The ability of guard cells to hydrate and dehydrate from the surrounding air was investigated using isolated epidermes of Tradescantia pallida and Vicia faba. Stomata were found to respond to the water vapour pressure on the outside and inside of the epidermis, but the response was more sensitive to the inside vapour pressure, and occurred in the presence or absence of living, turgid epidermal cells. Experiments using helium–oxygen air showed that guard cells hydrated and dehydrated entirely from water vapour, suggesting that there was no significant transfer of water from the epidermal tissue to the guard cells. The stomatal aperture achieved at any given vapour pressure was shown to be consistent with water potential equilibrium between the guard cells and the air near the bottom of the stomatal pore, and water vapour exchange through the external cuticle appeared to be unimportant for the responses. Although stomatal responses to humidity in isolated epidermes are the result of water potential equilibrium between the guard cells and the air near the bottom of the stomatal pore, stomatal responses to humidity in leaves are unlikely to be the result of a similar equilibrium.
Stomata respond to changes in the humidity of the air, but despite considerable controversy over the past 40 years, there is no consensus on the mechanism (Buckley 2005). Early studies have assumed that stomata respond to air humidity via a ‘hydroactive’ mechanism in which guard cell solute content is regulated based on leaf water potential (see Hall & Kaufmann 1975; Lange et al. 1975 for a discussion). However, Lange et al. (1971) showed that stomata in isolated epidermes could respond directly to humidity without the need for a signal based on leaf water potential. They suggested that guard cells simply hydrate from the intercellular air and dehydrate to ambient air through the external cuticle, and that the balance between these two processes determined the hydration state of the guard cells and hence the stomatal aperture. These studies have sparked debate on the relative roles of ‘hydroactive’ mechanisms and ‘hydropassive’ mechanisms. The former generally involves some sort of signal to the guard cells that originates in other cells and that is proportional to their water potential (Buckley 2005). The latter depends on water loss from the guard cell, either through the external cuticle (Maier-Maercker 1983) or from the inner walls (Dewar 1995), and a large hydraulic resistance between the guard cells and the surrounding epidermal cells. In these mechanisms, guard cell water potential is determined by the balance between water loss to the atmosphere and water gain from the epidermis.
The role of cuticular water loss in stomata responses to humidity has subsequently been addressed by several studies, with conflicting results. It has been shown that direct water loss through the cuticle can provide a mechanism for ‘feedforward’ responses of stomata to humidity – that is, responses that reduce transpiration rate in response to decreasing humidity (Farquhar 1978). Studies using water-sensitive paper (‘hydrophotography’) show that water is lost through the cuticle near the stomata more rapidly than through the cuticle for the rest of the epidermis (Maier-Maercker 1979), and this has been cited as evidence in favour of the importance of cuticular transpiration in the stomatal response to humidity (Maier-Maercker 1983). Further support for this idea comes from two studies that show an enhanced response of stomata to humidity in leaves that had been dipped in hexane to partially remove the cuticle (Meinzer 1982; Meinzer et al. 1990). However, a similar study, also using hexane to partially remove the cuticle, has shown no effect on the stomatal response to humidity (Kerstiens 1997), and Meidner (1986) concluded that cuticular conductance was too low to have a role in stomatal responses to humidity.
Water loss from the guard cells to the air in the intercellular spaces or stomatal pore has also been considered as a possible mechanism for decreasing the water potential of the guard cells in hydropassive mechanisms, and in many cases, this water loss has been assumed to be a major component of transpirational water flux (Sheriff & Meidner 1974; Meidner 1975). Theoretical analyses, however, reveal that the resistance between epidermal and guard cells would have to vary substantially over short periods of time to produce the observed dynamics of stomatal responses to humidity with such a mechanism (Buckley & Mott 2002). Finally, it has also been proposed that sucrose, produced by photosynthesis in the mesophyll, accumulates in the cell walls of the guard cells in proportion to the transpiration rate (Lu et al. 1997). The accumulation of sucrose raises the osmotic pressure of the water in the wall and withdraws water from the guard cells by osmosis.
Other data support hydroactive mechanisms. For example, stomata in leaf pieces showed a greater response to mannitol solutions than did stomata in isolated epidermes, suggesting a possible signal from the mesophyll in response to low water potential (Grantz & Schwartz 1988). In addition, occluding a single stoma on a leaf with oil did not change the response of that stoma to humidity, but blocking many stomata in a local area reduced the response to humidity (Kaiser & Legner 2007). It has been noted that hydroactive mechanisms provide a common mechanism for stomatal responses to changes in water supply to the leaf that tend to adjust stomatal conductance in parallel with changes in plant hydraulic conductance (Buckley 2005).
Interestingly, the most widely used mathematical model in current use for predicting stomatal responses to humidity is based on the relative humidity of the air near the leaf's surface (Ball, Woodrow & Berry 1987). Because this value depends on both ambient water vapour pressure and leaf temperature, it conflates stomatal responses to both parameters. Despite the predictive power of this model, experiments using helium in place of nitrogen in air have shown that stomatal responses to humidity are actually stomatal responses to the water loss rate from the leaf (Mott & Parkhurst 1991), and this appears to remove any possibility of a mechanistic basis for the correlation with relative humidity.
Some of the controversy over the mechanisms discussed earlier rests on the ability of the guard cells to exchange water with the air in the stomatal pore or the intercellular spaces. While some exchange is inevitable, hydropassive mechanisms depend on a relatively rapid exchange of water between guard cells and the vapour phase because guard cells must continuously lose water to the air to create passive closure. The classic study of Lange et al. (1971) suggests that guard cells can hydrate and dehydrate rapidly via the vapour phase, and can actually respond to humidity changes without liquid contact with the mesophyll. If such exchange occurs, it must be taken into account in formulating mechanisms for stomatal responses to humidity, and it could provide a basis for the apparent stomatal response to relative humidity (Ball et al. 1987) because the water potential of the air is temperature dependent.
This study was initiated to determine if water vapour exchange between guard cells and the surrounding air occurs rapidly enough to play a role in stomatal response to humidity. Experiments were conducted with isolated epidermes that were suspended in a chamber with no source of liquid water, and the responses of stomatal aperture to changes in water vapour pressure on both sides of the epidermis were determined.
MATERIALS AND METHODS
Plants were grown in a controlled environment greenhouse with day and night temperatures of 30 and 20 °C, respectively. Day length was extended to 16 h with high-pressure sodium lamps [photon flux density (PFD) approximately 1000 µmol m−2 s−1 at the top of the plant] when necessary. The plants were grown in 1 L pots containing peat:perlite:vermiculite (1:1:1 by volume) and were watered to excess daily with a dilute nutrient solution containing 9.1 mm N, 1.8 mm P, 2.7 mm K and 11 mm chelated Fe (Peter's 20-10-20; Grace Sierra Horticultural Products Co., Milpitas, CA, USA). The plants used for experiments were at least 4 weeks old, and the leaves chosen for use were fully mature but not senescing.
Isolated epidermes of Tradescantia pallida were prepared by carefully removing the upper epidermis and mesophyll from the lower epidermis by cutting and scraping with a razor blade. This method allowed the preparation of epidermes with living epidermal cells. Isolated epidermes of Vicia faba were prepared by peeling the lower epidermis away from the leaf or by the method described earlier. Microscopic examination of the epidermes prepared using either technique showed no intact mesophyll cells. The isolated epidermes were washed gently in distilled water, and then were spread across a filter paper saturated with 50 mm KCl, 1 mm CaCl2 and placed in a Petri dish. The lid was placed on the Petri dish, and it was placed in the dark for approximately 12 h. After the dark period, the Petri dish was placed under fluorescent lights at a PFD of approximately 100 µmol m−2 s−1 for 3 h.
The epidermes were removed from the Petri dish and anchored with vacuum grease over a hole (0.7 cm in diameter) in the middle of a thin piece of clear plastic (5 cm diameter). The piece of plastic was then placed between two aluminum blocks forming a (2.5 cm diameter, 0.5 cm deep) chamber on either side of the plastic/epidermis barrier. Water baths were used to control the temperature of the aluminum blocks (and therefore of the chambers). The temperatures of the epidermis, the air in the lower chamber and the aluminum blocks were measured with fine wire (135 µm), chromel–constantan thermocouples. Low-intensity light (PFD approximately 20 µmol m−2 s−1) was provided by a tungsten bulb that was filtered through 1 cm of water to remove infrared (IR) radiation. The two aluminum blocks that formed the chambers for the epidermis were mounted on a microscope stage, and the epidermis was viewed using a long focal length objective (Zeiss 6.3×, working distance 10.5 mm) on a Zeiss WL trinocular microscope (Zeiss, Jena, Germany). Digital images were taken at regular intervals with a digital camera and measured using digital image analysis software (ImagePro; Media Cybernetics, Bethesda, MD, USA). Figure 1 shows a diagrammatic representation of this experimental set-up.
The epidermes were initially very wet following removal from the Petri dish, and immediately after placement of the peel in the chamber, the stomata were usually occluded with water, and small droplets of water could be seen on the inner surface of the epidermis. We found that the stomata were relatively insensitive to changes in chamber humidity on either surface as long as this water was present. To remove this excess water and to clear the pores that were occluded with water, dry air was passed through the chamber for a short time (1–10 min). The dry air treatment was terminated when most of the pores were not occluded with water and epidermal temperature approached air temperature, indicating that water evaporation from the epidermis was near zero. Treatment of the epidermes following this dry air varied with the experiment and is explained further for each set of experiments.
Compressed N2 and O2 (80:20) was mixed with compressed 1% CO2 in N2 to produce 360 µmol mol−1 CO2 using electronic mass flow controllers. CO2 concentration was determined with an IR CO2 analyser (ADC, Hoddesdon, England). This mixture was then bubbled through distilled water at approximately 35 °C before it was divided into two streams, each flowing at 1.0 L min−1. Each stream was passed through the central helix of a 30 cm Graham condenser (a helical condenser, 30 cm long) and then through one of the two chambers around the epidermis. The temperatures of the two condensers were controlled independently, and each was monitored with a copper–constantan thermocouple threaded into the upper end of the condenser coils. This set-up allowed independent manipulation of the humidity of the gases in the two chambers. The dew point of the gas streams was determined using a chilled mirror dew-point hygrometer (DEW 10, General Eastern, Bellerica, MA, USA) or a thermocouple dew-point hygrometer (HR-33T; Wescor, Logan, UT, USA). The latter instrument could only be used for dew points that were within 0.5 °C of the ambient temperature. In all cases, the measured dew point was within 0.1 °C of the condenser temperature.
For aperture versus water potential measurements using osmotic solutions of mannitol and polyethylene glycol (PEG)-3200, epidermes were fixed in the bottom of a well slide and viewed using an inverted microscope. The solution bathing the epidermis was removed and replaced using a pipette, and the epidermis was washed once with each new solution. The osmotic pressure of the solutions was determined using a dewpoint hygrometer (HR-33T, Wescor).
It was possible to produce epidermes with either mostly dead or mostly live epidermal cells by subtle differences in how epidermes were prepared. Because the epidermal cells of T. pallida contain anthocyanins, it was easy to identify live and dead epidermal cells. The subsidiary cells of T. pallida looked identical in all preparations; they were determined to be alive and turgid in a few experiments using a cell pressure probe and were therefore assumed to be alive in all epidermes. It was possible to identify live and dead epidermal cells in V. faba by differences in their appearance. Live (turgid) cells could be seen to bow outwards, whereas dead (plasmolysed) cells did not bow outwards and remnants of the plasma membrane could be seen pulled away from the cell wall. For both species, dead epidermal cells were characterized by cell wall cavities containing little or no remnants of the cell. These ‘cells’ lacked an intact plasma membrane and had no turgor pressure. Live cells had clearly visible cellular contents and were visibly turgid at the beginning of the experiment. Stomata in all epidermes were found to be insensitive to light (Mott, Sibbernsen & Shope 2008), so illumination levels were kept low (PFD = 20 µmol m−2 s−1) to minimize the heat load on the epidermis and thermocouples.
Initial experiments were performed with epidermes that had almost no living epidermal cells and no extracellular water. It should be noted, however, that epidermes of T. pallida always had living subsidiary cells. These epidermes were assumed to have no extracellular water for several reasons. Firstly, during the initial dry air treatment (see Materials and Methods), it was possible to see water being replaced by air in the cavities formed by the cell walls of the dead epidermal cells, and evaporation caused the temperature of the epidermis to drop significantly below the chamber temperature. As these cavities became air filled, the temperature of the epidermis gradually increased to equal air temperature. Epidermes with no extracellular water will be hereafter termed ‘dry’.
To determine the response of the stomata to the water vapour pressure on the outside surface of the epidermis (ea), the vapour pressure on the inside surfaces (ei) was maintained at saturation, while ea was varied. For dry epidermes, changes in ea produced rapid and reversible changes in aperture, and a stable aperture was reached for each value of ea. Figure 2 shows typical results for one T. pallida epidermis; results for V. faba were similar (data not shown), and the experiment was repeated at least three times for each species using different epidermes from different leaves. Stomata in dry epidermes also responded to changes in ei, but the response was much larger than for an equivalent change in ea ( Figs 3 & 4). This extreme sensitivity made it necessary to keep ei very close to saturation to maintain open stomata. Stomatal responses to ei and ea were completely reversible for high values, but became irreversible for low values of either (data not shown). The kinetics for stomatal responses to ei were indistinguishable from those to ea, and all experiments showed approximately first-order kinetics with a time constant (τ) of between 10 and 15 min. Figures 3 and 4 show typical data for T. pallida and V. faba, respectively; the experiment was repeated at least three times for each species using different epidermes from different leaves.
To distinguish vapour-phase water transport from liquid-phase transport in these epidermis, we examined the effect of helox (79% helium, 21% O2) on the stomatal response to changes in ea in dry epidermes (Fig. 5). This experiment was performed twice with different epidermes from different leaves for both V. faba and T. pallida with similar results. Although there was sometimes a small transient change in aperture associated with the switch to helox, the steady-state aperture in helox was the same as that in normal air. This transient response was not always present and was probably caused by a small amount of dry air that was introduced into the chamber as the gas lines were switched. The change in aperture associated with a drop in ea from 2.64 to 2.20 kPa was the same in helox and air, but the kinetics of the change were substantially faster in helox (τ = 12 and 5 min for the data shown in Fig. 5).
To investigate the role of water loss from the cuticle in the observed responses to ea, we applied a thin layer of silicone vacuum grease to the lower surface of T. pallida leaves before isolating the epidermis. Grease was applied to leaves that had been in low light for several hours, and for which the stomata were mostly closed. Epidermes treated in this way were visibly shiny in appearance, and the grease layer could be seen through the microscope with appropriate lighting. When the stomata in these epidermes opened (in response to 50 mm KCl; see Materials and Methods), some were visibly occluded with grease, while others were not. Stomata that were occluded with grease did not respond to reductions in ea, but the response of stomata that were not occluded was indistinguishable from the response of an ungreased epidermis (Fig. 6).
We next performed experiments to determine if live epidermal cells were necessary for the response of stomata to ea. Although these epidermes were initially exposed to dry air like those discussed previously, the dry air treatment was terminated before epidermal temperature reached air temperature and before epidermal cells lost turgor. To keep the air exposed to the inner surface as close to saturation as possible, these experiments were performed with wet filter paper covering the bottom of the chamber for the inner surface and with no gas flow to that chamber. The chamber for the outer surface was maintained near saturation (ea = 2.81 kPa) for a period of time during which the apertures were constant. Water vapour pressure for the outer surface was then lowered to 2.37 kPa. In response to this decrease in ea, living epidermal cells could be seen to lose turgor and to plasmolyse, and stomatal apertures of T. pallida initially increased and then decreased, regardless of whether the epidermes contained live or dead epidermal cells (Fig. 7a) (but all stomata were surrounded by live subsidiary cells). In V. faba, stomata surrounded by live epidermal cells also opened before closing, but those with no adjacent live epidermal cells did not show an initial opening (Fig. 7b). Stomata where only one guard cell was adjacent to a live epidermal cell showed an intermediate response.
In the experiments with wet epidermes discussed earlier, there was a lag between the time that ea was decreased and the time that the stomata responded. The duration of this lag was variable among experiments, and temperature of the epidermis was substantially (several degrees) below air temperature during the lag. The lag was not present if an epidermis was subjected to low ea, returned to high ea and then lowered again, and it was not present when the stomata responded to an increase in ea. For these reasons, we concluded that the lag was caused by evaporation of water from the epidermis, and that only after most of the water was gone did the stomata respond rapidly to changes in ea.
To more quantitatively characterize the response of stomata in isolated epidermes to water vapour pressure and water potential, we performed a series of experiments with T. pallida in which ea and ei were equal and were varied in parallel. These experiments were performed with dry epidermes that showed no lag in their response to changes in ea or ei. Because there was no gradient in water vapour pressure across the epidermis, we assumed that the vapour pressure experienced by the guard cells (ep) was equal to the value of ea and ei. These experiments showed similar kinetics to those in Figs 2–4, and the water potential of the air in the pore adjacent to the guard cell (Ψp) was calculated from ep = ea = ei using the formula
where R is the ideal gas constant; T is the temperature of the air in the pore in kelvins; vL is the molar volume of liquid water, and es is the saturated vapour pressure of water in air at temperature T (Nobel 1983).
Assuming that the guard cells, epidermis and pore air are all isothermal, the closed symbols in Fig. 8 show the steady-state dependence of stomatal aperture on Ψp, as calculated from Eqn 1. Guard cells were visibly plasmolysed at the two lowest Ψ values, and aperture increased monotonically with Ψp for values above approximately −7.0 MPa The monotonic nature of the curve is consistent with the fact that all values were determined after epidermal and subsidiary cells had been plasmolysed, so there was no effect of the epidermal or subsidiary cell turgor pressure on aperture. The response of aperture to the Ψ of the guard cells (Ψg) was also determined using isolated epidermes submerged in mannitol or PEG solutions of known osmotic pressures (open symbols, Fig. 8). Solutions with osmotic pressures greater than about 6.5 MPa could not be used because these solutions could not be measured with the dewpoint hygrometer. For these experiments, epidermal cells and subsidiary cells were turgid at Ψ = 0, which caused the aperture to be smaller at that value than for lower values of Ψ.
It was also possible to vary Ψ of the air in the stomatal pore by raising epidermal temperature while maintaining ea and ei (and thereby ep) equal and constant. Assuming that the temperature of the air in the stomatal pore is close to that of the epidermis, then increasing epidermal temperature increases the value of es and decreases the water potential of the air at a constant value of ep (see Eqn 1). Figure 9 shows the response of stomatal aperture as epidermal temperature was varied by varying air temperature, and the open triangles in Fig. 8 show the steady-state apertures plotted versus Ψ as calculated from Eqn 1.
The data presented in this paper show that stomata in isolated epidermes can hydrate and dehydrate rapidly via the vapour phase, and that this exchange of water is responsible for stomatal responses to humidity in isolated epidermes. The ability of stomata to respond to humidity in isolated epidermes was first demonstrated by Lange et al. (1971), but it has never been subsequently explored to our knowledge. They showed that stomata of Polypodium vulgare and Valerianella locusta could respond to both ea and ei, and that the response to ei was more sensitive than that to ea. Our data confirm their findings, and we extend their findings with several new results and a different interpretation. In our experiments, a decrease in vapour pressure for either surface caused a reduction in stomatal aperture, and an increase in vapour pressure caused an increase in stomatal aperture. The effects of ea and ei on stomatal responses in isolated epidermes are clearest in experiments with dry epidermes (Figs 2–4), which show an immediate, reversible response the kinetics of which are approximately first order. Decreases in ea cause an increase in the vapour pressure difference (VPD) across the epidermis, and the resulting decrease in stomatal aperture is consistent with stomatal responses to a decrease in ambient humidity at a constant leaf temperature. Decreases in ei at a constant epidermal temperature result in a decrease in VPD, and the resulting decrease in stomatal aperture might seem inconsistent with the stomatal response to VPD in a leaf. However, it is generally accepted that the air inside the leaf is saturated at the temperature of the leaf, so ei in a leaf is determined by leaf temperature. In an isothermal leaf, it would therefore be impossible to decrease ei without also decreasing leaf and epidermal temperatures. Because this would decrease es as well, it is impossible to change ei in a leaf without changing es. Therefore, the situation in which ei was varied at constant epidermal temperature (Figs 3 & 4) cannot be duplicated in an isothermal leaf. Similarly, the experiment shown in which epidermal temperature was varied while holding ea and ei constant (Fig. 9) could also not occur in an isothermal leaf. If, however, epidermal temperature is not assumed to be equal to the mesophyll temperature, then ei and ep could vary independently of es, and this could have important implications for stomatal responses to humidity and temperature in leaves.
Our data also demonstrate that stomata respond to humidity in isolated epidermes with or without living or turgid epidermal cells. To maintain live, turgid epidermal cells, we found it necessary to start experiments with liquid water present on the epidermis. This was because epidermal cells plasmolysed at a Ψ of about −0.8 MPa, which corresponds to a vapour pressure so close to saturation that it was impossible to maintain without condensing water on the chamber walls. The data shown in Fig. 7a,b suggest that the initial increase in stomatal aperture following a decrease in ea was caused by loss of epidermal or subsidiary cell turgor, which reduces the back pressure on the guard cells and allows the pore to open (Buckley 2005). This is supported by the fact that the wrong-way response was not observed for any experiment in either species in which both the epidermal and subsidiary cells were flaccid when the experiment was started (i.e. Figs 2–4), and by the observation that the initial increase in aperture was correlated with visible plasmolysis of the epidermal cells. In V. faba, the wrong-way response was only observed for stomata that were surrounded by live epidermal cells, but in T. pallida, the wrong-way response was observed for stomata with live or dead epidermal cells. The latter finding is probably due to the fact that the subsidiary cells were still alive in these epidermes. The stomatal response to a change in ea was slower in wet-epidermis experiments than in dry-epidermis experiments. This was probably caused by water on the epidermis humidifying the air adjacent to the epidermis, which made it impossible to lower the vapour pressure adjacent to the epidermis in a step change.
Lange et al. (1979) interpreted their data with isolated epidermes to mean that the guard cells were hydrating and dehydrating entirely by vapour-phase transfer of water. Our data are consistent with this interpretation. It seems very unlikely that guard cells were continuously losing water in vapour phase and gaining water in liquid phase from the surrounding epidermal tissue for several reasons. Firstly, the epidermes were exposed to dry air at the beginning of each experiment. Initially, epidermal temperature dropped in response to dry air and the cell wall cavities of dead epidermal cells were visibly filled with water. After the dry air treatment, epidermal temperature was no longer affected by decreases in humidity, and the water in the cell wall cavities of dead epidermal cells had been replaced with air. These observations suggest that very little water was left on the epidermis, and it seems unlikely that sufficient water remained in the epidermal tissue to support water loss from the guard cells over the several hours required for each experiment. More convincingly, though, the effect of helox on stomatal apertures (Fig. 5) is inconsistent with a steady state involving water evaporation from the guard cells and liquid water transfer from the epidermal cells. Because water vapour diffuses 2.33 times faster in helox than in normal air (Parkhurst & Mott 1990), the rate of water exchange between the guard cells and the vapour phase should have been 2.33 times higher in helox than in air. This is supported by the fact that stomata responded approximately 2.33 times more rapidly to a decrease in ea in helox than in air (Fig. 5). However, the rate of liquid water transfer from the epidermal cells to the guard cells would be unaffected by helox. Thus, if the stomatal responses shown in this study were the result of a steady state involving water evaporation from the guard cells and liquid water transfer from the epidermal cells, then guard cell Ψ, and therefore stomatal aperture, should have declined in response to helox. Because aperture was unaffected by helox, but the kinetics of the response were affected, we conclude that stomata hydrated and dehydrated primarily through the vapour phase in these dry epidermes. The difference between the response of stomata to helox in leaves (Mott & Parkhurst 1991) and that in isolated epidermes is striking. To be sure that this difference was not the result of differences in plant material between the two studies, we tested the response of stomata to helox in the T. pallida leaves used in this study and found it to be consistent with the responses reported for Glycine max, V. faba and Phaseolus vulgaris (Mott & Parkhurst 1991) (i.e. stomata closed when air was replaced with helox at a constant ea).
Lange et al. (1979) interpreted their data to mean that guard cells were hydrating from the inside surface and dehydrating through the outside surface (through the cuticle). While there can be no doubt that some water is being lost through the cuticle, it is unclear to what extent the loss of water through the cuticle contributes to the stomatal response to ea. Experiments in which the lower epidermis was coated with silicone grease before isolating it from the mesophyll (Fig. 6) suggest that water exchange through the cuticle is probably small in comparison to water exchange through other areas of the guard cell. If the stomatal response to ea was caused by water loss from the cuticle, then reducing water loss from the cuticle should have altered the stomatal response to ea. Because apertures changed by approximately the same amount in the greased epidermes and in the controls, we suggest that stomatal responses to ea in this study were not caused by changes in cuticular water loss. Although it could be argued that the silicone grease did not substantially alter the rate of water loss through the cuticle, stomata that were occluded with silicone grease did not respond at all to changes in ea, suggesting that the silicone grease did substantially reduce water vapour movement.
We suggest that in the dry epidermes used in this study, the guard cells were in near equilibrium with the air in the stomatal pore. The response of aperture to parallel changes in ei and ea and the response of aperture to changes in epidermal temperature are consistent with equilibrium between ep and the guard cells (Fig. 10). The data for varying epidermal temperature show slightly smaller apertures than those for parallel changes in ea and ei or for osmotic solutions. This difference arises because stomata in different epidermes show slightly different average apertures when opened on KCl solutions. In addition, as discussed earlier, responses of aperture to helox support an equilibrium mechanism rather than a steady-state mechanism.
From the difference in sensitivity between ea and ei, we deduce that the point at which guard cells equilibrate with the air in the pore (Ψp) must be closer to the bottom of the pore than to the top. The vapour pressure of the air in the pore at the point of equilibrium (ep) can be determined by ea and ei as follows:
where σ = Ri/(Ri + Rp) is the ratio of the resistance to water vapour diffusion in the internal space between evaporating site and pore to the total resistance of water loss from the leaf (Fig. 10); σ therefore measures the sensitivity of guard cells to external humidity. At equilibrium, therefore, the water potential of the guard cell is given by Eqn 1. Because RT/vL is about 140 MPa in the temperature range of interest, Ψp is extremely sensitive to relative humidity. Based on the data shown in Fig. 8, we estimate that guard cells became flaccid when Ψg reached −7.0 MPa, which corresponds to a value of 0.92 for ep/es. Assuming that guard cells lost approximately 25% of their volume as they went from full turgor to zero turgor (Franks et al. 2001), then their osmotic pressure at full turgor (Ψ = 0) would have been approximately 5.0 MPa, which means that turgor pressure at maximum aperture would also have been approximately 5.0 MPa. This number is about 25% higher than the pressure values necessary for full aperture in Tradescantia virginia and V. faba (Franks, Cowan & Farquhar 1998), but this difference is consistent with the larger apertures observed in this study. This change in turgor implies a wall elasticity coefficient of 23 MPa, which is consistent with literature data for guard cells of V. faba (Franks et al. 2001).
Although it seems probable that stomatal responses to ea and ei in isolated epidermes are caused by water vapour exchange between the guard cell and air in the stomatal pore, the role of this water vapour exchange in leaves is unclear. There is no reason to assume that water is not exchanged between the guard cells and air in a leaf, so this process needs to be considered in any investigation of stomatal responses to humidity. Although it is possible that the response in leaves is the result of a near equilibrium in water potential between the air in the stomatal pore and guard cells, this seems unlikely for two reasons. Firstly, it would require that guard cells are completely hydraulically isolated from the epidermis, and there is no anatomical evidence to support this idea. Secondly, in contrast to isolated epidermes, stomata in leaves close in response to helox (Mott & Parkhurst 1991). This suggests that the stomatal response in leaves is caused by an increase in water loss from some point in the leaf, rather than by an equilibrium process. It seems more plausible, therefore, to postulate that water moves from the epidermis to the guard cells and from the guard cells to the air in response to water potential gradients. Given the rapid hydration and dehydration kinetics of guard cells in isolated epidermis, it seems plausible that the dehydration of guard cells to the air in the pore might play a role in the stomatal response to humidity in leaves.
In summary, this study demonstrates that stomatal guard cells can hydrate and dehydrate rapidly through the vapour phase, and that this process can produce stomatal responses to ambient humidity in isolated epidermes that look similar to those in leaves. Our data further suggest that stomatal responses to humidity in isolated epidermes are the result of Ψ equilibrium between the guard cells and the air near the bottom of the stomatal pore. Although water vapour exchange between guard cells and the air is likely to occur in leaves, it is unlikely that stomatal responses to humidity in leaves are the result of similar equilibrium. Thus, the role of water vapour exchange between the guard cells and the air in stomatal responses to humidity in leaves remains unclear.
We thank Rand Hooper, Jason Jacobs, Erik Sibbernsen and Benjamin Sharples for excellent technical assistance. K.A.M. thanks Joseph Berry for many useful discussions on these ideas and for the first suggestion that guard cells might be in equilibrium with the vapour phase. This work was supported by grants from the National Science Foundation to K.A.M and D.P.