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The response of stomata to changes in humidity for a single surface of an amphistomatous leaf was investigated in Xanthium strumarium and Vicia faba using gas exchange and direct observation of stomatal apertures. The stomatal response to humidity for a given surface was found to be the same whether or not the humidity for the opposite surface was changed concurrently. Stomata on the surface for which humidity was constant showed no response to changes in humidity for the opposite surface. Despite large changes in epidermal turgor on the surface for which humidity was changed, there was no change in epidermal turgor for the surface with constant humidity. Measurements of transpiration and epidermal turgor as functions of the mole fraction gradient of water between leaf and air were used to calculate a value for leaf hydraulic resistance. The results suggest that in these species, the mechanism for the stomatal response to humidity resides in the epidermis or the mesophyll very close to the epidermis, and that most of the hydraulic resistance of the leaf occurs between the xylem and the evaporating sites.
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Transpirational water loss creates a water potential gradient from the soil to the evaporating sites in leaves. The magnitude of this gradient has important effects on the physiology of the plant. Low water potentials in the leaf result in stomatal closure and restrict photosynthetic rate. Furthermore, low water potentials in the xylem can lead to cavitation, which decreases the capacity of xylem to supply water to the leaves (Sperry 2000). Using an Ohm's Law analogy, it can be seen that for a given transpiration rate (E), the magnitude of the water potential difference between the soil and the evaporating site in the leaf is determined by the resistance to water flow through the plant (Rp). Although Rp is determined by the entire pathway for water through the plant, it is clear that a substantial portion of this resistance is located in the leaf (Sack & Holbrook 2006), and correlations between leaf hydraulic architecture and leaf physiology have been widely studied (e.g. Sack, Cowan & Holbrook 2003; Brodribb et al. 2005; Sack, Tyree & Holbrook 2005).
The hydraulic resistance of the leaf (Rl) has been measured in several ways (Sack et al. 2002), but all of these techniques ultimately rely on measurements of water flow and water potential gradient between two points. The first of these points is the water potential of the xylem at the base of the leaf; the second point is the water potential at or near the evaporating site. There is reasonable consensus on the values for Rl and how they vary among different life forms, but there is considerable controversy over where most of the resistance to water flow within the leaf is located (Sack & Holbrook 2006). Some studies have suggested that most of the resistance is outside the xylem (Rox) (Cochard, Nardini & Coll 2004), while others have suggested that the resistance of the xylem (Rx) is of a similar magnitude (Sack, Streeter & Holbrook 2004; Sack et al. 2005). Rx : Rox was shown to be greater than one for sun species and less than one for shade species (Nardini, Gortan & Salleo 2005), and Rox has been found to vary with irradiance (Tyree et al. 2005) and time of day (Nardini, Salleo & Andri 2005). The current consensus is that the two resistances are of similar magnitude, and that the ratio varies among species and with experimental conditions (Sack & Holbrook 2006).
It is also unclear where the evaporating sites within the leaf are located, and what pathway water takes to arrive at the evaporating sites. Epidermal turgor pressure shows large and rapid changes in response to changes in transpiration (Shackel & Brinckmann 1985; Mott & Franks 2001), and it is commonly assumed that the epidermis is the site of evaporation or is in close hydraulic contact with the site of evaporation (Buckley 2005). Because epidermal turgor affects stomatal apertures, it has important implications for stomatal responses to humidity. For example, when humidity is lowered, transpiration immediately increases and this draws down the water potential of the epidermis. The decrease in epidermal turgor pressure relieves backpressure on the guard cells and allows the stomatal pore to open. This rapid response is in the opposite direction to the slower steady-state response, and is therefore often called the ‘wrong-way’ response.
The previous discussion raises some interesting questions for water pathways in leaves with stomata on both surfaces (amphistomatous leaves) and for the effects on stomatalconductance. It seems likely that the resistance network in an amphistomatous leaf will approximate that shown in the following diagram:
where Ψx and Ψ0 are the water potentials of the leaf xylem, and some arbitrary point upstream from the leaf, respectively, and Ψue and Ψle are the water potentials of the upper and lower epidermes (or evaporating sites), respectively. This diagram represents the situation at steady-state and does not account for water storage.
From this resistance network, it can be seen that if humidity is decreased for the upper surface while maintaining humidity constant for the lower surface, there are two general possibilities for the effect on the epidermal turgor of the lower surface assuming Rx and Rox are constant during the experiment. These two possibilities depend on the relative magnitudes of Rx and Rox. For both possibilities, decreasing the humidity for the upper surface will increase the transpiration rate for that surface and lower its epidermal turgor. If, in the first possibility, most of the resistance to water transport is in the xylem (i.e. Rx is large compared to Rox), then most of the water potential drop will occur between Ψ0 and Ψx, and Ψx will decrease in response to a decrease in Ψe for the upper surface. If E is constant for the lower surface (because humidity is constant), then the difference between Ψx and Ψle must stay constant. Thus, the decrease in Ψx will have a proportional effect on Ψle, and this should cause an effect on the stomatal conductance of the lower surface. On the other hand, if Rox comprises most of the Rl (i.e. if Rox is large compared to Rx), then, decreasing the humidity on the upper surface should lower the Ψue with only a minimal effect on the Ψx. This should produce only a small effect on Ψle and stomatal aperture for the lower surface. Intermediate ratios of Rx and Rox will have intermediate effects, and the same line of reasoning can be applied to changes in humidity for the lower surface while maintaining the humidity for the upper surface constant.
In this study, humidity was changed for one surface of an amphistomatous leaf and was maintained constant for the other surface. The effects of this treatment on the stomatal conductances and water relations for the two surfaces were measured and interpreted within the theoretical framework discussed previously to provide insight into hydraulic resistances in leaves and potential mechanisms for the stomatal response to humidity.
MATERIALS AND METHODS
Plants were grown in a controlled environment greenhouse as described previously (West et al. 2005). Gas-exchange measurements were made using two standard, single-pass gas-exchange systems that have also been described previously (West et al. 2005). The two gas-exchange systems were used to control two leaf chambers; one for the upper surface of the leaf, and one for the lower surface. The leaf chambers were clamped to the leaf with the leaf forming the barrier between them. The temperature of each chamber was controlled with a water bath that circulated water through the walls of the chamber, and the gas in the chamber was stirred using small fans. Leaf temperature was measured with a fine wire thermocouple pressed to the underside of the leaf, and was maintained at 25 ± 0.2 °C by adjusting air temperature.
For all gas-exchange experiments, photosynthetic photon flux density (PPFD) was held constant at 1200 µmol m−2 s−1, [CO2] was held constant at 360 µmol mol−1, and [O2] was held constant at 21 mmol mol−1. The mole fraction of water in air (w) was varied in the separate gas streams by adjusting the ratio of dry gas to humidified gas, and measuring the resultant gas stream with a dewpoint hygrometer.
In most experiments, the leaf was clamped between two rectangular chambers as described by West et al. (2005). In some experiments, however, the leaf was clamped between two circular (6.67 cm diameter) chambers that were surrounded by a larger, circular outer chamber (20 cm diameter). The leaf formed a complete barrier between the upper and lower halves of the inner chamber, but not the outer chamber. This resulted in three chambers: two central chambers (one for each surface of the leaf) and one outer chamber (enclosing both surfaces of the rest of the leaf). The experiment was begun by allowing the leaf tissue in all three chambers to reach a steady state at Δw of 10 mmol mol−1. For the two central chambers, the difference in water vapour mole fraction between the ingoing and outgoing gas streams was measured, allowing the calculation of transpiration and stomatal conductance for these chambers. However, for the large outer chamber, only the outgoing water mole fraction was measured, so only Δw could be calculated.
Direct observations of stomatal aperture were made by clamping a leaf to a modified microscope stage and observing the stomata with a long-focal-length objective. Light (PPFD = 600 µmol m−2 s−1) was applied to the adaxial surface of the leaf. This PPFD was lower than that used for gas-exchange experiments, but was still nearly saturating for photosynthesis (data not shown). It was necessary to use the lower PPFD to avoid overheating the leaf because there was no temperature-controlled chamber enclosing the leaf. Gas of the same composition as described previously was directed across each of the two leaf surfaces. The humidity of the gas streams for each surface could be controlled independently. Measurements of epidermal turgor pressure were made with a cell pressure probe as described previously (Mott & Franks 2001) using this same microscope system.
To estimate the hydraulic conductance to the evaporating site, two sets of parallel experiments were conducted in Vicia faba. In both sets of the experiments, a leaf was brought to a steady state with the difference in mole fraction of water between the leaf and air (Δw) between 7 and 8 mmol mol−1. Δw was then increased sequentially to approximately 11, 15 and 20 mmol mol−1. This protocol allowed us to obtain several data points at different values of transpiration (E) and Δw for the same leaf in a short period of time. The first set of experiments was carried out with the leaf mounted on the microscope stage. The pressure probe was inserted in an epidermal cell while Δw was between 7 and 8 mmol mol−1, and the pressure was recorded continuously as Δw was sequentially increased. The second set of experiments was carried out in a gas-exchange chamber, and the transpiration rate was recorded at each value of Δw. To make these values comparable with those of the pressure probe, measurements of E were taken after the wrong-way response of the stomata was complete, but before stomata began to close substantially in response to the change in Δw.
Stomatal responses to humidity in Xanthium strumarium and V. faba were first characterized using parallel changes in the humidity for both surfaces of the leaf. In these experiments, humidity is reported as the difference in the mole fraction of water between the intercellular air spaces of the leaf and the outside air (Δw). Thus, an increase in Δw represents a decrease in humidity. When Δw was increased in parallel for both surfaces, stomatal conductances for the two surfaces responded in parallel. Both surfaces showed a rapid, transient increase in stomatal conductance, followed by a slower decline to a steady-state value that was less than the starting value (Fig. 1, only the data for X. strumarium are shown; the data for V. faba were qualitatively similar). Decreasing Δw had the opposite effect.
When Δw was increased for one surface only, the stomatal conductance for that surface showed the same response as described previously, but the stomatal conductance for the other surface remained essentially unchanged (Fig 2a,b). Four replicate experiments were performed on each species, and in all cases, (1) the response of stomatal conductance for the surface with increased Δw was indistinguishable from the response of individual surfaces when Δw was increased for both surfaces; and (2) although small changes in conductance for the unperturbed surface were often observed, the response was not large enough to unequivocally distinguish it from experimental noise, and the direction of these small changes was not consistent among experiments.
In an effort to amplify the effect of the change in humidity on overall leaf water relations, Δw was increased for both surfaces of an entire X. strumarium leaf, except for a small area in the centre of the leaf where Δw was held constant for one surface (see Materials and Methods). At time zero, the Δw for the upper central chamber (i.e. for the upper surface of the central portion of the leaf) was increased from approximately 10 to 20 mmol mol−1. Also at time zero, the humidity for the outer chamber (serving both surfaces of the outer portion of the leaf) was lowered in parallel with the upper central chamber (as discussed in the Materials and Methods, Δw could not be precisely calculated for the outer chamber). The Δw for the lower central chamber (serving the lower surface of the central portion of the leaf) was maintained constant at 10 mmol mol−1. The net result of this manipulation was that Δw was increased for the entire leaf except for the lower surface of the central chamber. Stomatal conductance for the portion of the leaf in the upper central chamber showed a typical stomatal response to an increase in Δw, while that for the portion of the leaf in the lower central chamber showed no response (Fig. 3). This experiment was repeated twice for each surface, and although small changes in stomatal conductance for the surface with constant humidity were occasionally observed, the response was negligible compared with that on the surface for which humidity was changed.
To confirm that the value of stomatal conductance calculated from transpiration rate was an accurate indicator of stomatal aperture in these experiments, stomatal apertures on one surface of a leaf were observed directly with a standard transmission microscope, while Δw was altered for either the observed surface or the opposite surface (Fig. 4, the data shown are for V. faba; the data for X. strumarium were similar). The experiment was started by exposing both surfaces to Δw of 10 mmol mol−1 for several hours. At time zero, Δw for one surface was increased to approximately 25 mmol mol−1. Apertures of stomata on the surface for which Δw was constant were unaffected by the treatment. In contrast, apertures on the surface for which Δw was increased showed a rapid increase in aperture followed by a slower decline (Fig. 4).
Epidermal turgor pressure for one surface was unaffected by changes in humidity for the other surface (Fig. 5). When Δw for the upper surface was increased, the turgor pressure of the lower epidermal cells remained constant. The initial small excursion in the data associated with the change in humidity for the upper surface (Fig. 5) is an artifact caused by leaf movement associated with the shrinking of the upper epidermal surface in response to the change in humidity and transpiration for the upper surface. When Δw for the lower surface was subsequently increased, the turgor pressure of the lower surface showed a large and immediate decrease.
When Δw was increased sequentially to estimate the hydraulic conductance to the evaporating site, epidermal-cell turgor pressure decreased rapidly for a few minutes after each increase in Δw, and then became constant. This constant value of epidermal turgor pressure was found to be approximately linear with Δw (Fig. 6). Parallel experiments with gas-exchange data showed that the relationship between E and Δw was approximately linear with a slope of 0.286 mol m−2 s−1 (Fig. 7).
In both V. faba and X. strumarium, the stomatal responses to humidity were completely independent for the two surfaces. This conclusion is supported by two findings. Firstly, the large changes in humidity on one surface of the leaf had essentially no effect on the stomatal conductance of the other surface. Although small changes in conductance for the surface with constant humidity were occasionally observed, these were very rapid responses, and they were probably caused by small fluctuations in leaf temperature associated with the change in transpiration for the other surface. Secondly, the stomatal response to humidity for a given surface was the same regardless of whether or not the humidity was changed for the other surface. Indeed, this result held true despite attempts to magnify the effect by changing the humidity for both surfaces of the whole leaf while holding humidity constant for one surface in a small area in the middle of the leaf.
The complete insensitivity of stomata on one surface to large perturbations in the transpiration rate of the other surface is remarkable, given the physical proximity of the two surfaces (leaves of both species were less than 500 µm thick). Because of this, the gas-exchange results were verified with direct observations of stomatal apertures. Although stomatal conductance is considered a good indicator of stomatal aperture under most conditions, it could be argued that the large gradient in humidity across the leaf might somehow invalidate this assumption. This possibility was refuted by direct observations of stomatal apertures for one surface while humidity was changed on the other.
In addition to having independent humidity responses, the two surfaces of the leaf were also hydraulically isolated from one another. This can be deduced from stomatal response to humidity and from direct measurements of epidermal turgor. Because the initial rapid stomatal response to humidity is known to be caused by changes in epidermal turgor, the fact that stomatal conductance remained constant for surfaces with constant humidity despite the large changes in transpiration for the opposite surface suggests that epidermal turgor for the surface with constant humidity was not affected by changes in transpiration for the other surface. This conclusion was supported by direct measurements of epidermal turgor pressure using a cell pressure probe (Fig. 5).
Interpreting the results of this study within the framework of the resistance diagram discussed in the Introduction suggests that Rox must be very large compared with Rx, and that most of the water potential gradient in the leaf exists between the xylem and the evaporating site. This conclusion is supported by the fact that Ψx was apparently unaffected by large changes in E and Ψe for one surface. If Ψx had been affected by changes in E and Ψe for one surface, the change in Ψx would have affected the Ψe for the other surface. Because this was not observed in our experiments, most of the water potential drawdown caused by transpiration must occur between the xylem and the evaporating site. This analysis, of course, assumes that Rx and Rox are constant throughout the experiment. Rox has been shown to vary diurnally and with irradiance, it is also possible to envision changes in Rox occurring with the right kinetics and magnitude to produce the results described in this study.
By measuring Ψe and E as functions of Δw, and making a few assumptions, it was possible to estimate Rl (Rl = Rox + Rx). The first assumption is that the Ψ for the epidermis is close to that of the evaporating site. This is a common assumption for which there is an ample supporting evidence (Buckley 2005), and seems reasonable based on the kinetics of the initial wrong-way response to humidity (which is caused by changes in epidermal turgor). The second assumption is that reductions in Pe can be roughly equated with changes in Ψe. This assumption is supported by measurements of cell wall elasticity (ε) for epidermal cells (Steudle, Zimmermann & Luttge 1977; Cosgrove & Steudle 1981), which suggest that at moderate to high turgor pressures, most of the change in water potential will be due to turgor pressure changes with only minor contributions from changes in osmotic pressure.
Using the linear relationships between Pe and Δw, and between E and Δw (Figs 6 & 7), it was possible to develop a linear relationship between Pe and E. Thus,
The conductivity of the hydraulic pathway is the slope of the relationship between E and Pe (Eqn 3), and is equal to 15.7 mmol m−2 s−1 MPa−1. This value compares well with the range of 3.9–36 mmol m−2 s−1 MPa−1 given by Brodribb et al. (2005) for angiosperm leaves, and is close to the average for crop plants of 25 mmol m−2 s −1 MPa−1 given by Sack & Holbrook (2006).
In summary, several conclusions are possible based on the data presented in this paper. Firstly, in the species studied, the mechanism for the stomatal response to humidity resides in the epidermis (including the guard cells) and/or the mesophyll adjacent to the guard cells. Secondly, in V. faba and X. strumarium, the hydraulic pathways for the two surfaces are largely isolated from one another, and large perturbations in transpiration and water potential for one surface have essentially no effect on the water relations or stomatal conductance for the other surface. Thirdly, in these two species, most of the hydraulic resistance to transpiration resides between the leaf xylem and the evaporating sites. V. faba and X. strumarium are not closely related phylogenetically, but they both have thin, bifacial leaves. It seems possible that the last two conclusions will apply to many leaves with similar anatomy.
I thank Rand Hooper for his excellent technical assistance.