Vascular Transport in Plants

completion.

Part I

Fundamentals of transport

Outline

Chapter 1: Perspectives on the Biophysics of Xylem Transport

Chapter 2: Physiochemical Determinants of Phloem Transport

Chapter 3: Pathways and Mechanisms of Phloem Loading

Chapter 4: Stomatal Control and Water Transport in the Xylem

1

Perspectives on the Biophysics of Xylem Transport

William F. Pickard and Peter J. Melcher

Publisher Summary

This chapter provides a brief discussion on cohesion-tension theory. The chapter also focuses on factors that affect hydraulic resistance in plants. It addresses how sap is extruded into the empty tracheary element even though the free energy gradient from soil to xylem seems often to be in the wrong direction, how the element is hydraulically isolated during refilling to avoid its new contents being sucked away in the transpiration stream, and how a refilled vessel ultimately reestablishes hydraulic contact with neighboring conduits in such fashion as to become once again useful in water transport. For xylem sap to flow through the plant, the negative hydrostatic pressure gradient must be large enough to overcome the resistive force of sap viscosity and the force of gravity. Because the rate of xylem sap flow through plants is relatively slow, the laminar flow pattern within an individual vessel consists of layers of sap that flow past each other in a sheetlike fashion, with the result that neither eddies nor vortices occur and turbulence is absent. However, in both laminar and turbulent flow, viscosity leads to internal friction, which produces energy dissipation. The cohesion-tension theory still stands as the hegemonic model for understanding long-distance sap transport in plants, but a controversy related to it propelled the development of new ideas and possible alternative paradigms to replace the postulates of cohesion-tension theory.

I’m sorry to say that the subject I most disliked was mathematics. I have thought about it. I think the reason was that mathematics leaves no room for argument. If you made a mistake, that was all there was to it. (X, 1992, p. 35)

The fundamentals of the cohesion-tension theory of sap ascent are now well covered in textbooks (Taiz and Zeiger, 2002; Fisher, 2000) with which most in our potential readership are familiar. Moreover, many deeper questions of xylem biophysics were covered in now hoary reviews (Pickard, 1981; Zimmermann, 1983) and have been updated by Tyree in a substantial monograph (Tyree and Zimmermann, 2002); there is no need to be encyclopedic. Nevertheless, any review of xylem biophysics must include at least some discussion of the cohesion-tension theory. This will be followed by a rather more extensive discussion of how embolisms within the transpiration stream might be formed and, more important, resorbed. Since cohesion-tension theory has only recently survived a significant assault on its hegemony, we present a brief discussion of this controversy, including what has been learned. Finally, we focus on factors that affect hydraulic resistance in plants. In each of these endeavors, some effort will be expended to define each concept with clarity and to frame each explanation rigorously.*

The Biophysics of Sap Ascent in the Xylem

Fundamentals of Cohesion-Tension Theory

In broad outline, the cohesion-tension theory is familiar to virtually every worker in plant biology. We shall therefore treat it as an exercise in dogmatics requiring but little exposition.

Dogma I (Hales, 1727, expt. XXXV):* The rise of the xylem sap during transpiration is due to the transpiration itself, the capillary… orifices in the leaves being able as any sap is evaporated off to supply the great quantities of sap drawn off by perspiration by their strong attraction (assisted by the genial warmth of the sun). In modern scientific terminology†: the capillary menisci of the cell walls within the substomatal cavities, being evaporatively depleted by solar heating, contract and draw the sap upwards. Ascribing this core dogma of cohesion-tension theory to Hales is uncommon (but cf. Floto, 1999), it being more usual (e.g., Dixon, 1914; Pickard, 1981) to ascribe it instead to Dixon and Joly (1895) or (e.g., Steudle, 2001) to Böhm (1893).

Dogma II (cf. Dixon, 1914): Water possesses considerable tensile strength, which allows it to be drawn upward by capillary menisci in the leaves. Evidence for this has been reviewed, for example, by Dixon (1914), Pickard (1981), and Steudle (2001).

Dogma III (cf. Haberlandt, 1914): Nevertheless bubbles can form in tracheary elements and interrupt sap flow. This was reported early on (Dixon, 1914; Haberlandt, 1914) and has been much commented upon since (Tyree and Sperry, 1989; McCully, 1999; Canny et al., 2001; Facette et al., 2001).

Dogma IV: Fortunately, plants have developed ways of coping with the challenges posed by emboli. These include (1) anatomical adaptations such as cavitation-resistant conduits and redundancy of hydraulic capacity, (2) new growth, (3) and global pressurization of the xylem as by root pressure. Within the past decade, evidence has appeared for embolus resorption despite the existence of nearby water putatively under tension (e.g., Canny, 1997; McCully et al., 2000; Cochard et al., 2000; Facette et al., 2001; Pickard, 2001; Tyree and Zimmermann, 2002); however, there is as yet no consensus on the precise mechanism(s) by which this might occur (e.g., Hacke and Sperry, 2003; Konrad and Roth-Nebelsick, 2003; Pickard, 2003a; Vesala et al., 2003; Chapter 18, this volume).

The Etiology of an Embolism

Despite the long awareness of tracheary bubbles, there has always been some uncertainty about the mechanism by which they got there, especially after the discovery within the stem of acoustic (Ritman and Milburn, 1988) and electric (Pickard, 2001) transients, which were assumed to arise from putative* cavitation events within the tracheae.

There are four obvious ways by which tension within a liquid-filled pipe could produce an embolism (Pickard, 1981; Zimmermann, 1983; Tyree, 1997).

Homogeneous Nucleation

Consider a spherical bubble of radius r [m] in a liquid of pressure P [Pa] and surface tension γ [N•m−1]; and let the amount of ideal vapor in the bubble be N [mol]. It then follows from the Young-Laplace equation (Pickard, 1981) and the perfect gas law that the pressure differential across the interface is

(1)

where R [= 8.314 J•mol−1•K−1] is the gas constant and T [K] is the absolute temperature. If the net pressure begins positive, then the bubble will shrink to zero as liquid vapor condenses on its inner surface and other gas is pressurized into solution. If it begins negative and P < 0, then the bubble should expand and fill the available space. What is required is an initial bubble radius such that the net pressure across the interface is negative; and we do not know why that might occur. Detailed theoretical treatments have traditionally yielded tensile limits for water greater than 50 MPa (Pickard, 1981) and continue to do so (Xiao and Heyes, 2002). Similar results continue to be obtained experimentally (Zheng et al., 1991; Williams et al., 1999).

Heterogeneous Nucleation from a Hydrophobic Surface

Water, being less firmly bonded to a hydrophobic surface than a hydrophilic one (or to itself), should more easily be pulled loose from a hydrophobic patch to nucleate an embolism. Various theories of this process exist (e.g., Fisher, 1948; Blander and Katz, 1975; Brereton et al., 1998), of which the least inaccessible seems to be the first. All predict that the most important determinant of the bubble generation rate at a surface will be an exponential term of the form exp(−Wmax/kT), where Wmax [J] is the energy required to expand the bubble to critical radius, kB [1.381 × 10−23 J•K−1] is the Boltzmann constant, and T [K] is the absolute temperature. The expansion energy is given as:

(2)

where γ [~70 mN•m−1] is the surface tension of the sap-embolus interface, ψ [dimensionless] is a geometric factor thought to lie between 0 and 1 and to be smaller for less hydrophilic surfaces, PV [Pa] is the vapor pressure of the sap, and P [Pa] is the pressure within the sap. That is, the nucleation rate should rise appreciably as a vessel’s walls become less hydrophilic. On the other hand, compared to the bulk of the fluid, there must be many orders of magnitude fewer potential sites of heterogeneous nucleation at the sap-wall interface than there are potential sites of homogeneous nucleation in the bulk sap. How these two factors balance out is unclear as we know of no relevant experimental data or molecular dynamics simulations.

Heterogeneous Nucleation from a Preexistent Bubble

Because the interior surface of the xylem element is assumed to be hydrophilic, it is presumed that no such bubbles would have been formed during xylem development (Pickard, 1981; Steudle, 2001). Furthermore, if one had commenced to form, it should have been reabsorbed by the same processes that are thought to defeat homogeneous nucleation.

Heterogeneous Nucleation from an Air-Water Interface (Air-Seeding)

This and the other three mechanisms all predict embolus formation under sufficient sap tension. But this alone predicts that the determining variable is (P – PA), where PA [Pa] is the pressure of ambient air. Thus, it would appear that the key to embolization in this scenario is making (P – PA) sufficiently negative, and this is done experimentally either by inducing tension within the sap or by pressurizing the air at the interface. Experiments validating this prediction have been reviewed elsewhere (Tyree, 1997).

It would seem therefore that air-seeding, the currently hegemonic explanation of embolism formation (Tyree, 1997; Steudle, 2001), might be satisfactory if only one could finesse the anatomical observation that the location of such interfaces could well be the pores of the boundary pit membranes (Shane et al., 2000b). Such pores have been observed to be ~3.5 nm in diameter (Shane et al., 2000a) or < 20 nm (Choat et al., 2003, 2004); hence meniscal failure should require a sap tension T [Pa], where (Pickard, 1981, Eq. 4)

(3)

and δ [m] is the effective pore diameter. For an air-water interface, this implies a failure tension well above 10 MPa and also above the upper limit of what is observed experimentally (Tyree, 1997; Sperry and Hacke, 2004). One obvious way of reconciling these observations is to postulate either (1) rare much larger pores (Choat et al., 2004) or (2) contact angle modulation of the bounding surfaces of the pit pores such that | cosθ| is reduced from unity by a factor of 10 or more, making the pore boundary quasihy-drophobic. At present, such hypotheses are speculative, although there is no firm evidence against either.

The Healing of an Embolism

The possibility of bubble resorption within tracheary elements has been the subject of considerable experimentation and speculation. As described by Holbrook and Zwieniecki (1999), a complete model of refilling an embolized vessel while the still functioning xylem is under transpiration-induced tension must at the least explain:

• How sap is extruded into the empty tracheary element even though the free energy gradient from soil to xylem seems often to be in the wrong direction.

• How the element is hydraulically isolated during refilling to avoid its new contents being sucked away in the transpiration stream.

• How a refilled vessel ultimately reestablishes hydraulic contact with neighboring conduits in such fashion as to become once again useful in water transport.

These issues will now be addressed sequentially, but the reader is warned that the answers provided, even if ultimately accorded enthusiastic acceptance, are more prolegomena to the desired explanation than the explanation itself.

Extruding Sap into an Embolized Vessel

How can a plant extrude water into an embolized xylem vessel from regions of apparently lower free energy (tissue and/or soil) ? This question is presently the focus of much theorizing, all of which predicts that such extrusion can occur. The relevant literature has recently been reviewed by Clearwater and Goldstein (see Chapter 18) who identified no winning candidate but helpfully subdivided the various models into four distinct categories. We shall not recapitulate their efforts here but rather point out that the question itself may infelicitously direct the reader’s thinking. That is, the focus on free energy may betoken a covert assumption that the process can be explained using an exclusively thermodynamic model, and this has not been proven. For example, extrusion through a pipe is commonly considered to be a hydraulic/hydrodynamic problem and is treated using the Navier-Stokes equations. Theoretically this leads to the Hagen-Poiseuille equation, which is much used by modelers of flow in the plant vasculature (e.g., Nonweiler, 1975), and which has been subject to extensive experimental tests. Moreover, molecular dynamics simulations uniformly show that the Navier-Stokes equations should work acceptably until the pipe diameter is less than a few diameters of the solvent molecule itself (e.g., Koplik and Banavar, 1995; Fan et al., 2002); over this entire size range, the flow down a pipe should be hydraulic, with a net solvent flux proportional to the hydraulic pressure difference between the ends of the pipe. Conversely, the passage of solvent (e.g., water) through a pipe is bounded from below by the diameter of the solvent molecule for water in the range 0.2 to 0.3 nm; this is the aquaporin realm where the flow is osmotic and proportional to the chemical potential difference between the ends of the pore (e.g., Fujiyoshi et al., 2002; Zhu et al., 2004). The transition regimen between the hydraulic and osmotic limits seems to be largely uninvestigated.

This distinction between hydraulic and osmotic flows has been discussed at greater length elsewhere (Pickard, 2003a), where it was essential to a quantitative model of root pressure. This model has been (1) specifically advanced as a mechanism for extrusion of sap into embolized vessels (Pickard, 2003a) and (2) shown to predict recent novel data on the stem exudation of detopped plants (Pickard, 2003b). Our belief is that a distinction between hydraulic and osmotic flows must be made in any successful biophysical explanation of vessel refilling and that models that do not carefully treat this distinction (e.g., Hacke and Sperry, 2003) may be incomplete.

Hydraulic Isolation of an Embolized Vessel

Hydraulic isolation of an embolized vessel to permit luminal refilling has been hypothesized to be enabled by the surface chemistry of the vessel lumen and the pit channels connecting to it (Holbrook and Zwieniecki, 1999). Their idea was that water extruded into a lumen would preferentially wet the hydrophilic walls, forcing the gas (1) into a pressurized luminal bubble from which it resorbed or (2) into pit channels that somehow did not at once refill with water. At issue is the mechanism by which a pit channel might contrive to function like a valve, opposing the entry of water (but not gas), thereby meeting the challenge for the plant to fill the vessel lumen completely before filling pathways to adjacent vessels under tension. How this might be accomplished is illustrated in Fig. 1.1A. For an arbitrary liquid-gas interface in a cylinder of radius ρλ [m], it is easy to show that the equilibrium pressure difference is,

Figure 1.1 Schematic representations of (A) the final stage of refilling a vessel lumen and (B) an intermediate stage of refilling a pit; in each case, cylindrical symmetry is assumed. (A) Surface tension forces in a refilling lumen of radius ρλ [m] and contact angle θλ [deg] will suck fluid along the lumen with a force [2πρλ] [γcosθλ that must be balanced by the liquid pressure Pliq [Pa] and the gas pressure Pgas [Pa] on the gas-liquid interface, which give an axial force resisting compressing of [Pgas – Pliq] [πρλ²]. (B) Schematic (after pit membrane.

(4)

for water and a cylinder radius of 1 μm, 2γ/ρλ is roughly 150 kPa. As water continues to be extruded into the lumen, the luminal gas will be compressed and go gradually into (supersaturated) solution. If the pit channels are even mildly hydrophobic (cosθch < 0), their capillary repulsion (associated with their negative cosθch and their relatively small radii) will resist filling and inhibit pit bubble resorption until the vessel lumen is full and continuing extrusion of water into it can increase Pliq appreciably. The postulated resistance of an embolized pit to refilling would of course be augmented by (1) the energy required to increase the sap/gas interfacial area once the interface reenters the chamber and (2) the compression of the intrachamber embolus. That is, successful refilling to this stage will depend critically on the microstructure of the cell wall and especially on local variations in its surface chemistry. Hydrophobic inhibition of pit refilling might even be advantageous in that it could help to synchronize pit refilling and pit sap reconnection to the transpiration stream.

Reestablishment of Hydraulic Connections

Once the vessel lumen has been refilled and sap forced past a postulated hydrophobic band in the pit channel, refilling of the pit chamber should proceed swiftly as the pressurized sap flows up the presumedly hydrophilic walls of the pit chamber; this is shown schematically in Fig. 1.1B. After the initial capillary repulsion of the advancing air/sap interface has been overcome, hydraulic flow through a narrow hydrophobic pipe is actually easier than through a hydrophilic pipe (Vinogradova, 1999). Within the pit chamber, it would be desirable for the sap to make an optimal contact with the presumedly hydrophilic pit membrane (Zwieniecki and Holbrook, 2000), and this we define to occur when the sap surface and the pit membrane are as parallel as possible at first contact because this will leave the smallest volume of residual embolus (Holbrook and Zwieniecki, 1999). Referring to the idealization of Fig. 1.1B, parallelism will be achieved when

(5)

Zwieniecki and Holbrook (2000) provided typical experimental values of α (~75°), of θ (~50°) for the walls of vessels (but none for the walls of pit chambers), and none for β; if we assume pit chamber symmetry and a taut pit membrane, then β ~ 0° and we estimate θchamb ~ 15°, roughly approximating that of cellulose (Luner and Sandel, 1969).

Challenges to the Cohesion-Tension Theory

Anomalous Results from the Xylem Pressure Probe

The pressure probe, broadly defined, is a tool for measuring the internal pressure of a tissue or a single cell; reviews have been provided for tissue by Milburn and Ranasinghe (1996) and for single cells by Tomos and Leigh (1999) and Wei et al. (2001). The challenge to cohesion-tension arose when, from intravessel pressure probe studies, it became apparent that the negative hydraulic pressures actually experimentally detected within xylem elements often failed to support the predictions of the cohesion-tension theory (Zimmermann et al., 1993, 1995, 2002, 2004). These data have generated persisting uncertainty (Tyree, 1997; Meinzer et al., 2001; Steudle, 2001; Zimmermann et al., 2004).

Because experimental data have primacy in science, an obvious first step in resolving the conundrum is to examine the reliability of the data themselves. First, Holbrook et al. (1995) applied tension to the xylem water in a leaf by spinning its branch in a centrifuge and found that the calculated centrifugal tension agreed with pressure bomb measurements on the leaf after centrifugation. Second, Pockman et al. (1995) found using centrifugal force to induce negative pressure between –0.5 MPa and –3.5 MPa in intact stems … that xylem conduits remained water-filled and conductive. Third, negative pressures down to about –2 MPa have been demonstrated directly using mercury penetrometry, implying that serious limitations arise from the pressure probe method itself (Milburn, 1996, p. 399). Fourth, Sperry et al. (1996) have offered a harsh critique of the xylem pressure probe at pressures more negative than a few hundred kilopascals: (1) the xylem conduit wall will not necessarily seal tightly around the glass probe, a problem that should not occur at positive pressures if the seal behaves in the manner of a flapper valve like the tricuspid, and (2) the internal surfaces of the probe itself may provide heterogeneous nucleation sites more failure prone than those of the xylem conduit wall. Fifth, there is an obvious need for groups other than Zimmermann’s to make measurements with the xylem pressure probe (Tomos and Leigh, 1999); in general, it is scientifically desirable for controversial results to be confirmed by workers outside the orbit of their originator.

Simultaneous Comparison Between the Xylem Pressure Probe and the Pressure Chamber

The discrepancy between the pressure probe data reported by U. Zimmermann’s group and the pressure chamber measurements obtained by others was resolved by Melcher et al. (1997, 1998) and Wei et al. (1999). They demonstrated that the two techniques agreed when measurements were made on nontranspiring leaves (i.e., when water potential gradients within the leaf were eliminated).

The Compensating Pressure Theory of Martin Canny

The existence of U. Zimmermann’s disquieting pressure probe measurements prompted Canny (1995, 1998) to reconsider other possibly contrary data and to develop the compensating pressure theory of sap ascent, which would replace cohesion-tension with cohesion sustained by tissue pressure (Canny, 1995, p. 348), where tissue pressure is the mutual pressure of cells (Canny, 1998, p. 897). These proposals have generated significant controversy but little support (Milburn, 1996; Tyree, 1997; Wei et al., 1999). In the absence of detailed mathematical realizations of Canny’s proposals, we find it hard to give an informed evaluation. Similar difficulties are encountered in trying to assess U. Zimmermann’s multi-force theory (Zimmermann et al., 2004).

What Good Came Out of the Debate?

Although the cohesion-tension theory still stands as the hegemonic model for understanding long-distance sap transport in plants, the controversy propelled the development of new ideas and possible alternative paradigms to replace the postulates of cohesion-tension theory had it been abandoned (Canny, 1998; Zimmermann et al., 2002). Such challenges forced overdue reexamination of the biophysical bases of the cohesion-tension theory (e.g., Tyree, 1997). Nevertheless, significant unknowns in long-distance water transport in plants still remain.

Factors That Affect Hydraulic Conductance

For xylem sap to flow through the plant, the negative hydrostatic pressure gradient must be large enough to overcome the resistive force of sap viscosity and the force of gravity. Because the rate of xylem sap flow through plants is relatively slow, the laminar flow pattern within an individual vessel consists of layers of sap that flow past each other in a sheetlike fashion, with the result that neither eddies nor vortices occur and turbulence is absent.* However, in both laminar and turbulent flow, viscosity leads to internal friction, which produces energy dissipation. If both the viscosity and the vorticity of the fluid are nonzero, then viscous dissipation occurs. For laminar flow at zero gravity through cylindrical pipe, the flow rate is given by the Hagen-Poiseuille relation:

(6)

where Jv [m³•s−1] is the volumetric flow rate, V [m³] is the volume, t [s] is time, ΔP [Pa] is the difference in pressure between the upstream and downstream end of the pipe; r [m] is the radius of the pipe; and η [Pa•s] is the dynamic viscosity of the solution in the pipe and l [m] is the length of the pipe. Small changes in the radius of a pipe can greatly affect flow rate through the pipe, an increase of 19% being sufficient to double the flow. Evaporation from the surfaces of leaf menisci provides both the gravitational potential energy stored within the elevated sap and the energy lost to viscous dissipation.

The Hagen-Poiseuille equation works well for idealized systems. For example, recent measurements made using individual vessels in short sections of Fraxinus americana stems were in agreement with the Hagen-Poiseuille predictions (Zwieniecki et al., 2001a). Nevertheless, the Hagen-Poiseuille equation is unlikely to hold without taking into account some of the anatomical deviations of the xylem from idealized tubes: (1) xylem radii are seldom constant for the length of a conduit, (2) the sap must pass through bordered pit junctions that are separated by fibrous membranes, (3) the distances between these bordered pit membranes vary greatly (Zimmermann, 1983), (4) vestures and warts (protrusions into the xylem and in bordered pit channels) occur, (5) sap concentration and temperature may change along the flow path and changes in the ionic strength of the xylem sap alter flow rates (Zimmermann, 1978; van Ieperen et al., 2000), thus raising the possibility of a regulatory role for nonliving cells in the xylem (Zwieniecki et al., 2001b), and (6) molecular dynamics simulations show that unexpected flow patterns can, on the nanoscale, occur near boundary irregularities (Fan et al., 2002). Our conclusion is that simplified macroscopic hydrodynamics, artfully applied, enables useful qualitative estimates of behavior and that real anatomical variations within the xylem virtually preclude exact quantitative predictions.

Discussion

Plants, being sessile, are generally plastic in response to the changing environment around them, implying that continuous opportunistic growth occurs to optimize capture of local resources. Plants are always growing and always overcoming the challenge of connecting new growth with extant structures while maintaining performance. Long-distance transport of both mass and messages must occur in sap-filled conduits, driven either by pressure (phloem) or tension (xylem) but not by a mechanical pump (heart). Thus, it is imperative that we strive for understanding how plants connect up their hardware as they add new growth while maintaining distributed control of their internal resources, both local and nonlocal. That is, how is the design of the plant’s water transport subsystem influenced by (1) the constraints of biophysics, (2) the egregious pure delay in information-transfer imposed by the lack of a nervous system, (3) the limitations of a plant’s algorithms for growth, and (4) its typical longevity? The best water transport system in the universe will not guarantee a taxon’s reproductive success over multiple generations. Nature awards survival to those taxa that function best as integrated systems, and the cost of the water transport subsystem had better not be exorbitant. Thus, understanding the flow path of water through a plant’s intricate plumbing connections from the root to leaf is of great importance.

A major obstacle to deep understanding of plant water transport is the lack of a detailed wiring diagram of the xylem. Together with up-to-date information on the hydraulic resistances of the conducting elements, this would greatly add to our understanding. Classic measurements of conduit length (Handley, 1936; Skene and Balodis, 1968) and simple diagrams (Huber, 1935; Vité and Rudinsky, 1959, Kozlowski and Winget, 1963, Zimmermann and Brown, 1971) deserve updating. Nevertheless, the motion pictures created by Martin Zimmermann and Mattmüller (1982) provide a fundamental appreciation of how xylem elements/tracheids grow into each other and how plants ensure reliability of their water transport capacity by overbuilding connections along the path. However, developing a useful three-dimensional wiring diagram of the xylem from extant literature and movies is problematic because (1) it seems unwise to assume either linearity or time-invariance, (2) the resistance is located at bordered pit connections between xylem conduits (Lancashire and Ennos, 2002), and (3) the degree to which plants use xylem located in older tissue is understudied, thus making it unclear whether measurements that include older xylem yield underestimates of hydraulic resistance (Melcher et al., 2003). This may lead to real problems in interpreting resistance determinations, especially how one maps them onto the observed xylem microanatomy.

Nevertheless, the future of plant water relations seems bright as we profit from the burgeoning development of new tools and methods for studying the transport of both water and minerals within the transpiration stream such as the following:

• Nano-scale observational techniques, such as scanning electron microscopy with EDAX

• Confocal microscopy

• Atomic force microscopy, potentially applicable to the study of the luminal surface chemistry within conducting elements

• Magnetic resonance imaging, which should enable three-dimensional maps of the hydraulic connections in wood

• New infrared imaging techniques, which enable better mapping of surface temperatures and promote insight into water distribution, evaporation, ice formation, and even sap flow

• Abundant computing power, which facilitates the exploration of more realistic models of sap movement

We assert that opportunities now confronting the biology of mass transport in plants can best be exploited by interdisciplinary collaborations of people who are judged by the novelty of the questions they ask and by ingenuity of the techniques they bring to bear rather than the orthodoxy of their training or viewpoint. We conclude with a list of areas where major work is needed.

• The interdependencies and interactions of mass transport in xylem and phloem

• The nature and phenomenology of the distributed control under pure time delay, which seemingly characterizes the integration and regulation of plant processes

• The phenomenology and biophysics of embolism repair across the plant kingdom

• The detailed understanding of hydraulic architecture, both anatomically and mechanistically

• The ontogeny, ultrastructure, and biophysics of plasmodesmata, pits, and perforation plates

• The fate of the transpiration stream, the pathways by which its component water moves from the leaf xylem to the substomatal chamber, and the way in which the leaf handles the stream’s nonvolatile solutes

• The biology and biophysics of living wood cells

• The phenomenology and biophysics of lesser studied pipelike structures in plants such as laticifers and resin ducts

• The transport and exchange of metabolically important gases in apex, stem, and root

References

Blander, M., Katz, J. L. Bubble nucleation in liquids. AIChE J. 1975; 21:833–848.

Böhm, J. Capillarität un saftsteigen. Berichte der Deutschen Botanische Gesellschaft. 1893; 11:203–212.

Brereton, G. J., Crilly, R. J., Spears, J. R. Nucleation in small capillary tubes. Chem Physics. 1998; 230:253–265.

Canny, M. J. A new theory for the ascent of sap: Cohesion supported by tissue pressure. Ann Bot. 1995; 75:343–357.

Canny, M. J. Vessel contents during transpiration: Embolisms and refilling. Am J Bot. 1997; 84:1223–1230.

Canny, M. J. Applications of the compensating pressure theory of water transport. Am J Bot. 1998; 85:897–909.

Canny, M. J., McCully, M. E., Huang, C. X. Cryo-scanning electron microscopy observations of vessel content during transpiration in walnut petioles. Facts or artefacts? Plant Physiol Biochem. 2001; 39:555–563.

Choat, B., Ball, M., Luly, J., Holtum, J. Pit membrane porosity and water stress-induced cavitation in four co-existing dry rainforest tree species. Plant Physiol. 2003; 131:41–48.

Choat, B., Jansen, S., Zwieniecki, M. A., Smets, E., Holbrook, N. M. Changes in pit membrane porosity due to deflection and stretching: The role of vestured pits. J Exp Bot. 2004; 55:1569–1575.

Cochard, H., Bodet, C., Thierry, A., Cruiziat, P. Cryo-scanning electron microscopy observations of vessel content during transpiration in walnut petioles. Facts or artifacts? Plant Physiol. 2000; 124:1191–1202.

Dixon, H. H. Transpiration and the Ascent of Sap in Plants. London: Macmillan; 1914.

Dixon, H. H., Joly, J. On the ascent of sap. Philos Trans R Soc Lond. 1895; B186:563–576.

Facette, M. R., McCully, M. E., Shane, M. W., Canny, M. J. Measurement of time to refill embolized vessels. Plant Physiol Biochem. 2001; 39:59–66.

Fan, X. J., Phan-Thien, N., Yong, N. T., Diao, X. Molecular dynamics simulation of a liquid in a complex nano channel flow. Physics Fluids. 2002; 14:1146–1153.

Fisher, D. B. Long distance transport. In: Buchanan B., Gruissem W., Jones R.L., eds. Biochemistry & Molecular Biology of Plants. Rockville, MD: American Society of Plant Physiologists, 2000.

Fisher, J. C. The fracture of liquids. J Appl Phys. 1948; 19:1062–1067.

Floto, F. Stephen Hales and the cohesion theory. Trends Plant Sci. 1999; 4:209.

Fujiyoshi, Y., Mitsuoka, K., de Groot, B. L., Philippsen, A., Grubmüller, H., Agre, P., Engel, A. Structure and function of water channels. Curr Opin Struct Biol. 2002; 12:509–515.

Haberlandt, G. Physiological Plant Anatomy. London: Macmillan; 1914.

Hacke, U. G., Sperry, J. S. Limits to xylem refilling under negative pressure in Lauris nobilis and Acer negundo. Plant Cell Environ. 2003; 28:303–311.

[Reprinted by Scientific Book Guild, London, UK] Hales, S. Vegetable Staticks. London, UK: W. & J. Innys and T. Woodward; 1727.

Handley, W. R.C. Some observations on the problem of vessel length determination in woody dicotyledons. New Phytologist. 1936; 35:456–471.

Holbrook, N. M., Burns, M. J., Field, C. B. Negative xylem pressures in plants: A test of the balancing pressure technique. Science. 1995; 270:1193–1194.

Holbrook, N. M., Zwieniecki, M. A. Embolism repair and xylem tension: Do we need a miracle? Plant Physiol. 1999; 120:7–10.

Huber, B. Die physiologische bedeutung der ring- und zerstreutporigkeit. Berlin Deutsche Botanical Gazette. 1935; 53:711–719.

Konrad, W., Roth-Nebelsick, A., The dynamics of gas bubbles in conduits of vascular plants and implications for embolism repair. JTheoret Bot 2003; 224:43–61

Koplik, J., Banavar, J. R. Continuum deductions from molecular hydrodynamics. Annu Rev Fluid Median. 1995; 27:257–292.

Kozlowski, T. T., Winget, C. H. Patterns of water movement in forest trees. Botan Gazette. 1963; 124:301–311.

Lancashire, J. R., Ennos, A. R. Modelling the hydrodynamic resistance of bordered pits. J Exp Bot. 2002; 53:1485–1493.

Part C Luner, P., Sandel, M. The wetting of cellulose and wood hemicellulose. J Polymer Sci. 1969; 28:115–142.

McCully, M. E. Root xylem embolisms and refilling. Relation to water potentials of soil, roots, and leaves, and osmotic potentials of the root xylem sap. Plant Physiol. 1999; 119:1001–1008.

McCully, M. E., Shane, M. W., Baker, A. N., Huang, C. X., Ling, L. E.C., Canny, M. J. The reliability of cryoSEM for the observation and quantification of xylem embolisms and quantitative analysis of xylem sap in situ. J Microsc. 2000; 198:24–33.

Meinzer, F. C., Clearwater, M. J., Goldstein, G. Water transport in trees: Current perspectives, new insights and some controversies. Environ Exp Bot. 2001; 45:239–262.

Melcher, P. J., Meinzer, F. C., Yount, D. E., Goldstein, G., Measuring high tensions in plantsBennett P.B., Demchenko I., Marquis R.E., eds. High Pressure Biology and Medicine. Papers presented at the Vth International Meeting on High Pressure Biology. University of Rochester Press, New York, 1997:93–101.

Melcher, P. J., Meinzer, F. C., Yount, D. E., Goldstein, G., Zimmermann, U. Comparative measurements of xylem pressure in transpiring and non-transpiring leaves by means of the pressure chamber and the xylem pressure probe. J Exp Bot. 1998; 49:1757–1760.

Melcher, P. J., Zwieniecki, M. Z., Holbrook, N. M. Vulnerability of xylem vessels to cavitation in sugar maple. Scaling from individual vessels to whole branches. Plant Physiol. 2003; 131:1775–1780.

Milburn, J. A. Sap ascent in vascular plants: Challengers to the cohesion theory ignore the significance of immature xylem and the recycling of Münch water. J Exp Bot. 1996; 78:399–407.

Milburn, J. A., Ranasinghe, M. S. A comparison of methods for studying pressure and solute potentials in xylem and also in phloem laticifers of Hevea brasiliensis. J Exp Bot. 1996; 47:135–143.

Nonweiler, T. R.F. Flow of biological fluids through non-ideal capillaries. Encyclopedia Plant Physiol (N.S.). 1975; 1:474–477.

Ormières, D., Provansal, M. Transition to turbulence in the wake of a sphere. Phys Rev Lett. 1999; 83:80–83.

Pickard, W. F. The ascent of sap in plants. Prog Biophys Mol Biol. 1981; 37:181–229.

Pickard, W. F. A novel class of fast electrical events recorded by electrodes implanted in tomato shoots. Aust J Plant Physiol. 2001; 28:121–129.

Pickard, W. F. The riddle of root pressure. I. Putting Maxwell’s demon to rest. Functional Plant Biol. 2003; 30:121–134.

Pickard, W. F. The riddle of root pressure. II. Root exudation at extreme osmolalities. Functional Plant Biol. 2003; 30:135–141.

Pickard, W. F. The role of cytoplasmic streaming in symplastic transport. Plant Cell Environ. 2003; 26:1–15.

Pockman, W. T., Sperry, J. S., O’Leary, J. W. Sustained and significant negative water pressure in xylem. Nature. 1995; 378:715–716.

Ritman, K. T., Milburn, J. A. Acoustic emissions from plants: Ultrasonic and audible compared. J Exp Bot. 1988; 39:1237–1248.

Shane, M. W., McCully, M. E., Canny, M. J. Architecture of branch root junctions in corn: Structure of the connecting xylem and porosity of boundary pit membranes. Ann Bot. 2000; 85:613–624.

Shane, M. W., McCully, M. E., Canny, M. J., The vascular system of maize stems revisited: Implications for water transport and xylem safety. Ann Bot 2000; 86:245–258

Skene, D. S., Balodis, V. A study of vessel length in Eucalyptus obliqua L’Hérit. J Exp Bot. 1968; 19:825–830.

Sperry, J. S., Hacke, U. G. Analysis of circular bordered pit function. I. Angiosperm vessels with homogenous pit membranes. Am J Bot. 2004; 91:369–385.

Sperry, J. S., Saliendra, N. Z., Pockman, W. T., Cochard, H., Cruiziat, P., Davis, S. D., Ewers, F. W., Tyree, M. T. New evidence for large negative xylem pressures and their measurement by the pressure chamber method. Plant Cell Environ. 1996; 19:427–436.

Steudle, E. The cohesion-tension mechanism and the acquisition of water by plant roots. Annu Rev Plant Physiol Plant Mol Biol. 2001; 52:847–875.

Taiz, L., Zeiger, E. Plant Physiology, 3rd ed., Sunderland, MA: Sinauer Associates, 2002.

Tomos, A. D., Leigh, R. A. The pressure probe: A versatile tool in plant cell physiology. Annu Rev Plant Physiol Plant Mol Biol. 1999; 50:447–472.

Tyree, M. T. The cohesion-tension theory of sap ascent: Current controversies. J Exp Bot. 1997; 48:1753–1765.

Tyree, M. T., Sperry, J. S. Vulnerability of xylem to cavitation and embolism. Annu Rev Plant Physiol Mol Biol. 1989; 40:19–38.

Tyree, M. T., Zimmermann, M. H., Xylem structure and the ascent of sap. Springer Series in Wood Science. 2nd Ed. Springer-Verlag, New York, 2002.

van Ieperen, W., van Meeteren, U., van Gelder, H. Fluid composition influences hydraulic conductance of xylem conduits. J Exp Bot. 2000; 51:769–776.

Vesala, T, Hölttä, T., Perämäki, M., Nikinmaa, E. Refilling of a hydraulically isolated embolized xylem vessel: Model calculations. Ann Bot. 2003; 91:419–428.

Vinogradova, O. L. Slippage of water over hydrophobic surfaces. Int J Mineral Processing. 1999; 56:31–60.

Vité, J. P., Rudinsky, J. A. The water-conducting systems in conifers and their importance to the distribution of trunk-injected chemicals. Contributions Boyce Thompson Institute. 1959; 20:27–38.

Wei, C., Steudle, E., Tyree, M. T. Water ascent in plants: Do ongoing controversies have a sound basis? Trends Plant Sci. 1999; 4:372–375.

Wei, C., Steudle, E., Tyree, M. T., Lintilhac, P. M. The essentials of direct xylem pressure measurement. Plant Cell Environ. 2001; 24:549–555.

Williams, P. R., Williams, P. M., Brown, S. W.J., Temperley, H. N.V. On the tensile strength of water under pulsed dynamic stressing. Proc R Soc London A. 1999; 455:3311–3323.

X, M. The Autobiography of Malcolm X. New York: Ballantine Books; 1992.

Xiao, C., Heyes, D. M. Cavitation in stretched liquids. Proc R Soc London A. 2002; 458:889–910.

Zheng, Q., Durben, D. J., Wolf, G. H., Agnell, C. A. Liquids at large negative pressures: Water at the homogeneous nucleation limit. Science. 1991; 254:829–832.

Zhu, F., Tajkhorshid, E., Schulten, K. Theory and simulation of water permeation in aquaporin-1. Biophys J. 2004; 86:50–57.

Zimmermann, M. H. Hydraulic architecture of some diffuse-porous tress. Can J Bot. 1978; 56:2287–2295.

Zimmermann, M. H. Xylem Structure and the Ascent of Sap. Berlin: Springer-Verlag; 1983.

Zimmermann, M. H., Brown, C. L. Trees Structure and Function. New York: Springer-Verlag, 1971; 169–220.

(Films C1404 and D1418). Gottingen. Zimmermann, M. H., Mattmüller, M., The vascular pattern in the stem of the palm Rhapis excelsa I. The mature stem. II. The growing tip. Publications of the Institute for Scientific Films Series 15 Nos. 22 and 23, 1982.

Zimmermann, U., Haase, A., Langbein, D., Meinzer, F. Mechanisms of longdistance water transport in plants: A re-examination of some paradigms in the light of new evidence. Philos Trans R Soc London, ser. B. 1993; 341:19–31.

Zimmermann, U., Meinzer, F., Bentrup, F-W., How does water ascend in tall trees and other vascular plants? Ann Bot 1995; 76:545–551

Zimmermann, U., Schneider, H., Wegner, L. H., Haase, A. Water ascent in tall trees: Does evolution of land plants rely on a highly metastable state? New Phytologist. 2004; 162:575–615.

Zimmermann, U., Schneider, H., Wegner, L. H., Wagner, H-J., Szimtenings, M., Haase, A., Bentrup, F-W. What are the driving forces for water lifting in the xylem conduit? Physiol Plantarum. 2002; 114:327–335.

Zwieniecki, M. A., Holbrook, N. M. Bordered pit structure and vessel wall surface properties. Implications for embolism repair. Plant Physiol. 2000; 123:1015–1020.

Zwieniecki, M. A., Melcher, P. J., Holbrook, N. M. Hydraulic properties of individual xylem vessels of Fraxinus Americana. J Exp Bot. 2001; 52:257–264.

Zwieniecki, M. A., Melcher, P. J., Holbrook, N. M. Hydrogel control of xylem hydraulic resistance in plants. Science. 2001; 291:1059–1062.

*Martin Zimmermann’s observation that discussions of sap ascent have become so mathematical that they are not read by many plant anatomists (1983, p. 1) may be no less apt today than it was twenty years ago. But it misses the take-home lesson of Malcolm X’s dictum quoted at the beginning of this paper that mathematical rigor does tend to squelch wrangling. And if it can’t quite do that, it may at least pinpoint the root causes of the dispute.

*Where appropriate, pointers will be given to page (p.), section (s.), chapter (ch.), equation (eq.), figure (fig.), appendix (app.), table (tab.), or experiment (expt.) of the pertinent reference.

†Hales’ use of the term perspiration was correct usage for his era.

*The modifier putative has been used because unresolved doubts persist concerning the validity of the indirect methods (cf., McCully 1999, p. 1005).

*The labelynolds number in subcellular biological flows has been discussed by Pickard (2003c), whose calculation can be extended to this case to show that it should not exceed 1, even for sap moving at 1 mm•s−1 in a vessel 1 mm in diameter. But the transition to eddy formation is typically well above 10 (Ormières and Provansal, 1999).

2

Physiochemical Determinants of Phloem Transport

Aart J.E. van Bel and Jens B. Hafke

Publisher Summary

This chapter identifies several questions as to the physiochemical determinants of phloem transport. There is a general quantitative unawareness with regard to physiochemical parameters. Moreover, one must be thoughtful of potentially large differences between plant species due to disparate structural/functional conditions. The steepness of the hydraulic gradient depends on the physiological activities along the whole sieve tube stretch. Thus, diversity in modes of phloem loading, release/retrieval along the transport pathway, and in modes of phloem unloading, may have a strong impact on generation of the hydraulic pressure gradient. This recognition requires detailed studies on deployment and functioning of proteins involved in shuttling osmotic equivalents and water through the sieve element/companion cell (SE/CC) plasma membrane of the successive phloem zones and the structural frame of the SE/CCs in various plant groups. Furthermore, the reciprocal feedback regulation of sources and sinks and the compensatory and buffering activities of transport phloem must be investigated on the cell-biological level.

Structure-Functional Basics of Phloem Transport

Driving Forces of Mass Flow in Sieve Tubes

In 1930, Ernst Münch published the first coherent concept of phloem transport in his brilliant book Die Stoffbewegungen in der Pflanze. According to his ideas, mass flow (Jv) in the phloem was essentially driven by a pressure gradient (ΔP), the difference in turgor pressure (= hydraulic pressure) between source and sink tissues. As has been demonstrated later, turgor pressure differences between source (Psource) and sink ends (Psink) of the sieve tubes are actually responsible for the pressure gradient. In sources and sinks, local turgor pressures are established by differences in chemical water potential (mainly osmopotential) between the sieve tube content and the apoplast. The hydraulic pressure gradient (Psource exceeds Psink) thus essentially depends on physiological determinants of the sieve-tube osmotic potentials at either end of the phloem system (Fig. 2.1). These physiological activities mainly include metabolic processing of carbohydrates in the tissues bordering the phloem transport channels and the activities of plasma membrane-bound carriers, pumps, and channels lining the conducting channels.

Figure 2.1 The driving force for pressure flow depends on the turgor pressure difference between the sieve tube ends in source and sinks. The difference in turgor pressure (ΔP) between the source (Psource) and sink (Psink) results from the difference in water potential between the local apoplasm (P°) and the adjacent sieve element (Pi). Mass flow is further determined by the radius (r) of the tube (in this instance the functional radii of the sieve pores) and the viscosity (η) of the solute (mainly the viscosity of the sugars translocated) and length of the path (L).

Furthermore, the mass flow is directly proportional to the radius (r) of the sieve tubes and inversely proportional to the distance (L) between source and sink, and the viscosity of the phloem sap (η) as formulated in the Hagen-Poiseuille equation:

The Münch concept encountered fierce opposition with the advent of electron microscopy. Deposits residing on the sieve pores were thought to be incompatible with mass flow. As a number of investigators (Kollmann, 1973; Johnson et al., 1976; Sjolund and Shih, 1983) showed that the deposits were virtually absent in several plants after careful fixation, the resistance crumbled, and physiological logic regained dominance.

Mathematical calculations using translocation data mainly obtained with radiolabeled photoassimilates built a strong platform supportive of mass flow (e.g., Goeschl and Magnuson, 1986; Magnuson et al., 1986; Murphy, 1989a–d; Thompson and Holbrook, 2003a, b). Moreover, nuclear magnetic resonance (NMR) applied to intact plants (Köckenberger et al., 1997) and confocal laser scanning microscopy (CLSM) imaging of real-time displacement of phloem-mobile fluorochromes (Knoblauch and van Bel, 1998) provided conclusive optical evidence in favor of mass flow through the sieve tubes. Even clots of proteins covering the sieve pores in intact sieve tubes did not prohibit mass flow owing to the presence of narrow corridors in the protein deposits (Knoblauch and van Bel, 1998; Ehlers et al., 2000). Despite all this, fine-regulation between source supply and sink demand (Bancal and Soltani, 2002; Minchin et al., 2002) and quantification of physiochemical determinants of mass flow are still a matter of debate (Henton et al., 2002; Thompson and Holbrook, 2003a,b).

Having adopted mass flow as the mode of translocation, we will deal here with the structural and physiological origin of its physiochemical determinants. Significance and size of these variables associated with various types of phloem software and hardware (van Bel, 1996a) are discussed. Major emphasis is laid on ΔP, since this parameter is subject to complex physiological regulation.

Phloem Zones and Generation of a Turgor Pressure Gradient

Photoassimilates are transported through the sieve tubes, the conducting channels of angiosperm phloem, which are arrays of sieve element/companion cell (SE/CC) modules. Phloem consists of three successive functional zones (or phloem sections), each with a specific task (Fig. 2.2; see also Color Plate section) (e.g., van Bel, 1996a). In the first phloem section (collection phloem), photoassimilates are loaded into the SE/CCs of leaf minor veins after production in the leaf mesophyll. From the minor vein sieve tubes, photoassimilates are translocated via the second phloem section (transport phloem), which is located in leaf major veins, petioles, stems, and roots, to the sinks. There, photoassimilates are unloaded from the SEs in the third phloem section (release phloem) into growing or storage cells, where they are metabolized or sequestered.

Figure 2.2 A dynamic version of the Münch pressure flow model, the local fluxes of photoassimilates (violet arrows) and water (blue arrows) and the relative proportion of sieve elements (SE) and companion cells (CC) in the respective phloem zones. Photoassimilates are translocated via the phloem through essentially leaky instead of hermetically sealed pipes (sieve tubes). The solute content and implicitly the turgor are controlled by release/retrieval systems located on the plasma membrane of the sieve element/companion cell complexes (SE/CC-complexes). The retrieval mechanisms are energized by the proton-motive force. Differential release/retrieval balances along the pathway control the influx/efflux of sugars and water in the respective phloem zones. In collection phloem (where phloem loading takes place), the retrieval or uptake dominates; in the release phloem (where phloem unloading takes place) the release dominates. In transport phloem having a dual task (nourishment of axial sinks along the pathway and terminal sinks), the balance between release and retrieval varies with the requirements of the plant. The gradual loss of solute and the commensurate amounts of water have been ascribed to a slightly decreasing proton-motive gradient along the phloem pathway. Alternatively, the relative size reduction of companion cells along the source-to-sink path may explain a decreasing retrieval capacity of the SE/CC-complexes in the direction of the sink. The massive photoassimilate delivery in the sinks is assigned to symplasmic and/or apoplasmic loss of photoassimilates from the SEs driven by the high consumption and/or storage rates in the sink tissues.

Shifting physiological properties of SE/CCs along the phloem pathway, also inferred from the decreasing volume ratio between SEs and CCs towards the sinks (Fig. 2.2), have important consequences for their functional output. The SE/CCs in sources and sinks mostly generate a one-way traffic into and out of the sieve tubes, respectively. In contrast, the SE/CCs of the transport phloem have a dual function (Fig. 2.2) (van Bel, 2003a). Transport phloem carries photoassimilates to terminal sinks such as root/shoot tips. Concurrently, transport phloem is responsible for maintenance and growth (e.g., cambium) of heterotrophic tissues in the plant axis (axial sinks). This is reflected by a continuous leak and retrieval along the phloem pathway (Aloni et al., 1986) as has been demonstrated in bean plants, in which 6% of ¹¹C-labeled photoassimilates was lost and 3.4% retrieved every centimeter along the phloem pathway (Minchin and Thorpe, 1987). This solute exchange, which requires some degree of symplasmic isolation of SE/CCs (Kempers et al., 1998), goes along with appreciable water fluxes dependent on the dominance of release or retrieval (Fig. 2.2). We next sketch the structural and physiological factors involved in turgor pressure generation in the successive zones of the phloem system.

Generation of a Hydraulic Pressure Gradient in Collection Phloem

Modes of Phloem Loading; Consequences for Generation of Hydraulic Pressure?

Particularly in collection phloem, the ultrastructure of CCs is diverse (Fig. 2.3; see also Color Plate section). Three classes of CCs, all having a dense cytoplasm, have been distinguished: intermediary cells (ICs) with numerous vesicles of unknown nature, transfer cells (TCs) with numerous cell wall invaginations, and simple companion cells (SCs) without special features (Gamalei, 1989; Turgeon, 1996a). Plasmodesmal densities between CCs and adjacent parenchyma vary by a factor 1000. The SE/IC complexes (with many plasmodesmata (PD)s towards the mesophyll are mostly associated with symplasmic phloem loading against a sugar gradient (e.g., Turgeon, 1996a) and the SE/TC complexes (very few PDs) with apoplasmic phloem loading against a sugar gradient (Turgeon and Wimmers, 1988; van Bel et al., 1992, 1994). The latter mode of phloem loading is associated with apoplasmic transfer of sucrose; the production of RFOs (raffinose family oligosaccharides) is an integral part of the symplasmic phloem loading mechanism (Turgeon, 1996a).

Figure 2.3 Relationship between ultrastructure of the companion cell (CC) in the minor veins and the mode of phloem loading in dicotyledons. Three types of companion cells are presented here. (a) Intermediary cells (ICs) characterized by numerous cytoplasmic vesicles and many plasmodesmata at the wall interface between mesophyll and companion cells (MC/CC interface). Simple companion cells (SCs), without particular properties, have a moderate plasmodesmal density at the MC/CC-interface. Transfer cells (TC) possess cell wall invaginations to increase plasma membrane surface and have virtually no plasmodesmata at the MC/CC-interface. The structural differences between ICs (b) and TCs (c) are clearly visible in electron-micrographs (courtesy of S. Dimitrovska). V vesicles, * wall