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0 Research School of Biological Sciences, Australian National University, PO Box 475, Canberra 2601, Australia
Received for publication October 21, 1999. Accepted for publication March 31, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONTHEORETICAL OBJECTIONS OF...PRACTICAL TESTS OF STILLER...LITERATURE CITED |
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Key Words: cavitation • cohesion theory • compensating pressure theory • embolism • tissue pressure • water transport
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONTHEORETICAL OBJECTIONS OF...PRACTICAL TESTS OF STILLER...LITERATURE CITED |
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), I knew well that it touched upon many topics in plant biology wherein I had little expert knowledge. I wrote: "The full implications of the Compensating-Pressure Theory, and a just appreciation of its usefulness, must await the concerted criticism of a wider community." I am very pleased that this criticism is beginning to be published, both theoretical objections (Comstock, 1999
) and experimental tests (Stiller and Sperry, 1999
). I welcome the chance to respond to this criticism and to develop further some of the theoretical and experimental aspects of the theory. All criticisms, even invalid ones, help me to clarify my understanding of the theory and to find clearer ways of explaining it and new ways of testing it. They often show me how a whole new set of puzzle pieces can be fitted in to the growing picture of the theory.
There is not space here to provide all the relevant arguments and facts from the beginning, and I assume that readers are familiar at least with the two articles quoted above and with the paper that stimulated the writing of them (Canny, 1998
). They should consult the original statement (Canny, 1995
) for the discussion of its fig. 12 (p. 352). The two critical articles (Comstock, 1999
; Stiller and Sperry, 1999
) will be discussed separately, treating the main topics raised by each.
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THEORETICAL OBJECTIONS OF COMSTOCK |
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TOPABSTRACTINTRODUCTIONTHEORETICAL OBJECTIONS OF...PRACTICAL TESTS OF STILLER...LITERATURE CITED |
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) gives the tension in the wall (S) as
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) that all nonwoody plant organs have their vascular systems confined within sheaths (endodermis, fibrous sheaths, etc.). The pressure-containing elements of such organs are not primarily the outer surface, but the much smaller sheathed vascular bundles. They have smaller ratios of R/T, and so will have lower tensile stresses in their sheaths. Three of the four pressure-containing systems I illustrated in figs. 9–11 of Canny (1995)
are of this kind. The fourth (figs. 7, 8) is wood, which is a much more complex solid—a coherent composite of radially oriented pressurizing plates (rays) and pressurized sheets of tracheary elements, which cannot be described by the La Place equation (above). Using measurements of the stele of the wheat root in the fig. 9, R/T = 7, so a pressure of 2 MPa within the stele would require a tensile strength of 14 MPa in the endodermis, which seems modest enough.
The balance of pressures/water potentials in Fig. 12 of Canny (1995)
My reading of Comstock's discussion of my failings and inconsistencies in this figure is that he has resolved them himself, and finally thought himself around to the point of view I started from. He says towards the end of this section, "one could define a system in which the xylem and parenchyma were, together, within a common constraining outer layer." This is just what I was trying to explain, obviously ineptly, and "in misleading and unnecessary new terminology." The terminology is not new, but old. It was standard for 50 yr. I do not apologize for it. My mind moves slowly and simply, and more assuredly with linear positive variables and the old Höfler diagram. He objects to my distinguishing two components of turgor pressure, but the distinction is important. Only part of the tissue turgor pressure can do external work (e.g., burst pavements or epidermis, drive extension growth, squeeze water out of reservoirs). The other part is wasted internally in stretching cell walls.
I have recently tried to translate these ideas in a new and simple statement, which uses the accepted terminology (Canny, 1999
). Within the one compartment there are several pressure-generating engines: osmotic pressure, wall pressure, tissue pressure, xylem pressure; and the resultant of all these forces is the pressure within the compartment. By arranging the appropriate signs, the resultant may be turned into a water potential. Comstock's failure to treat the compartment as a whole has led him to misinterpret the part of fig. 12 which forms section A of his redrawn version (his fig. 1). The water potential of the composite system is zero, and all the fluid space within it is at a pressure of +0.8 MPa, parenchyma and vessel sap. There are no gradients of pressure, and thus no movements of water, no changes of volume or concentration with time, and no matter or energy moving through it. It is indeed at equilibrium. It is not helpful to isolate parts of the system mentally and try to assign them water potentials. Dissecting the system will change the potential of all the components. For example, if a parenchyma cell is removed from its constraints without change of volume, its water potential will go from zero to -0.8 MPa.
In section B of Comstock's fig. 1 he is right and I am wrong. I see now, as I did not at the time I was devising fig. 12, that as work is done by the system the usable energy decreases and the unusable energy increases. Water moves into the cells, and their volume increases and their walls are further stretched. Tissue pressure decreases and wall pressure increases pari passu. This transfer between the two components was fully understood by Höfler (1920)
and is obvious from his fig. 3. In section B of Comstock's fig. 1 I should have set wall pressure (WP) = 1.0 MPa. All the osmotic pressure is balanced by the wall pressure. In section B, the values of the variables are such that mental dissection of the parts of the system to seek individual water potentials of components is innocuous. Physical dissection does not alter the forces produced by the separate engines. This set of values of variables was chosen as the steady-state example for fig. 12 because they illustrate the limiting condition of usable tissue pressure. At stresses beyond this, the components of the tissue cease to interact with one another and the cohesion theory takes over.
Before leaving the discussion of fig. 12, I should remind the reader of a point that Comstock does not bring out, but which will be important when I come to discuss the article by Stiller and Sperry. The object of devising fig. 12 was to investigate what in fact the water potential of a tissue is a measure of. Having arrived at the conclusion that it was not a measure of tension in the xylem sap, I tried to imagine the changes in water contents, pressures, and volumes within a tissue during life, sample collection, and measurement. The conclusion was that the water potential measured that part of the turgor pressure that had been doing work in the intact plant—the compensating pressure.
The phase-change valve in the leaf
For my theory to work, water movement into the pipes at the bottom and out of the pipes at the top needs to be independent of the pressure in the pipe. Comstock finds this impossible to imagine and focuses his disbelief on the top valve, where I claim that evaporative water flux out of the leaf is not changed by changing the absolute pressure in the xylem. This is because the energy to effect the phase change from liquid water to water vapor is large compared with the pressures in the xylem. It is a valve also in the sense that the water movement through it cannot be reversed. Comstock has not convinced me that this argument is invalid. He justifies his disbelief in the valve by reference to an experiment in which the valve is nonexistent. "The lack of regulating valve anywhere in the leaf mesophyll is made particularly obvious by hydraulic conductance measurement techniques in which water is pushed into the xylem of a severed twig under positive pressure. Under these conditions, water readily moves through the leaf tissue, floods the normally gas-filled intercellular spaces, and drips as a liquid from each stomatal pore." He has abolished the phase change (and therefore the valve). It is not sensible to argue from the behavior of water flow under positive pressure with no phase change to what might be expected with evaporation, where there is a phase change, and which generates a negative pressure. The squeezing of water out of cells into intercellular spaces, noted by Comstock for the the leaf mesophyll, is minimal within vascular bundles. Their lack of intercellular spaces makes good sense when they are viewed as pressure-containing ducts, as discussed above. Within the bundles, pressure pushes water into the main space available, the vessels, repairing embolisms.
I am disappointed that Comstock did not help me with the operation of the hypothesized lower valve (pump) in the roots. That pump is much more complicated and interesting than the leaf valve, and relies on more esoteric and less familiar principles. Since publishing an account of it I have been expecting someone to point out some fatal error or misconception in my reasoning, or some experimental evidence against it. The experimental evidence we have collected ourselves is consistent with it (Enns et al., 1998, 2000
).
Measuring the usefulness of a theory
Comstock's confidence in the cohesion theory, that it has proved "itself robust and extremely consistent to a wide range of experimental and technical approaches" is clearly legitimate, in the sense that the water relations of plants have been comfortably studied within its constraints for 35 yr. However, this has been possible only because of a tacit agreement to ignore large bodies of experimental fact about water transport that were once well known and widely believed to be essential. To take a single example, Haberlandt (1914)
, after a long discussion of the known facts of water transport and the tissues that carry the stream, makes a summary, which he starts with what he presumably thought was the most important fact.
"At this stage it will be desirable to summarize in half a dozen sentences those facts concerning the conduction of water which may be regarded as definitely established: (1.) Vessels and tracheides normally contain both air and water, the relative amounts of the two substances varying according to the season and the time of day." He means here, not just that wood contains gas as well as water, but that individual vessels and tracheids contain simultneously gas and liquid. The body of evidence supporting this statement is vast and various, and spans 200 yr. But because it is not possible to believe simultaneously this statement and the cohesion theory, that evidence and that fact are never discussed. Now that independent evidence of the fact is appearing, collected by modern techniques (Canny, 1997a, b
; McCully, Huang, and Ling, 1998
; Buchard, McCully, and Canny, 1998
; McCully, 1999
; Pate and Canny, 1999
; Shane and McCully, 1999
), resistance to its publication is fierce (see McCully et al., 2000
).
I am frequently asked, how do you tell whether one theory is better than another? My answer is very simple. The usefulness of a theory is measured by the range and diversity of the phenomena that it explains, the number of pieces in the completed puzzle-picture. A theory that the World is flat explains most of what is necessary to be known by a community living in the same territory for many generations. It does not suffice for a group that sets out resolutely to find the edge of the World. The cohesion theory satisfies those who are content not to look beyond "the wide range of experimental and technical approaches" within which it works. I find the compensating pressure theory more useful, because it explains that range and much else besides.
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PRACTICAL TESTS OF STILLER AND SPERRY |
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TOPABSTRACTINTRODUCTIONTHEORETICAL OBJECTIONS OF...PRACTICAL TESTS OF STILLER...LITERATURE CITED |
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. 1) The fundamental question (not addressed) is whether the water potential is a measurement of tension in the liquid in the xylem vessels. There are two derivative questions.
2) Whether embolized vessels can refill during measurement of the hydraulic conductance of a plant organ when this is assessed by the rate of flow of solution through it down a pressure gradient (addressed but not answered satisfactorily).
3) If the proposed refilling does occur during conductance measurement, whether this is due to the activity of living cells in the xylem (addressed and answered for woody tissue—living cells are not necessarily involved).
The connection between the three questions is that Pockman, Sperry and O'Leary (1995)
used the conductance method to measure "vulnerability curves" and claimed that they had measured the tensions in the xylem liquid, providing the answer to the fundamental question. I objected at the time that the demonstration was invalid because the presence of continuous liquid threads under tension in the vessels was not proved. All that was proved was that three different drying stresses (as measured by the change in water potential) changed the hydraulic conductance along a sigmoid path. One interpretation of this was what Pockman, Sperry, and O'Leary (1995)
said, that the drying stresses produced stretched liquid, which broke at the point of inflection of the curve. I pointed out that there were other interpretations that would produce similar curves, but that the crucial test was to show that the liquid was there up to the inflection, and was not there beyond the inflection. This has still not been shown.
A diagram is necessary to clarify the arguments (Fig. 1). This is a simplified version of fig. 3 of Stiller and Sperry (1999)
, a plot of percentage loss of hydraulic conductance vs. a stress produced by centrifugation. The abscissa of the original figure by Pockman, Sperry, and O'Leary (1995)
was a generalized drying stress, which included drying on the bench, and drying by forcing pressurized air through the stem, as well as the centrifugal stress. All three stresses were translated into values of water potential (MPa) and produced similar sigmoid curves like that in Fig. 1. We all agree that the conductance decreases along such a sigmoid path, that there is little change in conductance in the region A to B, a sudden loss of conductance in the region of B, and near complete loss of conductance in the region C.
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My interpretation is that the water threads break and are interrupted by gas even in region A. But the conductance is little changed because the process of perfusing the xylem with solution dissolves the gas. The greater the stress the more gas is produced, but it is not until the gas content of the vessels reaches a critical volume around B that this refilling ceases to be significant, the measured conductance falls, and the vessels contain mostly gas. The crucial test of my interpretation is to study the gas/liquid content of the vessels in region A, first before conductance measurement, and then after conductance measurement.
One mechanism by which this refilling might happen preoccupied me because it derived from my proposal (explained above) that the compensating pressure could be a major component of the water potential, and because it made easily tested predictions (Canny, 1998
). This mechanism depended on living cells to provide sufficient pressure to dissolve the embolisms. This is the interpretation (Question 3) that Stiller and Sperry have tested by doing two of the four tests I asked for on young stems of birch. Their advance is important, and a valuable contribution to the derivative question. My fanciful interpretation of water potential as measuring compensating pressure in these particular experiments was clearly wrong. They have shown that neither the turgor of the xylem parenchyma cells, nor the live state of the xylem parenchyma, influences the shape of the curve in this woody tissue.
They also attempted a partial answer to Question 2 by testing the effect of living/dead cells on the conductance measurement (their fig. 4). But they chose to do this, not in the critical region A where I suggest refilling happens, but in the region B to C where, in my interpretation, no refilling would be expected. The drying stress in this experiment was not centrifuging, but a perfusion of the xylem with air for 10 min. A cursory reading suggests that this was a fairly mild stress, but in their fig. 4 the ordinate is percentage conductance. The stressed stems had 30% of the control conductance, i.e., 70% loss of conductance. This is the level of stress (Fig. 1) beyond the inflection of the sigmoid curve where I would expect no refilling during measurement, whatever the mechanism may be.
I am grateful to Stiller and Sperry for resolving the question of the role of living cells, but I am not satisfied that they have answered Question 2, that my interpretation of the region A to B is wrong. Having been proved wrong in one of my interpretations of "vulnerability curves," I am not shy to propose another. Even with no activity of living cells, during the conductance measurement small bubbles of gas mixed with liquid in the vessels would be subjected by the technique to a small positive pressure (approximately +5 kPa). Small bubbles of gas in water at atmospheric pressure are inherently unstable and collapse. As Sperry has already shown himself (Alder et al., 1997
: Fig. 6), centrifuged birch stems can take up water and increase their conductance by 50% in 10 min. I need more evidence to convince me that bubbles of gas are not there in region A of Fig. 1, and that they do not partially dissolve during conductance measurement. Even if living cells of woody stems do not contribute to changes in xylem conductance during these manipulations, herbaceous organs, whose structural rigidity relies strongly on tissue turgor, should also be tested. It seems quite possible that in them living cells may provide the forces that, in wood, are provided by imbibition and surface tension.
The presence and disappearance of the bubbles (Question 2), and the answer to the fundamental question, Question 1 (the presence/absence of a continuum of liquid in the vessels up to the inflection), can be answered simultaneously. The obvious way to test the state of liquid in the vessels is by snap-freezing of stems centrifuged at different speeds and examining the frozen vessels in the cryoscanning electron microscope (McCully et al., 2000
). The same technique can be used to explore the content of the vessels at different stages of conductance measurement. I issue this public invitation to Sperry to take advantage of our microscope and expertise to answer the question. We can arrange that he freezes the stems in his laboratory, transports them frozen to our laboratory, and examines the liquid and gas content of the frozen vessels.
Without going to all this trouble, a very simple test to decide between the two interpretations would be to measure (e.g., by weighing) how much water comes out of the ends of the stems at different radial accelerations. In Sperry's interpretation little water should come out in region A, but when region B is reached there should be a sudden loss of water over a small range of increasing acceleration. In my interpretation there would be a linear relation between water loss and radial acceleration over the whole range of A to C.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONTHEORETICAL OBJECTIONS OF...PRACTICAL TESTS OF STILLER...LITERATURE CITED |
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Buchard, C. J. A., M. E. McCully, and M. J. Canny. 1999 Daily embolism and refilling of root xylem vessels in three dicotyledonous crop plants. Agronomie 19: 97–106[CrossRef][ISI]
Canny, M. J. 1995 A new theory for the ascent of sap. Cohesion supported by tissue pressure. American Journal of Botany 75: 343–357[CrossRef]
———. 1997a Vessel contents after excision—a test of Scholander's assumption. American Journal of Botany 84: 1217–1222[Abstract]
———. 1997b Vessel contents during transpiration—embolisms and refilling. American Journal of Botany 84: 1223–1230[Abstract]
———. 1998 Applications of the compensating pressure theory of water transport. American Journal of Botany 85: 897–909[Abstract]
———. 1999 The forgotten component of plant water potential. Plant Biology 1: 595–597[CrossRef][ISI]
Comstock, J. P. 1999 Why Canny's theory doesn't hold water. American Journal of Botany 86: 1077–1081
Enns, L. C., M. J. Canny, and M. E. McCully. 2000 An investigation of the role of solutes in the xylem sap and in the xylem parenchyma as the source of root pressure. Protoplasma 22: 506–515
———, M. E. McCully, and M. J. Canny. 1998 Solute concentrations in xylem sap along vessels of maize primary roots at high root pressure. Journal of Experimental Botany 49: 1539–1544
Haberlandt, G. 1914 Physiological plant anatomy. Translated by M. Drummond, Macmillan, London, UK
Höfler, K. 1920 Ein Schema für die osmotische Leistung der Pflanzenzelle. Berichte der deutschen botanischen Gesellschaft 38: 288–298
McCully, M. E. 1999 Root xylem embolisms and refilling. Relation to water potentials of soil, roots and leaves and osmotic potentials of root xylem sap. Plant Physiology 119: 1001–1008
———, C. X. Huang, and L. E. C. Ling. 1998 Daily embolism and refilling of xylem vessels in the roots of field-grown maize. New Phytologist 138: 327–342[CrossRef][ISI]
———, M. W. Shane, A. N. Baker, C. X. Huang, L. E. C. Ling, and M. J. Canny. 2000 The reliability of cryoSEM for the observation and quantification of xylem embolisms and quantitative analysis of xylem sap in situ. Journal of Microscopy 198: 24–33
Pate, J. S., and M. J. Canny. 1999 Quantification of vessel embolisms by direct observation: a comparison of two methods. New Phytologist 141: 33–44[CrossRef][ISI]
Pockman, W. T., J. S. Sperry, and J. W. O'Leary. 1995 Sustained and significant negative water pressure in xylem. Nature 378: 715–716[CrossRef][ISI]
Shane, M. W., and M. E. McCully. 1999 Root xylem embolisms: implications for water flow to the shoot in large, field-grown maize plants with only one root. Australian Journal of Plant Physiology 26: 107–114
Stiller, V., and J. S. Sperry. 1999 Canny's compensating pressure theory fails a test. American Journal of Botany 86: 1082–1086
Strasburger, E. 1891 Über den Bau und die Verrichtungen der Leitungsbahnen in den Pflanzen. Gustav Fischer, Jena, Germany
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