap continues to take place. I shall now proceed to examine these arguments, and to show that the objections raised, though apparently so strong, are not really valid.

We shall first refer to the argument which has been based upon the fact that in trees conduction takes place very rapidly through woody portions which are regarded as dead. It is not, it must be noted, implied by this that the presence of wood is essential to the ascent of sap, in- asmuch as even in trees there are tracts of living cortical tissue in the roots which have to be traversed before the water can reach the woody tissues. In seedlings of Gramine

only one or two days old, again, water ascends, and is ex- creted at the tip of the yet unopened leaf. Transpiration is here at its minimum, and the fibro-vascular elements at this early stage cannot be regarded as ‘dead wood.’ Finally, in herbaceous plants, where woody elements are ee Ane the ascent of sap is seen to take place. the tree, the mass of wood in the interior were dead, it might. still conceivably be of use in the irrigating system as a central reservoir. This would certainly be advantageous to rapidity of transit. At the lower end of the tree, the wood abuts upon the delicate parenchymatous tissues of the root, and at the upper upon those of the leaves. According to the physiological theory, then, it might be supposed that it was by the multiple activity of the cells of the root that water was pumped into the wood ; and that at the other end the central reservoir was able to furnish a supply to make up for the constant loss by transpiration. Laterally also, in the stem itself the cortical tissues could draw upon this central supply. Under such an arrangement no part of the _ plant could be very far away from the reservoir.

As a matter of fact, this sketch corresponds roughly to the working of the tree as an hydraulic machine. The system is, however, somewhat more complex than has been indicated. Besides the central, we have also to remember the presence of lateral reservoirs, in the parenchymatous tissues of the cortex. But the transport of water through these is not, of course, so rapid as through the central, more specifically conducting, system. In the case of herbaceous plants, where the quantity of wood is insignificant, we may regard the central channels as abolished. Here we have soft cortical tissues extending continuously from root to leaves through the stem, and it is obviously through these that the ascent of water takes place. In woody trees, then, there is no reason to suppose that the cortical tissues could not play a similar part in the conveyance of water. The difference is, that in this case there is also an added and

better channel available, which will naturaily come into requisition where quick transit is required. In a woody trunk, then, we have (1) the outer cortical cylinder of water-conducting tissues, by which the ascent of sap takes place slowly. We have (2) the highly-conducting central woody tissue, which not only allows of water ascend- ing rapidly through it, but is also (3) in lateral communi- cation with the outer cylinder. The hydraulic system thus consists of a large central canal, as it were, connected with innumerable lateral reservoirs, which are the cells of the cortex. When a demand arises for rapidity of water-supply on account of transpiration, we can now see that no less than three different factors are brought into requisition. First there is the rapid upward transit through the wood ; secondly, the slow ascent through the cortex ; and thirdly, the lateral supply from the cortex by way of the nearest wood. :

As regards the last of these, the cortical tissues in contact with the wood act ina manner not very unlike that of the roots towards the soil. That is to say, under different cir- cumstances, they absorb water from it, and excrete water into it, these alternating processes being by no means accidental, but guided by appropriate excitatory reactions. Turning our attention for a moment to the movements of Mimosa \eaf, we find that on excitation the expelled water makes its way to the fibro-vascular tissue. There is here, in the excitable tissue, unlike the case of secretory organs, no external vent, and we see the necessity of a central reservoir to which water excitatorily expelled may find access. On the subsidence of excitation, the water is re-absorbed by the organ, causing expansion and re-erection of the leaf. Such movements of inflow and outflow evidently take place in the trunk of the tree itself. Under the stimulus of sunlight, the excited cortical tissue will squeeze water inwards into the central reservoir. If this takes place, the effect will be seen in a diametric contraction. At the time when transpiration is most rapid, under the action of sunlight, there is thus

besides the water coming from the roots, an additional supply available from the lateral reservoirs. The loss of water thus sustained by the cortex during the day is made up again at night, when it will suck water outwards from the central reservoir. We have here a case analogous to the action of the excitatory tissue of the pulvinus of J/imosa expelling water into the wood on excitation, and re- absorbing it on the cessation of excitation. The occurrence |

of these reactions in the cortex explains the observation made by Kraus that the organs of the plant diminish in bulk from morning to afternoon, the reverse seo taking place from afternoon to morning. We have thus seen how important a factor is excitatory reaction in the observed movements of water, even on the supposition that the woody tissue, being dead, is a merely passive agent. The question has still to be attacked, however, whether this assumption, so generally made, is correct, that the wood used for conduction of water is dead. This supposition has arisen from the chemical transformation undergone by the protoplasm in woody vessels. We have seen, however, in the case of the epidermal cells of the skin, that it is possible for chemical transformation to occur, without necessarily being accompanied by the death-change.

Before proceeding to inquire whether the conducting woody channels are really dead, it is desirable to say a few words as to the particular tissues in the wood, which are most effective for this purpose. Many experiments have been carried out to determine this. Among other things various staining fluids have been employed. But an objec- tion raised in the case of some of these has been that the water of such solutions travels faster than the dye dissolved in it. For my own part, I have found the employment of dilute solution of phenolpthaline to be exceedingly delicate and useful for the purpose of this investigation. It is perfectly colourless, and the staining appears only after appropriate development. ‘The cut end of the stem is placed in this dilute solution and left for some time. Transverse

sections of the stem at different heights are then made and placed in the field of a microscope. There is now nothing distinguishing to be seen, as the solution sucked up by the stem was colourless. Dilute solution of potash, however, will act on this as a developer. The particular tissues, therefore, through which the solution has been conducted, on now being subjected to the action of this agent, become of a rich crimson colour. By this means it is easy to see, as already determined by various observers, that it is the younger, or ‘sap-wood, which is concerned in the work of the rapid conduction of water, the older, or ‘heart-wood,’ being ineffective for this purpose. If, however, the wood taking part in the ascent of sap had been dead, and acted as a passive agent merely, it is difficult to understand the reason of this selective action of the younger, and presumably more living, woody tissues. |

It occurred to me, finally, that as electrical response is an indubitable concomitant of the excitatory reaction of living tissues, the question as to whether sap-wood was alive or dead could be subjected to a decisive test. For this purpose I took various strips of sap-wood from different woody plants. The cortical tissue was in each case carefully removed, and the specimens were placed in water, allowing them a period of rest. The first experiment was to observe whether local stimulation by the Rotary Mechanical Stimulator did or did not evoke electrical response. I found from this, that mecha- nical excitation of the sap-wood induced considerable excita- tory response of galvanometric negativity. I then subjected the same tissue to the action of boiling water for: a length of time, and again tested its electrical reaction by the same method. The wood was found to be very resistant to the action of heat, and it was only after long immersion that the responses were entirely abolished. Drying was in fact found, significantly enough, to be an easier method than the application of heat, to kill, and therefore to. abolish the responsiveness of, the wood. If the wood be first dried, and then soaked in water, it entirely ceases to manifest electrical

response. The ordinary wood of commerce exhibits no response. The familiar fact that the cut end of a woody stem, when not placed in water immediately, ceases to suck up water, has been supposed to be due solely to the intervention of air-bubbles. From the experiment which I have described, however, it would appear that the death of the exposed tissue by drying must be included here as a factor in this abolition of suction. With sap-wood I was also able to obtain the in- dication of galvanometric negativity in response to thermal stimulation. Hot platinum wire was applied at a distance of 5 mm. from the proximal contact. Response was thus due to the transmitted effect of excitation.

Electrical Response of Sap-wood ied Sells of living wood The normal negative responses seen in the and its variations under | first series are depressed after application chemical agents. For this purpose I employed both the electrical and vibrational modes of stimulation. For the first of these, the strip of sap-wood was cut in the form of a two-pronged fork, of which one prong was killed by exposure to the drying influence of the air, while the other was kept alive by immersion in water. The specimen was now placed in water as a whole, in order to moisten the dried half. After this, electrical connections were made in the usual manner with the killed and unkilled ends of the speci- men. On next subjecting it to equi-alternating shocks’ response was obtained as induced galvanometric negativity of the living prong. These responses are seen in fig. 216 in the first series of records to the left. Chloroform was next

applied, and we observe the consequent depression of re- sponse; when the chloroform was blown off the responses were found to undergo revival. In the next experiment, a specimen of living wood was mounted in the vibrational apparatus, and its normal re- sponses taken. I next applied copper sulphate, and the record shows the consequent abolition of response (fig. 217). I have thus been able to establish the fact that the woody vessels of the sap-wood are not dead but living, and hence fully susceptible of physiological reaction. This will, I think, be found to dispose of one of the difficulties raised in regard to the physiological theory of the ascent of sap.

We come, secondly, to the objections that have been based on the ground of the ascent of sap through a tree whose roots have been killed by boiling water, and, further, on the ex- showing Normal Responses ot : * Living Wood to Vibrational periments of Hartig and Stras- Stimulus, and the Abolition of burger. These observers set cut Response by a Toxic Dose of ends of trees in tubs of poisonous Wei eee solutions, such as copper sulphate, which were found, in spite of their toxic character, to ascend to the leaves. It is clear that if such violent protoplasmic poisons ascend the trunk, they must kill all the cells lying in their path. And from this it was inferred that the living cells in the stem could not be necessary to the rise of sap. Strasburger was thus led to the conclusion that ‘the supposition that the living elements in any way co-operate in the ascent of the transpiration current is absolutely precluded.’’

It does not, however, appear that this inference on the part of Strasburger was justified, for we must remember the fact that any cut piece of stem when placed in water is found to exhibit suctional activity. Hence the active cells con- cerned—if the process is to be regarded as due to such— must be distributed throughout the plant. Death of a given zone, then, would arrest the activity of that particular zone but not that of another zone higher up. Thus a poisonous solution would only abolish activity in those cells which it had already reached. The activity of cells above would remain unaffected. That the death of cells below offers no resistance to the passage of water, when suctional activity is

again show in the course of the next chapter, in cases in which the lower part of the plant was killed by boiling water. Under the action of poisons, similarly, I have been able to show that a poison can: pass easily through killed tissues owing to the suctional activity of cells higher up. This was demonstrated by means of experiments on Des- modium gyrans, where the cut end of the petiole was placed in copper sulphate solution. It is fortunate that in this case, during the ascent of poison, we have areas whose activity is manifested visibly by the rhythmic motile indications of the pulvini of the inserted lateral leaflets. That copper sulphate solution arrests rhythmic activity, and induces death, is seen by the rapid stoppage of pulsation when we apply it directly on the pulvini of the pulsating leaflets. When it is applied, however, at the cut end of the petiole, the arrest of pulsation only takes place after sufficient time has elapsed for the poison to ascend through the intervening distance. This shows clearly that successive zones are killed one after another, and that the death of a point below does not stop the suction above. From this experiment it is evi- dent that the application of poison, at the root, or the cut end of a stem, need not be expected to arrest suction until the whole plant has been killed, and from Strasburger’s account of his experiment it is evident that the movement of water did come to a stop when the poison reached the top of the tree.

We thus see that the objections which have been raised, with regard to the physiological nature of the ascent of sap, are not valid. I shall therefore proceed in the next chapter to describe crucial experiments in demonstration of the fun- damentally excitatory character of this process, Propagation of excitatory wave in plant attended by progressive movement of water — Hydraulic response to stimulus—The Shoshungraph—Direct and photo- graphic methods of record—Responsive variations of suction under physiological modifications induced by various agents—Effects of lowesing and raising of temperature—Explanation of maintenance:of suction, when root killed— Effect of poison influenced by tonic condition—Effect of anzesthetics on suctional response—Excitatory verszs osmotic action—-Stimulation by alter- nating induction-shocks—Terminal and sub-terminal modes of application— Three modes of obtaining response-records, namely (1) the unbalanced, (2) the balanced, (3) the over-balanced—Renewal of suction previously at standstill, by action of stimulus—Reponsive enhancement of suction by stimulus—After-effect of stimulus —Diminution of latent period as after-effect