Humidity in a small area of a Vicia faba L. leaf was perturbed with a flow of dry air from an 80 µm (inside diameter) needle, while the remainder of the leaf was maintained at high and constant humidity. The influence of the needle flow on the humidity at the leaf surface was quantified by using a spatially explicit dewpoint hygrometer to observe condensation patterns. When the dry air from a needle was applied to the leaf, stomata within the influence of the needle opened within the first few minutes of the perturbation, and local epidermal turgor pressure declined within the same time frame. When the needle flow was removed from the leaf, these responses were reversed, but with more variable kinetics. Stomata and epidermal cells outside the influence of the needle flow, which were exposed to a constant and high humidity, showed similar, but smaller, responses when the needle flow was applied to the leaf. Since the opening of these stomata should have had only a small effect on transpiration (because of the high humidity), it is likely that the reduction in epidermal turgor was the cause (rather than the result) of the stomatal opening. The magnitude of the turgor response was only loosely related to the distance from the needle flow up to distances of almost 400 µm. The data support the idea that neighbouring stomata can interact through the influence of transpiration on epidermal turgor.
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Measurements of leaf gas exchange are used to calculate conductance for gas diffusion through stomata. Although values of stomatal conductance for an entire leaf are often reported, considerable spatial heterogeneity in this parameter can exist for a single leaf. On the smallest scale, stomatal apertures have been shown to vary in an approximately random fashion (Laisk, Oja & Kull 1980; Gorton, Williams & Binns 1988; Weyers & Lawson 1997; Lawson, Weyers & A’Brook 1998), with the average aperture determining the measured value of stomatal conductance for a particular area of the leaf. On a larger scale, this heterogeneity can take a variety of forms, ranging from broad gradients in conductance across the entire leaf to more sharply defined patches of varying size that have different stomatal conductances to adjacent areas (Weyers & Lawson 1997; Mott & Buckley 1998). The latter phenomenon has been called patchy stomatal closure or patchy stomatal conductance.
Conductance patches can show complicated spatial and temporal patterns (Cardon, Mott & Berry 1994; Siebke & Weis 1995), with individual patches showing oscillatory, stable or unpredictable behaviours. These behaviours imply the existence of both mechanisms that coordinate the behaviour of stomata within a patch and mechanisms for discoordinating the behaviour of different patches (Mott & Buckley 1998). Hydraulic interactions among stomata have been proposed as a mechanism for the coordination of stomata within a patch (Haefner, Buckley & Mott 1997). These proposed hydraulic interactions depend on the interactions between epidermal turgor, stomatal aperture and transpiration. Specifically:
3an increase in stomatal aperture causes an increase in transpiration rate, and therefore causes a reduction in epidermal turgor.
These relationships can be used to construct a mechanism by which movements of stomata could be coordinated within an areole. If, under constant environmental conditions, the aperture of one stoma were to increase while the apertures of other stomata in the areole initially remained constant, there would be a reduction in water potential and turgor pressure of the epidermal cells near the stoma that opened. This reduction in water potential and turgor would be transmitted to the surrounding epidermal cells, causing nearby stomata to open slightly. This in turn would increase the transpiration rate from the nearby stomata, further lowering the epidermal turgor, and thereby propagating the effect. The magnitude of the effect is limited because the effect of epidermal turgor on stomatal aperture is smaller than the effect of stomatal aperture on epidermal turgor – otherwise once a stoma began to open, it would continue to open until epidermal turgor reached zero.
Evidence for such ‘hydraulic coupling’ among stomata has been provided by two studies thus far (Mott, Denne & Powell 1997; Mott, Shope & Buckley 1999). In one of these studies, the time for stomata to overcome epidermal backpressure during light-induced opening was assessed at high and low humidities (Mott et al. 1999). In the other study, the humidity of a small area of a leaf was perturbed, and the response of stomata adjacent to this area was measured (Mott et al. 1997). That study showed that the adjacent stomata, for which there was no perturbation in humidity, responded in the same direction as those for which humidity was perturbed. Although the responses of adjacent stomata were consistent with the proposed hydraulic coupling mechanism, no direct evidence for hydraulic interactions was obtained. The present study provides this evidence by showing that reductions in turgor pressure caused by local decreases in humidity can be propagated along the epidermis and are correlated with movements of stomata that do not experience the decrease in humidity.
Materials and methods
Vicia faba L. plants were grown in temperature-controlled greenhouses with day and night temperatures of 30 and 20 °C, respectively. Supplemental lighting from high-pressure sodium lamps was used when necessary to extend the day length to 16 h. Plants were grown in 1 L pots containing a sterile soilless media (1 : 1 : 1, perlite : vermiculite : peat moss), which was watered to excess daily with a nutrient solution containing 9·1 mm N, 1·8 mm P, 2·7 mm K and 11 µm chelated Fe (Peter’s 20–10–20, Grace Sierra Horticultural Products Co., Milpitas, CA, USA). Mature leaves that were not senescing were chosen for measurement.
The overall approach in these experiments was similar to that used in a previous study (Mott et al. 1997). Leaves to be used for measurements were clamped to the stage of a compound microscope using a metal plate covered with closed cell neoprene foam. The metal plate had a hole (2·5 cm diameter) in the centre, through which the leaf could be viewed with the microscope. A 32× long-focal-length objective was used to allow experimental manipulations on the leaf surface. A 2·0 L min−1 gas stream containing 21% O2, 79% N2 and 0·0036% CO2 was mixed from compressed pure gases and humidified to different levels (noted for each experiment) by bubbling through water. The dewpoint of this gas stream was determined with a dewpoint hygrometer (DEW-10; General Eastern, Woburn, MA, USA) and was recorded once a second with a datalogger (CR-21X; Campbell Scientific, Logan, UT, USA). This gas stream, hereafter called the ‘primary stream’, was passed over the leaf surface using a 0·5 cm (inside diameter) tube that was mounted at a shallow angle approximately 1 cm from the area under the microscope objective. To perturb a small region of the leaf, a second gas stream of approximately 0·01 L min−1 was applied to the leaf from a 80 µm (inside diameter) needle that was positioned at a 45° angle, almost touching the leaf surface. This gas stream was of the same composition as the primary stream, except that it was not humidified, and its water content was therefore constant at 0·76 mmol mol−1. The needle could be raised away from the leaf surface when necessary. The leaf temperature just outside the area influenced by the needle stream was measured with a fine-wire chromel constantan thermocouple and recorded every second by the datalogger.
The influence of the needle stream on the humidity at the leaf surface was determined as described in a previous study (Mott et al. 1997). Briefly, the primary and needle flows were applied to a spatially explicit dewpoint hygrometer, which consisted of a glass cover slip mounted to an aluminium block with heat sink compound. Water from a temperature-controlled bath was circulated through water channels in the aluminium block to control its temperature, and light was applied laterally to increase the visibility of water droplets on the surface. To determine humidity isobars on the surface of the hygrometer, the humidity of the primary stream was set to 27·8 mmol mol−1 with the flow initially turned off, and the needle stream was lowered to the surface of the hygrometer. The primary stream was then turned on briefly while the patterns of condensation were observed through the microscope. When the patterns of condensation stabilized (within a few seconds of turning on the primary stream), a digital image was captured, and the primary stream was turned off to allow the condensation to evaporate. By changing the temperature of the block in 1°C increments and tracing the extent of condensation, a series of humidity isobars were determined to show the effect of the needle stream. Selected isobars are shown in Fig. 1.
A cell pressure probe was used to monitor epidermal turgor at various distances from the area perturbed by the needle flow. The position of the probe relative to the needle was recorded by capturing digital images. Stomatal apertures were determined by capturing digital images at regular intervals and measuring aperture with image processing software as described previously (Mott et al. 1997).
The effect of the needle stream on the humidity at the surface of the spatially explicit dewpoint hygrometer (see Materials and methods) is shown in Fig. 1. Numbers beside the isobars show the humidity as a percentage of the total difference between the needle stream (dry) and the primary stream (humid). Percentages higher than 63% were impossible to determine because the dryness of the needle stream (0·76 mmol mol−1) made condensation temperatures too low. The points a and b indicate the positions of the stomatal aperture and epidermal cell turgor measurements described below. Each experiment was performed on a different leaf.
To be certain that stomatal apertures outside the influence of the needle stream were reacting to the needle stream in a manner consistent with previous results, stomatal apertures were measured over time after lowering the needle to the surface of the leaf for 16 min. Two experiments using different leaves were conducted, and the results are shown in Fig. 2. In both experiments, the mole fraction gradient for water vapour between the leaf and air (Δw) in the area unaffected by the needle stream was very small (4·3 and 0·1 mmol mol−1, respectively), and in both experiments the measured stoma was located at approximately point b shown in Fig. 1. When the needle was lowered to the leaf (time zero), stomatal apertures increased rapidly by about 1 µm over a period of several minutes. When the needle was raised away from the leaf surface, stomatal apertures declined, but they did not reach the original aperture within the time they were tracked. Stomatal apertures under the needle showed larger responses (data not shown, but see Mott et al. 1997 for examples).
To test whether changes in epidermal turgor pressure were responsible for the stomatal movements observed in Fig. 2, turgor pressure was measured for epidermal cells in areas of the leaf that were either within the influence of the needle stream or at various distances away from the influence of the needle stream. Most of these experiments were performed with the humidity of the primary stream very high, for reasons that are explained in the discussion section. However, a few experiments were performed with the humidity for the primary stream at lower levels. In many experiments, the seal between the pressure probe and the cell was lost as turgor responded to the imposition of the needle flow. However, a total of 23 experiments were performed in which turgor pressure was successfully monitored for at least 5 min after the needle was lowered to the leaf. In five of these experiments, the seal between the probe and cell lasted long enough to remove the needle flow and track pressure for another 5 min or so. Figures 3 and 4 show data from two such experiments. In the experiment shown in Fig. 3, the probe was inserted in an epidermal cell at position b, and Δw for the area of leaf not affected by the needle stream was 1·3 mmol mol−1. When the needle was lowered, epidermal turgor pressure dropped rapidly over a period of several minutes and stabilized at a value approximately 0·1 MPa lower than the starting value. When the needle was raised after about 10 min, pressure returned to its original value with similar kinetics. Figure 4 shows the response of epidermal turgor at position a when the Δw for the area of leaf influenced by the primary stream was 4·7 mmol mol−1. Although the Δw for the area under the needle was 32 mmol mol−1, the Δw for position a cannot be known precisely because of limitations in determining the isobars shown in Fig. 1– but the Δw was at least 20·2 mmol mol−1 (63% of the maximum value directly under the needle). Again, epidermal turgor pressure dropped rapidly following the application of the needle flow, and recovered when the needle flow was removed. Note that turgor pressure dropped by almost 0·3 MPa in this experiment, compared to approximately 0·1 MPa for position b. In both of these experiments, turgor pressure recovered almost completely when the needle flow was removed, but in general the magnitude and kinetics of this recovery were more variable than the initial decline.
To investigate the attenuation of this turgor effect with distance, the change in turgor pressure 3 min after the needle was lowered was plotted against the distance from the needle for all 23 experiments (Fig. 5). Only data for which the Δw outside the needle flow was below 5 mmol mol−1 are included in this figure. The distance from the needle was measured as the orthogonal distance from the line defined by the front of the needle, as illustrated in Fig. 1. All but one experiment showed a reduction in turgor pressure associated with the application of the needle flow, and none of the experiments showed an increase in turgor pressure. Although there was a great deal of scatter in the data, linear regression gave a negatively sloped line with an r2 value of 0·17.
The experiments described here are similar to those reported earlier (Mott et al. 1997), in that the humidity for a small area of a leaf was perturbed using a stream of gas emitted from a needle that was very close to the leaf surface. As in the previous study, the effect of the needle gas stream on the spatial patterns of humidity at the leaf surface was estimated by examining condensation patterns on a smooth, temperature-controlled surface, which we have termed a spatially explicit dewpoint hygrometer. In this study, however, the needle gas was dry and the main flow was humid, whereas in the previous study the situation was reversed. This change was made primarily because of the technical difficulty in accurately determining the humidity of the very small needle flow. Because the needle flow was extremely dry (0·76 mmol mol−1, the humidity of the tank gas), it was impossible to determine humidity isobars corresponding to the very low humidities near the tip of the needle because the spatially explicit hygrometer could not be cooled enough to condense water from such dry air. This was not a problem in this study because we were primarily interested in determining the extent of needle flow influence so that we could identify epidermal cells and stomata that were not influenced by the needle flow.
The general shape and size of the humidity isobars determined in this study (Fig. 1) are similar to those in the previous study. For example, in this study, the effect of the needle flow was reduced to 63% at about 150 µm from the tip of the needle, whereas in the previous study, this value occurred around 100 µm from the tip of the needle. The effect of the needle flow on ambient humidity was reduced to 15% at about 250 µm in both studies. In this study, the isobars were slightly wider than in the previous study and they were slightly skewed away from the direction of the primary flow, whereas in the previous study, the isobars were almost perfectly symmetrical. In addition, the effect of the needle flow extended between 50 and 100 µm behind the tip of the needle, whereas in the previous studies, the isobars did not extend behind the needle appreciably. These small differences may have been caused by the differences in technique (i.e. reversing the humidity gradient between the needle and primary flows). Alternatively, they may have been caused by differences in the positioning of the primary flow or differences in needle flow rate, needle angle and needle position. These aspects of the experiment were kept constant within each study but not between the studies. The usefulness of these isobars in predicting the humidity for the leaf surface is open to question, but V. faba has a glabrous epidermis and it seems reasonable to assume that the isobars for the leaf are similar to those determined with the spatially explicit hygrometer.
When stomata are exposed to a sudden decrease in atmospheric humidity, transpiration rate increases immediately. This increase in transpiration causes a reduction in epidermal turgor (Shackel & Brinckmann 1985; Nonami & Schulze 1989; see also Fig. 5), reducing the backpressure on the guard cells and causing the stomatal pore to widen (Cowan & Farquhar 1977; Schulze 1994). This ‘wrong-way’ response to humidity is well-documented in the literature (e.g. Lange et al. 1971; Kappen, Andresen & Losch 1987; Kappen & Haeger 1991), and is usually followed by a slower and larger response in the correct direction, i.e. stomata close in response to a decrease in humidity. In this study, the needle stream was not applied to the leaf long enough for stomata to begin to close in response to the decrease in humidity, but, consistent with the above explanation, turgor pressure declined for epidermal cells within the influence of the needle flow when the dry needle flow was applied to the leaf surface (Fig. 5).
Stomata that were outside the influence of the needle stream also showed a rapid and almost immediate opening response (Fig. 2), despite the fact that they experienced no change in humidity. These data are shown to verify that the stomata outside the influence of the needle stream were opening, as was demonstrated in a previous study (Mott et al. 1997). It is, however, noteworthy that in this study the response occurred when Δw for the observed stoma was very low (i.e. primary flow humidity was very high). At low values of Δw, the opening of these stomata should have very little effect on their transpiration rate, and therefore should have little effect on the turgor of surrounding epidermal cells. Nevertheless, these surrounding epidermal cells showed rapid decreases in turgor pressure in response to the needle flow, and these decreases in turgor occurred concurrently with the opening of the adjacent stomata (Figs 2, 3 and 4). The most parsimonious explanation for this result is that the reduction in turgor pressure for epidermal cells outside the influence of the needle was caused by the increase in transpiration rate for stomata within the influence of the needle flow, and that the opening of stomata outside the influence of the needle was caused by the reduction in turgor pressure of the surrounding epidermal cells.
An alternative explanation for these data is that some sort of chemical or electrical signal was produced in the area influenced by the needle flow and caused the stomata outside the influence of the needle to open. Then, as these stomata opened, they caused the local epidermal turgor pressure to drop. In this explanation, therefore, the drop in epidermal turgor pressure outside the influence of the needle would have been caused by the increase in stomatal aperture rather than vice versa. This explanation is unlikely because for the very low primary flow Δw values in these experiments, the opening of stomata outside the influence of the needle would have had a very small effect on transpiration and thus on epidermal turgor.
The scatter in the relationship between turgor change and distance from the needle (Fig. 5) is somewhat surprising. It is possible that this scatter reflects differences in water transport properties along the epidermis among the individual experiments. Although major veins were avoided, the minor veins in V. faba are surrounded by mesophyll and were difficult to locate using the microscope set up for the pressure probe. Thus, no attempt was made to avoid minor veins, and no attempt was made to orient the leaf or probe in a particular direction for each experiment. The effective hydraulic conductance along the epidermis could therefore have varied among experiments based on the proximity and orientation of nearby minor veins and could have influenced the propagation of the turgor change.
In summary, these data support the hypothesis that neighbouring stomata can interact through the effects of transpiration on epidermal turgor pressure. These interactions will be stronger at low humidities than at high humidities (Mott et al. 1999), and could serve to coordinate the movements of stomata in a localized region of a leaf. We speculate that such coupling occurs regularly when humidity is moderate or low, but the emergence of distinct conductance patches requires that the movements of one locally coordinated patch become discoordinated from those of other patches. The latter criterion occurs only under certain environmental conditions, and hence patchy stomatal conductance occurs only under these conditions. Although not always resulting in patchy stomatal conductance, the existence of such coupling among stomata may have important implications for the regulation of stomatal conductance in intact leaves.
We thank Rand Hooper for excellent technical assistance.
Received 20 June 2000;received inrevised form 30 January 2001;accepted for publication 1 February 2001