This Article Abstract Full Text (PDF) All Versions of this Article: 143/2/1068    most recent pp.106.089334v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Physiol. Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Kaiser, H. Articles by Legner, N. Search for Related Content PubMed PubMed Citation Articles by Kaiser, H. Articles by Legner, N. Agricola Articles by Kaiser, H. Articles by Legner, N. Related Collections Biology of Transpiration

WHOLE PLANT AND ECOPHYSIOLOGY

Localization of Mechanisms Involved in Hydropassive and Hydroactive Stomatal Responses of Sambucus nigra to Dry Air¹

Botanisches Institut der Christian-Albrechts-Universität, D–24098 Kiel, Germany

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
The response of stomata to a reduction of air humidity is composed of a hydropassive opening followed by active closure. Whereas the mechanisms behind the hydropassive opening are largely understood, the location and physiological basis of the sensing mechanisms leading to active closure are not yet known. This study attempts to evaluate the importance of a single pore's transpiration on its own response and that of adjacent pores. Selected stomata on attached intact leaves of Sambucus nigra were sealed with mineral oil and the response to a reduction of humidity was continuously observed in situ. Blocking a pore's transpiration had no appreciable effect on hydropassive opening and subsequent stomatal closure. If the adjacent stomata were additionally sealed, the closing response was reduced, but not the hydropassive opening. On the other hand, sealing the entire leaf surface, except a small area including the observed stomata, also reduced stomatal closure. These results indicate that strictly local processes triggered by a pore's own transpiration are not required to induce stomatal closure. To describe the effect of one pore's transpiration on the hydropassive and hydroactive responses of neighboring stomata, a simple spatial model was constructed. It suggests that 90% of the closing effect covers an area of approximately 0.5 mm², whereas the effect on hydropassive opening affects an area of approximately 1 mm². This divergence may suggest mechanisms other than or in addition to those involving changes of local leaf water potential.

This is mainly due to the fact that investigation of stomatal humidity responses must be done on leaves with unspoiled water relations. This excludes many of the recently developed methods of molecular and cell biology.

The typical stomatal response to a sudden decrease in air humidity is composed of a transient opening caused by hydraulic mechanisms, which is followed by a delayed closing movement (Kappen et al., 1987; Buckley and Mott, 2002). The initial hydropassive opening occurs because of the mechanical advantage (Sharpe et al., 1987; Franks et al., 1998) of the epidermal cells over guard cells. This leads to stomatal opening when guard cell and epidermis turgor equally decline. The subsequent closing response, however, is difficult to explain by purely hydraulic mechanisms. The most parsimonious explanation is an osmotically driven decrease in guard cell turgor. Although direct evidence for active closure is still lacking, it is supported by much circumstantial evidence (Buckley, 2005).

The search for possible mechanisms for stomatal closure in dry air has, for a long time, been motivated by the hypothesis of a so-called feed-forward response, which stands for a direct response to atmospheric humidity (Schulze et al., 1973; Farquhar, 1978).

Feed-forward responses produce a decrease in transpiration rate despite an increase in the driving gradient. To mechanistically explain the feed-forward response, a direct sensing of atmospheric humidity via transpiration through the external guard cell cuticle (peristomatal transpiration) was suggested (e.g. Cowan, 1977; Farquhar, 1978; Grantz, 1990). According to some authors, the guard cell cuticle is more permeable for water than the epidermal cuticle (Kerstiens, 1997a). Until now, it has not been shown convincingly that the effect of the rather small cuticular transpiration is sufficiently large to predominate over the much larger water losses through the stomatal pore. On the contrary, experimentally increased permeability of the cuticle did not sensitize stomata to dry air (Kerstiens, 1997b). Maier-Maercker (1983, 1999) used the term peristomatal transpiration in a broader sense, also including evaporation into the substomatal cavity, and suggests that total water loss from guard cells may trigger stomatal responses to dry air. Other mainly hydraulic explanations assume large hydraulic resistance for the flow to the evaporating site in the vicinity of the guard cells (Farquhar, 1978; Nonami et al., 1990; Dewar, 2002), leading to direct sensing of ambient humidity partly independent of whole-leaf water loss. All these hypotheses assume that water loss of guard cells triggers their responses to changing air humidity. Until now, there is no clear evidence favoring or disproving these different hypotheses, which claim that guard cells are the primary sensor for changes in transpiration rate.

The feed-forward hypothesis has been challenged by a reevaluation of previous reports of feed-forward responses (Monteith, 1995) and new data (Franks et al., 1997), which suggest that the decline of transpiration rate in spite of increasing humidity gradient is irreversible, possibly caused by water stress to the leaf. Thus, it is more appropriate to use the term apparent feed-forward response. Moreover, a number of studies suggested feedback control of transpiration, which keeps transpiration below a target value (Mott and Parkhurst, 1991; Franks et al., 1997). This could either involve localized sensing mechanisms, which are confined to the guard cells or their direct vicinity, or spatially distributed mechanisms involving other leaf tissues. Transpiration-dependent accumulation of substances in the apoplast of guard cells (Lu et al., 1997; Zhang and Outlaw, 2001) could form a locally confined sensing mechanism. Apoplastic solutes are assumed to be transported to the sites of evaporation in the guard cell apoplast and to become concentrated. This effect has been confirmed for Suc (Outlaw and De Vlieghere-He, 2001).

Much of the evidence derived from gas exchange and water status measurements points to control of stomatal responses by local leaf water potential (_l; Buckley, 2005). The precise location and nature of the putative sensing mechanisms, however, are not yet identified, but could involve mechanosensitive channels (Cosgrove and Hedrich, 1991; Garrill et al., 1996) or osmosensing (Yoshida et al., 2006).

Knowing the location of the involved mechanisms could enhance the understanding of transpiration sensing, but only little and mostly indirect evidence is available.

Microscopic observations of oscillating stomata revealed that stomata of Sambucus nigra with a spacing of 2 mm oscillated independently and with different frequencies, which resulted in a phase shift of individual responses (Kaiser and Kappen, 2001). This pointed to localization of the involved mechanisms of feedback control on a spatial scale smaller than 2 mm. The phenomenon of stomatal patchiness also helps to pin down the spatial scale of transpiration-sensing mechanisms because it relies on locally coordinated actions of stomata. Thermal imaging and imaging of chlorophyll fluorescence, which is taken as a measure of CO₂ supply to the leaf and corresponds to stomatal opening, suggests that stomata inside an areole may behave similarly (West et al., 2005). This could point to a distributed sensing mechanism, which coordinates neighboring stomata. Because direct microscopic observations are lacking, these methods, like other integrating methods, are not able to assess the actual degree of the proposed conformity. Consequently, they remain inconclusive with respect to the question of spatial localization of transpiration-sensing mechanisms.

Therefore, it seemed necessary to assess spatial aspects of humidity sensing on the level of single stomata by direct microscopic observation rather than by integrating methods. In previous, similar attempts, small streams of dry air applied by capillaries were used to modify the local transpiration rate (Lange et al., 1971; Mott et al., 1997; Mott and Franks, 2001). In contrast, we used the new method of applying small amounts of mineral oil, which, by capillary force, are sucked into the pores and, as a barrier of hydrophobic liquid, block transpiration. If processes driven by a pore's transpiration, which are located in the guard cells or in their immediate vicinity, were involved, this should lead to a decrease of the active closing response of oil-treated pores. Additionally, the inverse experiment was performed: Transpiration of the entire leaf was blocked by sealing it with adhesive foil, leaving only small areas to transpire. In the case of strictly local sensing mechanisms, relying on processes related to the transpiration stream through individual pores, the humidity response of stomata in these areas should be the same as before. These predictions were to be tested.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Assessment of the Effectiveness of Blocking Pore Transpiration and Possible Side Effects of Oil Treatment and Covering the Leaf with Adhesive Foil

To assess the effect of treating pores with oil on leaf conductance, the entire lower surface of a leaf was gently brushed with an oil-saturated wiper in the middle of the day when stomata had their maximal opening. Possibly not all stomata were sealed by this treatment. Nevertheless, leaf conductance immediately decreased from 107 to 17.5 mmol m^–2 s^–1, which is comparable to conductance in the middle of the night (16.5 mmol m^–2 s^–1) when stomata were closed.

To detect short-term effects of the application of mineral oil, stomatal movements were recorded while oil was applied with micropipettes under conditions of high humidity (leaf-to-air mole fraction of water vapor [_W] = 2 mmol mol^–1, photosynthetic photon flux density [PPFD] = 500 µmol m^–2 s^–1, [CO₂] approximately 360 µmol mol^–1). The stomatal aperture did not change during oil application and in the following hour (Fig. 1 ). One week after the application of oil to the stomatal pore and their surrounding pores, the stomatal degree of opening under identical light, CO₂, and humidity conditions (_W = 2 mmol mol^–1, PPFD = 500 µmol m^–2 s^–1, [CO₂] approximately 70 µmol mol^–1) at the same time of day was virtually unchanged (Fig. 2 ). To check whether fixing adhesive foil to the lower leaf surface causes damage, stomatal responses to reduced air humidity were observed before attaching the foil, with attached foil, and after careful removal (Fig. 5). After foil removal, the response was the same as before, which indicates that no permanent damage had occurred.

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Short-term responses of stomata to the application of mineral oil. Ten pores were continuously observed while filling them with a micropipette under conditions of high humidity (W = 2, PPFD = 500 µmol m–2 s–1, [CO2] approximately 360 µmol mol–1). Individual responses were aligned to the time of oil application (arrow). The average initial pore area was 75.3 µm2 ± 38.9 (SD). For better comparison, the aperture of each stoma in the moment of oil application was arbitrarily set to 100%.

View larger version (7K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Long-term effects of oil treatment. Twenty guard cell pairs were measured under identical high humidity conditions before and after oil treatment. Box and Whisker plots show the difference of circularity prior to and 1 week after oil treatment of the observed pores and all adjacent pores from a circular area with a diameter of 0.5 mm. The control group was left untreated.

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. The effect of restricting leaf transpiration to small areas of 0.5 mm2 on the responses of stomata to dry air. The graph shows the responses (mean ± SD) of nine stomata to an increase of W from 2 to 18 mmol mol–1 before and after attachment of perforated foil to the lower leaf surface and, finally, after removal of the foil. Measurements were performed on three consecutive days.

When leaves were subjected to a sudden decrease of air humidity, stomata showed the well-known initial transient hydropassive opening response, followed by partial or total active stomatal closure (Fig. 3 ). To extract the most important characteristics of the humidity response, the amplitudes of the hydropassive increase of circularity (c_pass) and actively regulated decrease of circularity (c_act) were calculated as shown in Figure 3. Treating single pores with oil had no detectable effect on the stomatal response to a switch from high to low atmospheric humidity. Both c_pass and the subsequent course of the active closing movement remained unchanged (Figs. 4 and 6A). The 95% confidence interval for the difference between the treated and the untreated state is –0.671% to 0.86% for c_pass and –1.582% to 1.645% for c_act, which shows that even if an effect exists, it is rather slight.

View larger version (12K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Typical stomatal response to a sudden increase in W from 2 to 18 mmol mol–1 (at time = 0). Arrows indicate the amplitude of hydropassive opening (cpass) and hydroactive closure (cact). cpass is defined as the difference between circularity prior to the humidity change and the maximal circularity (cmax). cact is the difference between cmax and the final circularity 1.5 h after the humidity change.

View larger version (25K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Stomatal response to dry air before and after sealing pores with oil. The graph shows responses of six stomata (mean ± SD) to an increase of W from 2 to 18 mmol mol–1 (vertical line) before (A) and after (B) sealing the pores with mineral oil (black symbols) compared to an untreated control group (white symbols).

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Effect of different spatial restriction schemes on the stomatal response to an increase of W. This plot compares the amplitudes of the hydropassive opening and the active closing response between stomata in their untreated (white bars) and treated (gray bars) states. A, Effect of oil treatment of individual pores on their humidity response (26 individual responses from two leaves). B, Effect of oil sealing of pores and their adjacent pores (27 responses from three leaves). C, Effect of restricting the transpiration to circular areas of 0.8-mm diameter (21 responses from three leaves). A and B display the amplitude of the circularity of stomatal pores; C displays the amplitude of the circularity of the guard cell pairs. Insets show significance level (n.s., P > 0.05; ***, P < 0.001) and 95% confidence intervals for the difference of means between treatments.

Effect of Allowing Transpiration Only on a Small Area

The observation that occlusion of an area of approximately 0.2 mm² only partly inhibited the response to dry air suggested that a significant portion of the closing stimulus must have originated outside this area. To confirm this conclusion, the inverse experiment was performed by preventing transpiration on the whole leaf except small spots of 0.8-mm diameter, which were allowed to transpire freely (Figs. 5 and 6C ). Whereas the amplitude of the hydropassive opening response appeared to be only slightly reduced, the amplitude of the active closure was halved to 7.8%. After removal of the adhesive foil, the amplitude of the closing response was the same as before.

Model for the Distance-Effect Relationship of Stomatal Transpiration for Hydropassive Opening and Active Closing

Experimental data from the different treatments were pooled to construct a simple spatial model of the distance-dependent effect of a pore's transpiration on neighboring stomata. The model leaf was simplified in that it had evenly spaced stomata with a stomatal density typical for S. nigra of 46 mm^–2. It was assumed that the transpiration of each stomatal pore has distance (d)-dependent effects on c_pass and c_act of its own guard cells and that of the other stomata, which follow a Gaussian function. Thus, the stimulus acting on a stoma (S) is calculated as the sum of the distance (d_i)-dependent stimuli caused by the transpiration of its own and that of the surrounding pores.

The only parameter determining the shape of the function, , was estimated by the least-squares optimization procedure implemented in Excel (solver) using 89 individual stomatal responses to a step change from high to low humidity showing the effects of different treatments (26 responses from two leaves with only one pore sealed; 27 responses from three leaves showing the effect of treating a pore and its adjacent pores; 13 responses on three different leaves with only the surrounding stomata sealed [data not shown]; and 21 responses from three leaves from a circular transpiring area of 0.8-mm diameter, whereas the rest of the leaf was prevented from transpiring by adhesive foil). Confidence intervals for were estimated by a bootstrap approach (Press et al., 2002): The parameter estimation procedure was repeated 2,000 times for both the hydropassive and hydroactive responses with synthetic datasets generated by randomly drawing (with replacement) sets of stomatal responses from the original dataset. From the resulting set of simulated , the 0.025 and 0.975 percentiles were taken as limits of 95% confidence intervals. The simulation yielded a _hydr = 0.347 for the hydropassive opening movement with a 95% confidence interval from 0.276 to 0.501 and _act = 0.244 for the active closing movement with a 95% confidence interval from 0.215 to 0.283 (Fig. 7A ). The confidence interval for _hydr is larger because of the smaller amplitude of the hydropassive response, resulting in larger relative measurement errors. Bootstrap analysis showed that, with P = 0.002, _hydr was larger than _act. According to this parameter estimation, 90% of the effect of a stoma's transpiration covers an area of approximately 1 mm² (hydropassive response) and 0.5 mm² (active closure).

View larger version (33K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Modeled effect of a pore's transpiration on the responses of other stomata in dependence on the distance. The estimated effects together with their 95% confidence intervals on hydropassive opening (dashed line/gray fill) and active closure (black line/hatched fill) are displayed. Drop lines mark the radius of the areas that receive 90% of the total stimulus. For further explanation, see text.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Oil sealing of pores proved to be a reliable and rather simple method to block transpiration of selected pores without mechanical disturbance. In comparison to applying streams of air (Lange et al., 1971; Mott et al., 1997; Mott and Franks, 2001), oil treatment provides a spatially better-defined manipulation of transpiration. The oil, due to capillary force, stays in place even if stomata open and close for at least 1 week. Coating the entire leaf with oil reduced leaf conductance to the same degree as stomatal closure. Therefore, it is justified to assume that sealing a pore with oil effectively suppresses transpiration. Oil treatment did not seem to interfere with stomatal responses because, during application with a micropipette, stomata did not respond in either direction and in the long term did not change their response characteristics under nontranspiring conditions (Figs. 1 and 2). The same is true for covering the leaf with adhesive foil: After removal, the response was essentially the same as before shielding (Fig. 6). The drawback of these methods is the possible spatial inhomogeneity of leaf internal CO₂ concentration caused by hindered CO₂ diffusion into the photosynthesizing leaf. This problem was addressed by conducting all experiments at approximately the CO₂ compensation point when, regardless of stomatal opening, all leaf areas should have equal internal CO₂ concentration. The general degree of opening in these experiments is possibly somewhat higher than under ambient [CO₂], but this effect was equal in all treatments. Moreover, a control experiment comparing humidity responses under ambient and reduced [CO₂] revealed no difference in the course of the response to reduction in humidity (data not shown).

The most important result of these experiments is that blocking a single pore's transpiration does not reduce the amplitude of passive opening and active closure in response to a decrease in air humidity (Figs. 5 and 6A). The response of a guard cell pair to dry air does not require transpiration through its pore as long as the other pores on a leaf are transpiring. This suggests that transpiration from all other surrounding pores may still have reduced water potential locally to the same degree as for no oil treatment. This argues against a number of hypothetical mechanisms that suggest a response of guard cells to water loss from their own substomatal surface or substomatal cavity.

First of all, this result is incompatible with purely hydraulic mechanisms driven by differential transpiration between guard and adjacent epidermal cells. Different authors have suggested that direct evaporation from the substomatal face of the guard cells could draw guard cell turgor down relative to the epidermal turgor if there is a sufficiently large hydraulic resistance between guard cells and epidermis (Nonami et al., 1990; Dewar, 1995, 2002). This mechanism, however, requires complicated additional assumptions to be able to explain the initial wrong-way response to an increase of transpiration (Buckley and Mott, 2002; Buckley, 2005). Our results add evidence contrary to this hypothesis because stomata perform the unchanged biphasic response to reduced humidity even when their pores do not transpire and no direct evaporation from the guard cells apart from cuticular transpiration occurs.

Another possible mechanism acting in the immediate surrounding of the guard cells is the accumulation of substances in the guard cell apoplast caused by evaporation from the guard cell walls, which was proposed by Grantz (1990). For the apoplastic solute Suc, Outlaw and De Vlieghere-He (2001) found transpiration-dependent accumulation in the guard cell apoplast, which should result in a sufficient decrease of apoplastic osmotic potential to decrease stomatal aperture. Such an effect, however, appears unlikely because the neighboring epidermal cells should also lose turgor, thus at least nullifying the opening effect. In any case, this observation bolstered the idea of transpiration-driven accumulation of abscisic acid (ABA), which was also supported by microanalytical determination of ABA in the guard cell apoplast (Zhang and Outlaw, 2001; Zhang et al., 2001). Our results add evidence against such a distillation-dependent effect in S. nigra because the humidity response was not changed when direct evaporation from the guard cell apoplast was largely prevented.

However, the possibility remains that peristomatal transpiration from the outer surface of guard cells mediates the humidity response. This would explain the lacking effect of oil treatment of single pores on their humidity response. There are three reasons why this explanation does not appear valid.

First of all, stomata of S. nigra are known for their tendency to oscillate in dry air (Kaiser and Kappen, 2001), which in itself points to a prominent role of feed back versus feed forward. It has been shown in detail that, during oscillations, the onset of guard cell deflation strictly follows the moment of initial opening of the pore, whereas in the closed state, guard cells inflate even if the air is very dry. Closure, therefore, is initiated by pore transpiration rather than cuticular transpiration of guard cells.

Second, if peristomatal transpiration had been a major signal, sealing the adjacent pores or blocking transpiration outside a small area should have left the response unaffected, which it did not (Fig. 6B).

Third, in the presented experiments, stomata generally had a high degree of opening. The majority of stomata did not fully close in dry air, possibly due to lowered [CO₂]. Therefore, in the untreated leaf, the portion of cuticular (peristomatal) transpiration from guard cells must have been negligible compared to stomatal transpiration. The treatment had an unknown, but presumably small, effect on peristomatal transpiration, whereas it had a large and substantial effect on transpiration through the pore. Thus, it appears justified to assign treatment effects to the change in pore transpiration.

The responses of oil-treated pores indicate that transpiration-related events leading to stomatal closure in dry air are not located at single pores, but, to a greater or lesser degree, distributed in the leaf lamina. In other words, the transpiration of a pore must have a closing effect on neighboring pores. The other two experiments were designed to determine the lateral extension of this effect. Sealing the surrounding pores in addition to the observed pore led to a decrease of the amplitude of active closure by about 40% (Fig. 6B). Apparently, a significant portion of the total closing stimulus must have originated from the now sealed area of approximately 0.2 mm², but a larger part was still provided by the rest of the leaf.

This conclusion is confirmed by the inverse experiment: When transpiration was restricted to small areas of 0.5 mm² by attaching perforated foil, the amplitude of active closure was reduced by approximately 50% (Fig. 6C). This means that, in these experiments, roughly one-half of the closing stimulus originated inside the still transpiring leaf area and the other half was removed by preventing transpiration from the rest of the leaf.

Together, the experiments point to an extension of the lateral effect of a pore's transpiration smaller than 1 mm. To describe this spatial relationship more precisely, all datasets under the three different treatments were used to estimate the shape parameter of a spatial model (Fig. 7A). This model is based on the assumption that the effect of a pore's transpiration on its own guard cells and on other stomata decreases with distance. The function that best describes this relationship is unknown and open to discussion. The use of a bell-shaped function seemed appropriate because both the lateral spreading of water potential disturbances and potentially involved diffusion of molecules in the gaseous and aqueous phase of the tissue follow thermodynamic and therefore stochastic processes. As a first guess, the Gaussian function was used. The shape parameter describes the decreasing effect with distance to the transpiring pore, with a decline to 50% in the distance . According to the parameter estimation by this model, the lateral effect of a pore's transpiration on active closure of other pores on the leaf decreases to 50% in a distance of 0.24 mm and 90% of the total effect cover an area of 0.5 mm² and affects only approximately the 23 nearest stomata.

This result is in general agreement with Xanthium strumarium measurements by Mott et al. (1997), who subjected small groups of stomata to different humidity from the rest of the leaf by directing a small stream of air to the leaf surface by a needle. Similar to the results presented here, they found that, even if stomata were under constant humidity, they responded to reduced humidity in their vicinity. The extension of the lateral effects is roughly in the same range as reported here, although the small number of observed stomata, together with the less well-defined border between regions of high and low humidity, allows no exact estimation.

Comparable experiments were also performed by Lange et al. (1971), who directed streams of different humidity to closely neighboring regions of epidermal strips of Polypodium vulgare. Somewhat contradictory to our results, stomata strictly followed the local humidity conditions and even closely neighboring stomata showed opposite responses. The reason for the discrepancy could be the use of epidermal strips or differences between species. The epidermis of P. vulgare is connected to the mesophyll only at the main veins, which may result in different hydraulic conditions in the epidermis influencing properties of the stomatal humidity response.

The conclusion drawn, that the primary processes of humidity sensing must be small-scale distributed processes in the leaf tissue, seems to agree with the hypothesis that _l triggers stomatal responses to humidity. Much indirect or circumstantial evidence shows a close correlation between _l and stomatal responses (for review, see Buckley, 2005).

This hypothesis is supported by the frequent observation that any perturbation of the balance between water supply and loss that influences _l causes similar stomatal responses. Nonetheless, it is still uncertain whether this is a causal relationship. With this question in mind, we will now discuss the hydraulic aspects revealed by the hydropassive opening response.

Following the concept of the mechanical advantage of epidermal cells over guard cells (Sharpe et al., 1987; Franks et al., 1998), the amplitude of the transient wrong hydropassive opening can be taken as a surrogate measure for the decline of local _l if the hydraulic conductivity between guard and epidermal cells is sufficient for an equilibration during the dynamic changes in local _l caused by increased transpiration. Typical relaxation half times of plant cells are mostly around 1 to 60 s (Kramer and Boyer, 1995), which would be sufficient to allow hydraulic equilibration during the hydropassive opening, which lasts for 5 to 10 min. Apparently, hydraulic resistance of guard cell membranes has not yet been measured systematically, but K.A. Mott and J.C. Shope (unpublished data), reported by Buckley (2005), point to similar relaxation times in guard cells of Vicia faba. The theoretical possibility that hydraulic resistance of the guard cell membrane is temporarily increased (e.g. by regulation of aquaporins; Sarda et al., 1997; Huang et al., 2002), which would prevent quick equilibration, appears counterproductive because this would impede turgor loss of guard cells in a situation that demands stomatal closure.

Following this consideration, it seems justified to use the amplitude of the hydropassive response as a surrogate measure for the local decline of _l. Sealing the observed pore and its adjacent pores with oil had no effect on the hydropassive opening. This shows that direct evaporation from a pore's substomatal cavity or the adjacent substomatal cavities is not necessary to cause local disturbance in local _l, resulting in transient hydropassive opening. Obviously, hydraulic coupling of the epidermis is sufficient to allow lateral water transport, which equalizes these local differences in _l. According to the model estimation (Fig. 7), the hydraulic effect of stomatal transpiration declines to 50% in a distance of 0.35 mm. This agrees with epidermal turgor reductions measured by Mott and Franks (2001) in different distances to a small region influenced by a stream of dry air applied through a needle. Turgor pressure effects dropped to roughly 50% in a distance of 300 to 400 µm from the needle tip.

Conspicuously, the effect of a pore's transpiration on hydropassive opening extends further than the effect on active closure. This was most obvious when both the observed and the surrounding pores were treated with oil (Fig. 6B): In this case, the active closure was clearly reduced, although the constant hydropassive effect indicates virtually the same local reduction of _l as before. From the hypothesis that guard cells respond to local _l, we would expect, however, that both effects extend to the same distance. The observation that the lateral effect of a pore's transpiration on stomatal closure is more locally confined than the effect on _l suggests that guard cells may not exclusively respond to the disturbance of local _l, but may also utilize other sensing mechanisms driven by transpiration. These could act instead of or in addition to direct sensing of local _l. Another physical property related to transpiration that could be sensed is, for example, the rate of hydraulic flow through a tissue, which should better reflect local transpiration conditions than steady-state _l.

Although the mechanisms remain unclear, these results suggest an important role of the leaf tissue in humidity/transpiration sensing as other results also indicate (Cochard et al., 2002). One possibility for such an indirect sensing mechanism is the production of an intermediate signal that is transported to guard cells. This two-step mechanism is also consistent with the often extended lag phase, which precedes stomatal closure in response to dry air (H. Kaiser, unpublished data; Buckley and Mott, 2002). This lag phase can be attributed to the time necessary for producing and releasing the closing agent into the apoplast and for diffusion to the guard cells. Other stimuli that are sensed directly by the guard cells (e.g. blue light and CO₂), in contrast, generally elicit much faster responses.

The putative sensitive tissue and the intercellular signals are not yet identified. As the results presented here allow only indirect conclusions on the involved sensing mechanisms, they will be discussed only briefly. Much evidence suggests an important role of mesophyll cells (Lee and Bowling, 1995). Grantz and Schwartz (1988) found that guard cells need the presence of mesophyll cells to be able to respond to osmotic stress. Leaf tissues may produce signaling molecules that diffuse to the guard cells or modify the composition of the transpiration stream. This could involve changes in the pH (Roelfsema and Prins, 1998) and ionic composition (Atkinson et al., 1989; Ruiz et al., 1993) of the apoplastic solution or release and recompartmentation of ABA (e.g. Trejo et al., 1993; Hartung et al., 1998; Wilkinson and Davies, 2002). Although ABA is the favorite candidate for an intermediate signal, there is also evidence for an ABA-independent signaling pathway (Assmann et al., 2000; Luan, 2002; Yoshida et al., 2006).

In conclusion, by combining different experimental procedures, we have established that guard cells do not respond to their own transpiration, but to a locally averaged water loss in the surrounding of the pore. The observation that disturbances of _l as measured by the hydropassive opening response do not correlate with the hydroactive closure could point to the existence of controlling entities other than local _l. The results support the hypothesis that the primary and initial transpiration-sensing processes are not located in the guard cells, but involve transpiration-related processes in the leaf tissue.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Culture and Experimental Setup

Experiments were performed on attached leaves of potted Sambucus nigra plants of approximately 50 to 80 cm in size. Plants were drawn from cuttings and cultivated in 40-cm pots in a climatic chamber at a PPFD of 220 µmol m^–2 s^–1 (16-h light/8-h dark) and a temperature of 20°C. Plants were amply supplied with water and nutrients. Stomatal movements were observed on mature leaves in a gas-exchange chamber designed for simultaneous measurement of CO₂-water gas exchange and microscopic observation of stomatal movements under controlled light, humidity, temperature, and CO₂ conditions (Kaiser and Kappen, 2001). Temperature, leaf-to-air mole fraction of water vapor (_W) and [CO₂] in the cuvette were set to 20°C to 22°C, 2 mmol mol^–1, and approximately 360 µmol mol^–1, respectively. Irradiance (500 µmol m^–2 s^–1, 16-h light/8-h dark) was provided by a fiber-optic illuminator (Kaltlicht-Fiberleuchte FL-400 with Spezial Fiberoptik 400-F; Walz). After mounting the leaf in the gas-exchange cuvette, the plant was allowed to adjust to measuring conditions for at least 24 h. Leaves were fixed with the adaxial side to a Perspex plate with double-sided transparent adhesive tape (Tesa 56661–2; Tesa) to allow micromanipulation. Subsequently, the plate was mounted inside the cuvette, which allows observation of the lower leaf surface with a long-distance microscope lens (50x) led through the bottom of the gas-exchange cuvette (Kaiser and Grams, 2006). The microscope (Axiovert 25CFL; Zeiss) is mounted on a motorized translation stage, which allows repositioning of samples of selected stomata. Digital images of stomata were recorded with a video camera, digitized, and stored for subsequent measurement of aperture with custom image analysis software. The aperture of oil-treated pores can no longer be measured due to refraction of the oil. In these experiments, the area of the guard cell pair between the anticlinal walls was measured. This measure is linearly related to aperture when pores are open (Kaiser and Kappen, 2001) and can be taken as a surrogate measure of stomatal opening. Pore area or guard cell pair area were converted to circularity (c = width x 100/length) to allow comparison between differently sized stomata. The humidity control by a bypass compensation system was used to perform quick changes in air humidity from _W = 2 to approximately 18 mmol mol^–1 by switching the humidity of the incoming air to lower humidity. Within approximately 90 s, _W arrived at its new steady state (Fig. 3). One hour before increasing _W, [CO₂] was reduced to approximately 60 to 70 µmol mol^–1, which is approximately the CO₂ compensation point for C3 plants (von Caemmerer and Farquhar, 1982). This avoids intercellular [CO₂] gradients due to locally suppressed CO₂ diffusion into the mesophyll. Stomatal responses to a reduction in air humidity were observed before and after the treatment with oil or adhesive foil on subsequent days, always beginning at the same time 4 h after illumination was switched on. Stomatal apertures were observed from at least 30 min before to 1.5 h after reduction of air humidity. Images were taken every 3 to 4 min if apertures changed fast, otherwise at longer intervals of up to 10 min.

Oil Treatment of Pores

The cuvette allows micromanipulation on the lower leaf surface by micropipettes inserted through small holes in the cuvette wall. Micropipettes were drawn from 1.5-mm borosilicate glass capillaries with a pipette puller (L/M-3P-A; List). Tips were ground to a diameter of approximately 5 µm. Mineral oil (M8662; Sigma-Aldrich) was applied to pores by approaching the oil-filled pipette to a pore and gently pressurizing it manually by pressing a rubber ball. In each experiment, six to 20 pores with a spacing of at least 2 mm were selected. Either the observed pore or the observed pore plus the adjacent pores was sealed with oil. In an additional experiment, which was only used for the model parameter estimation, only the adjacent pores were sealed (leaving the observed central pore free). In all experiments, a control sample of the same size as the treatment sample was observed to detect and correct for interday variability, which, however, was found to be small.

Shielding of the Leaf with Perforated Adhesive Foil

To shield the entire leaf except circular areas of 0.8 mm, thin polyethylene plastic foil (20 µm) was punched with a 0.8-mm syringe needle, which was ground squarely and sharpened. The correct size of the holes was confirmed microscopically after attachment to the leaf. Very thin double-sided adhesive tape (Pritt permanent) was used to attach the foil to the leaf, allowing only the epidermis inside the circular holes to transpire. In some cases, the foil was not attached firmly to the epidermis at the edge of the punched hole, leaving a gap between leaf and foil. These stomata were excluded from the experiments. Up to six holes with a distance of at least 10 mm were punched into a foil in one experiment. The foil was at first attached provisionally in its final position to select stomata located inside the holes. The foil was then removed to observe a control response to a decrease in air humidity of selected stomata. Thereafter, the foil was attached firmly in the same position as before to measure the humidity response of the sample of stomata on the next day. In some experiments, the foil was carefully removed afterward and another control response was measured on the next day to test for permanent damage due to experimental treatment.

	ACKNOWLEDGMENTS

 
We want to thank Annika Hagemann for help with image analysis, Ludger Kapppen and three anonymous reviewers for helpful comments on a former version of the manuscript, and Patricia Schoone for language editing.

Received September 1, 2006; accepted November 27, 2006; published December 8, 2006.

	FOOTNOTES

 
¹ This work was supported by the Deutsch Forschungsgemeinschaft (grant no. KA1711/1–1).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hartmut Kaiser (hkaiser{at}bot.uni-kiel.de).

www.plantphysiol.org/cgi/doi/10.1104/pp.106.089334

^* Corresponding author; e-mail hkaiser{at}bot.uni-kiel.de; fax 49–431–880–5568.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Assmann SM, Snyder JA, Lee Y-RJ (2000) ABA-deficient (aba1) and ABA-insensitive (abi1-1, abi2-1) mutants of Arabidopsis have a wild-type stomatal response to humidity. Plant Cell Environ 23: 387–395[Medline]

Atkinson CJ, Mansfield TA, Kean AM, Davies WJ (1989) Control of stomatal aperture by calcium in isolated epidermal tissues and whole leaves of Commelina communis L. New Phytol 111: 9–17[CrossRef][Web of Science]

Buckley T, Mott K (2002) Dynamics of stomatal water relations during the humidity response: implications of two hypothetical mechanisms. Plant Cell Environ 25: 407–419[CrossRef]

Buckley TN (2005) The control of stomata by water balance. New Phytol 168: 275–292[CrossRef][Web of Science][Medline]

Cochard H, Coll L, Le Roux X, Améglio T (2002) Unraveling the effects of plant hydraulics on stomatal closure during water stress in walnut. Plant Physiol 128: 282–290[Abstract/Free Full Text]

Cosgrove DJ, Hedrich R (1991) Stretch-activated chloride, potassium and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L. Planta 186: 143–153[Web of Science][Medline]

Cowan IR (1977) Stomatal behaviour and environment. Adv Bot Res 4: 117–228

Dewar RC (1995) Interpretation of an empirical model for stomatal conductance in terms of guard cell function. Plant Cell Environ 18: 365–372[Medline]

Dewar RC (2002) The Ball-Berry-Leuning and Tardieu-Davies stomatal models: synthesis and extension within a spatially aggregated picture of guard cell function. Plant Cell Environ 25: 1383–1398[CrossRef]

Farquhar G (1978) Feedforward responses of stomata to humidity. Aust J Plant Physiol 5: 787–800[Web of Science]

Franks P, Cowan I, Farquhar G (1997) The apparent feedforward response of stomata to air vapour pressure deficit: information revealed by different experimental procedures with two rainforest trees. Plant Cell Environ 20: 142–145[Medline]

Franks P, Cowan I, Farquhar G (1998) A study of stomatal mechanics using the cell pressure probe. Plant Cell Environ 21: 94–100[CrossRef]

Garrill A, Findlay GP, Tyerman SD (1996) Mechanosensitive ion channels. In M Smallwood, JP Knox, DJ Bowles, eds, Plant Membranes: Specialised Functions in Plants. Bios, Oxford, pp 247–260

Grantz DA (1990) Plant response to atmospheric humidity. Plant Cell Environ 13: 667–679[CrossRef]

Grantz DA, Schwartz A (1988) Guard cells of Commelina communis L. do not respond metabolically to osmotic stress in isolated epidermis implications for stomatal responses to drought and humidity. Planta 174: 166–173[CrossRef][Web of Science]

Hartung W, Wilkinson S, Davies W (1998) Factors that regulate abscisic acid concentrations at the primary site of action at the guard cell. J Exp Bot 49: 361–367[Abstract]

Huang RF, Zhu MJ, Kang Y, Chen J, Wang XC (2002) Identification of plasma membrane aquaporin in guard cells of Vicia faba and its role in stomatal movement. Acta Bot Sin 44: 42–48

Kaiser H, Grams TEE (2006) Rapid hydropassive opening and subsequent active stomatal closure follow heat-induced electrical signals in Mimosa pudica. J Exp Bot 57: 2087–2092[Abstract/Free Full Text]

Kaiser H, Kappen L (2001) Stomatal oscillations at small apertures: indications for a fundamental imperfection of stomatal feedback-control inherent in the stomatal turgor mechanism. J Exp Bot 52: 1303–1313[Abstract/Free Full Text]

Kappen L, Andresen G, Lösch R (1987) In situ observations of stomatal movements. J Exp Bot 38: 126–141[Abstract/Free Full Text]

Kerstiens G (1997a) Cuticular water permeance and its physiological significance. J Exp Bot 47: 1813–1832[CrossRef][Web of Science]

Kerstiens G (1997b) In vivo manipulation of cuticular water permeance and its effect on stomatal response to air humidity. New Phytol 137: 473–480[CrossRef][Web of Science]

Kramer PJ, Boyer JS (1995) Water Relations of Plant and Soils. Academic Press, San Diego

Lange OL, Lösch R, Schulze E-D, Kappen L (1971) Responses of stomata to changes in humidity. Planta 100: 76–86[CrossRef][Web of Science]

Lee J, Bowling DJF (1995) Influence of the mesophyll on stomatal opening. Aust J Plant Physiol 22: 357–363[Web of Science]

Lu P, Outlaw WH Jr, Smith BG, Freed GA (1997) A new mechanism for the regulation of stomatal aperture size in intact leaves: accumulation of mesophyll-derived sucrose in the guard-cell wall of Vicia faba. Plant Physiol 114: 109–118[Abstract]

Luan S (2002) Signalling drought in guard cells. Plant Cell Environ 25: 229–237[CrossRef][Medline]

Maier-Maercker U (1983) The role of peristomatal transpiration in the mechanism of stomatal movement. Plant Cell Environ 6: 369–380[CrossRef]

Maier-Maercker U (1999) New light on the importance of peristomatal transpiration. J Plant Physiol 26: 9–16

Monteith JL (1995) A reinterpretation of stomatal responses to humidity. Plant Cell Environ 18: 357–364[CrossRef]

Mott K, Denne F, Powell J (1997) Interactions among stomata in response to perturbations in humidity. Plant Cell Environ 20: 1098–1107[CrossRef]

Mott KA, Franks PJ (2001) The role of epidermal turgor in stomatal interactions following a local perturbation in humidity. Plant Cell Environ 24: 657–662[Medline]

Mott KA, Parkhurst DF (1991) Stomatal response to humidity in air and helox. Plant Cell Environ 14: 509–515[CrossRef]

Nonami H, Schulze E-D, Ziegler H (1990) Mechanisms of stomatal movement in response to air humidity, irradiance and xylem water potential. Planta 183: 57–64[Web of Science]

Outlaw WH Jr, De Vlieghere-He X (2001) Transpiration rate: an important factor controlling the sucrose content of the guard cell apoplast of broad bean. Plant Physiol 126: 1716–1724[Abstract/Free Full Text]

Press WH, Teukolsky SA, Vetterling WT, Flannery BP (2002) Numerical Recipes in C: The Art of Scientific Computing. Cambridge University Press, Cambridge

Roelfsema MRG, Hedrich R (2005) In the light of stomatal opening: new insights into "the Watergate". New Phytol 167: 665–691[CrossRef][Web of Science][Medline]

Roelfsema MRG, Prins HBA (1998) The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification. Planta 205: 100–112[CrossRef][Web of Science][Medline]

Ruiz LP, Atkinson CJ, Mansfield TA (1993) Calcium in the xylem and its influence on the behaviour of stomata. Philos Trans R Soc Lond B Biol Sci 341: 67–74[CrossRef]

Sarda X, Tousch D, Ferrare K, Legrand E, Dupuis J, Cassedelbart F, Lamaze T (1997) Two TIP-like genes encoding aquaporins are expressed in sunflower guard cells. Plant J 12: 1103–1111[CrossRef][Web of Science][Medline]

Schulze ED, Lange OL, Evenari M, Kappen L, Buschbom U (1973) Stomatal responses to changes in temperature at increasing water stress. Planta 110: 29–42[CrossRef][Web of Science]

Sharpe PJH, Wu H, Spence RD (1987) Stomatal mechanics. In E Zeiger, GD Farquhar, IR Cowan, eds, Stomatal Function. Stanford University Press, Stanford, CA, pp 92–114

Trejo CL, Davies WJ, Ruiz L (1993) Sensitivity of stomata to abscisic acid (an effect of the mesophyll). Plant Physiol 102: 497–502[Abstract]

von Caemmerer S, Farquhar GD (1982) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 376–387[CrossRef][Web of Science]

West JD, Peak D, Peterson JQ, Mott KA (2005) Dynamics of stomatal patches for a single surface of Xanthium strumarium L. leaves observed with fluorescence and thermal images. Plant Cell Environ 28: 633–641[CrossRef]

Wilkinson S, Davies WH (2002) ABA-based chemical signalling: the co-ordination of responses to stress in plants. Plant Cell Environ 25: 195–210[CrossRef][Medline]

Yoshida R, Umezawa T, Mizoguchi T, Takahashi S, Takahashi F, Shinozaki K (2006) The regulatory domain of SRK2E/OST1/SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and osmotic stress signals controlling stomatal closure in Arabidopsis. J Biol Chem 281: 5310–5318[Abstract/Free Full Text]

Zhang SQ, Outlaw WH Jr (2001) Abscisic acid introduced into the transpiration stream accumulates in the guard-cell apoplast and causes stomatal closure. Plant Cell Environ 24: 1045–1054[CrossRef]

Zhang SQ, Outlaw WH, Aghoram K (2001) Relationship between changes in the guard cell abscisic-acid content and other stress-related physiological parameters in intact plants. J Exp Bot 52: 301–308[Abstract/Free Full Text]

Related articles in Plant Physiol.:

On the Inside

Peter V. Minorsky
Plant Physiol. 2007 143: 553-554. [Full Text]  

This article has been cited by other articles:

				Y. Kang, W. H. Outlaw Jr, G. B. Fiore, and K. A. Riddle Guard cell apoplastic photosynthate accumulation corresponds to a phloem-loading mechanism J. Exp. Bot., December 1, 2007; 58(15-16): 4061 - 4070. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 143/2/1068    most recent pp.106.089334v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in Plant Physiol. Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Kaiser, H. Articles by Legner, N. Search for Related Content PubMed PubMed Citation Articles by Kaiser, H. Articles by Legner, N. Agricola Articles by Kaiser, H. Articles by Legner, N. Related Collections Biology of Transpiration