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- Materials and Methods
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Stomata regulate the CO2 supply for photosynthesis and transpiration in the leaf (Zeiger, 1983; Willmer & Fricker, 1996). Stomatal responses (i.e. opening and closure) are controlled by various environmental parameters, including light and CO2 concentration; however, the mechanisms of stomatal responses to these factors have not been elucidated completely.
Stomata exhibit at least two types of response to light (Kuiper, 1964; Sharkey & Raschke, 1981; Zeiger, 1983). One type is termed the blue-light response, for which the action spectrum peaks at c. 450 nm. Blue light is efficient in stomatal opening (Hsiao & Allaway, 1973; Iino et al., 1985). Blue light induces H+ extrusion from the guard cells, and thereby the membrane potential of a guard cell becomes hyperpolarized (Assmann et al., 1985; Shimazaki et al., 1986; Roelfsema et al., 2001). It is well established that phototropins, the blue-light receptors in guard cells, absorb blue light and induce stomatal opening by sequentially causing the following events: activation of the plasma membrane H+-ATPases, hyperpolarization of the plasma membrane and activation of K+ uptake channels (Kinoshita & Shimazaki, 1999; Kinoshita et al., 2001; Shimazaki et al., 2007). In addition to the regulation of H+-ATPases, blue light inhibits S-type anion channels (Marten et al., 2007). Stomata have also been shown to open in green light, even when leaf photosynthesis is inhibited (Wang et al., 2011). Because the green light used in the study by Wang et al. (2011) would not be absorbed by phototropins, certain light receptors other than phototropins might be responsible for the stomatal opening in green light. The second type of stomatal response is termed the red-light response, in which the action spectrum resembles that of photosynthesis. The red-light response is strongly inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which is a potent inhibitor of photosynthesis. In addition, there is a strong correlation between stomatal conductance and the photosynthetic rate in red light. Accordingly, it is widely believed that photosynthesis is involved in the red-light response (Messinger et al., 2006; Wang et al., 2011). Red light also inhibits S-type anion channels when it is projected on a large area of a leaf (Roelfsema et al., 2002). It has also been shown that illumination of a single stoma and its surroundings with a red beam is not sufficient to induce stomatal opening (Mott et al., 2008).
CO2 is another environmental factor that regulates stomatal responses (Willmer, 1988). It has been shown that Nicotiana tabacum mitogen-activated protein kinase 4 (NtMPK4)-silenced plants lacking stomatal sensitivity to CO2 are less responsive to red light, indicating that both responses are interconnected (Marten et al., 2008). Guard cells in the epidermal strip respond directly to CO2 (Schwartz et al., 1988; Webb et al., 1996; Webb & Hetherington, 1997). Molecular studies using Arabidopsis thaliana have revealed that stomatal closure at high CO2 is mediated by carbonic anhydrases (CAs) localized in the plasma membrane of guard cells (Fabre et al., 2007; Hu et al., 2010; Kim et al., 2010). By contrast, several lines of evidence have indicated that the mesophyll is involved in stomatal responses to CO2.
Many studies use epidermal strips to evaluate stomatal responses to light and/or CO2. However, some studies have indicated that stomata in epidermal strips respond to light and/or CO2 much less than those in intact leaves (Lee & Bowling, 1992, 1995; Mott et al., 2008). Moreover, several studies have indicated that the role of photosynthesis in guard cells in controlling stomatal responses to red light is minor (Schwartz & Zeiger, 1984; Tominaga et al., 2001). For example, the stomatal guard cells of Paphiopedilum leeanum leaves have no chloroplasts; however, the stomata in the intact leaves open in response to red light (Nelson & Mayo, 1975). Furthermore, in Chlorophytum comosum, red-light-induced stomatal opening requires the mesophyll with active chloroplasts. The stomata over the chloroplast-less mesophyll do not respond to red light (Roelfsema et al., 2006). In a study by Mott et al. (2008), the stomata in epidermal strips exhibited a limited response to light or CO2, whereas those in epidermal strips placed on a mesophyll layer responded to light and CO2 in a manner similar to those in leaf segments (Mott et al., 2008). On the basis of these experiments, Mott et al. (2008) suggested that signals produced in mesophyll control the stomatal response. Their work is pioneering and highly suggestive in that they clearly showed the importance of the mesophyll in the stomatal response by a very straightforward method. However, in the experiment by Mott et al. (2008), the stomata in the epidermal strips might have opened widely in a hydropassive manner, as the stomata in the epidermal strips did not respond to environmental factors, such as light and CO2, but remained open. Therefore, it remains unclear whether the stomatal response in epidermal strips can be compared with that in leaves.
Sibbernsen & Mott (2010) found that stomatal opening decreased when various liquids were injected into the intercellular spaces of leaves, suggesting that mesophyll signals are gaseous. By contrast, Lee & Bowling (1993, 1995) showed that stomata responded to light when epidermal strips were floated on a solution containing illuminated mesophyll cells or chloroplasts. When the epidermis was floated on the same buffer without mesophyll or chloroplasts, the stomata did not respond to light (Lee & Bowling, 1992, 1995). Stomatal opening was also observed when epidermal strips were floated on the supernatant of a solution containing illuminated mesophyll cells (Lee & Bowling, 1992). The authors also observed that guard cell protoplasts swelled when suspended in the supernatant (Lee & Bowling, 1993). These studies indicate that mesophyll signals are aqueous.
In this study, referring to Mott et al. (2008), we devised a novel method to observe microscopically stomatal responses under more physiological conditions. By placing epidermal strips on a buffer-containing gel, rather than on a solution, we prevented the epidermal strips from being subject to extreme desiccation or hydration for up to 8 h. With this new system, we aimed to clarify whether the mesophyll plays an important role in stomatal responses to CO2 by comparing the stomatal responses of the leaf segments, epidermal strips and epidermal strips restored onto mesophyll segments. We used red light, in addition to white light containing a blue-light component. We also investigated how photosynthesis regulates stomatal responses using DCMU. Whether mesophyll signals move to the epidermis via the aqueous phase in the apoplast was further examined by inserting doughnut-shaped spacers made of polyethylene film or cellophane between the epidermal strip and the mesophyll segment.
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- Materials and Methods
- Supporting Information
In this study, we constructed a system to control the environment of leaf segments or isolated epidermis and to observe stomatal responses to CO2 (100 ppm or 700 ppm) on these materials microscopically. By using the buffer-containing gel rather than aqueous buffers, we were able to observe the stomatal response in a reasonably physiological state for a long time. We compared stomatal responses in a leaf segment, epidermal strip and epidermal strip placed on a mesophyll segment in red light or white light. The present results clearly indicate that the mesophyll is important for both stomatal opening and closure, supporting the existence of ‘mesophyll signals’, signals which have been proposed in previous studies (Lee & Bowling, 1992, 1993, 1995; Mott et al., 2008). We also investigated whether mesophyll signals are gaseous by inserting a polyethylene or cellophane spacer between the epidermal strip and the mesophyll segment. By comparing the stomatal responses between the polyethylene-inserted sample and the cellophane-inserted sample, we concluded that mesophyll signals are aqueous. We then treated the leaf with DCMU to inhibit photosynthesis throughout the whole leaf. It has been suggested that mesophyll signals inducing stomatal opening are dependent on photosynthesis at low CO2, whereas those inducing stomatal closure are independent of photosynthesis at high CO2.
Conditions for observing stomatal responses
Previous studies have reported that 1 mM CaCl2 in the incubation buffer tends to induce stomatal closure (De Silva et al., 1985; Schwartz, 1985; McAinsh et al., 1995). When the epidermal strip was placed on the gel, the epidermal cells were closely associated with the gel, whereas the guard cells located above the air-filled substomatal cavities were not directly associated with the gel. Epidermal cells are closely associated with the mesophyll in intact leaves, with mesophyll apoplastic Ca2+ concentrations probably ranging from 0.1 to 1 mM (Sattelmacher, 2001). The apoplastic Ca2+ concentrations in epidermal cell walls that are closely associated with the mesophyll are expected to be in the same range as that of the mesophyll apoplast. Therefore, in our system, 0.4–0.6 mM free Ca2+ in the gel would not severely inhibit stomatal opening. Certainly, stomatal opening was severely inhibited in white light when the substomatal cavities were filled with buffer from the gel and the guard cells were directly associated with the buffer (data not shown). We also investigated stomatal responses in the samples placed on a Ca2+-free gel (Fig. S2). In this gel, Mg2+ was used as cross-linker for the solidification of gellangum. With this gel, the stomatal opening was moderately speeded up, especially in white light (compare Fig. 7 and Fig. S2). In the epidermal strip, the induction of stomatal closure at high CO2 was slower under the Ca2+-free condition than under the 1 mM Ca2+ condition (compare Fig. 7e and Fig. S2e). Although the absolute value of the stomatal aperture and the speeds of opening and closure differed depending on the presence of Ca2+ in the gel, both stomatal opening and closure were greatly accelerated when the epidermal strip was placed on the mesophyll. Thus, we concluded that the extracellular Ca2+ concentration did not influence markedly the tendencies of stomatal responses, and the role of mesophyll in stomatal responses could be assessed adequately with 1 mM CaCl2 buffer-containing gel.
Ethylene is expected to arise from the cut surface of samples (Boller & Kende, 1980). Although ethylene is known to affect the regulation of the stomatal aperture, its effect remains unclear. In some species, ethylene induces stomatal closure (Desikan et al., 2006), whereas, in others, it mediates auxin-induced stomatal opening (Merritt et al., 2001). In our system, when compared with other environmental factors, the effects of ethylene on stomatal responses are considered to be small, because stomata in each sample responded to light and/or CO2 as in intact leaves (data not shown). If the effects of ethylene were stronger than those of other environmental factors, the effects of ethylene would be superimposed on the physiological responses to light and/or CO2.
Previous studies have supported the contention that photosynthesis in both guard cells and mesophyll is involved in the regulation of stomatal responses (Sharkey & Raschke, 1981; Messinger et al., 2006; Wang et al., 2011). It is also known that chloroplasts in guard cells exhibit high photosynthetic electron transport activity (Lawson et al., 2002, 2003; Lawson, 2009). When a leaf is illuminated with red light, photosynthesis occurs in both the guard cells and the mesophyll. The results of the current study showed that stomata in the epidermal strip of C. communis barely opened in red light at low CO2. By contrast, stomata in the leaf segment opened widely (Fig. 7a,b and Table 1). However, when the epidermal strip was placed on the mesophyll segment, stomata opened widely (Fig. 7c). Moreover, stomata in the leaf segment that had been treated with DCMU, which is a potent inhibitor of photosynthesis, barely opened in red light at low CO2 (Fig. 10b and Table 3). These findings indicate that stomatal opening in red light is more strongly dependent on photosynthesis in the mesophyll than in guard cells.
Unlike the stomatal response to red light, stomata in the epidermal strip opened widely when illuminated with white light (compare Fig. 7b and e). The blue-light receptors, phototropins, are localized in the guard cells of A. thaliana, and it has been established that blue light excites these phototropins and leads to stomatal opening (Kinoshita et al., 2001; Shimazaki et al., 2007). It is highly likely that the guard cells of C. communis also contain phototropins (Iino et al., 1985); hence, when the leaf was illuminated with white light, the blue component in white light strongly induced stomatal opening (Fig. 6d).
Stomata in the leaf segment treated with DCMU opened when illuminated with white light (Fig. 10d,e). When stomata open, H+-ATPase in guard cell plasma membranes consumes cytosolic ATP (Shimazaki et al., 2007). As photosynthesis was inhibited in the DCMU-treated leaf segment, the ATP for H+-ATPases would be produced by respiration, with carbohydrates stored in the guard cell being consumed (Mawson, 1993). However, stomata in the leaf segment treated with DCMU did not open in red light (Fig. 8b), even though carbohydrates would be available. Although blue light activates H+-ATPases in guard cell protoplasts (without mesophyll cells), red light has no such function in guard cell protoplasts (Taylor & Assmann, 2001). Hence, it is probable that mesophyll signals induced stomatal opening via the activation of H+-ATPase in guard cells.
In addition to the regulation of H+-ATPases, the regulation of S-type anion channels in guard cells would be important for fast stomatal opening. In intact plants, the inhibition of S-type anion channels in guard cells occurs in red light or at low CO2 (Roelfsema et al., 2002; Marten et al., 2008). Loss of SLAC1, a major S-type anion channel in guard cells, leads to slow stomatal closure in the dark (Negi et al., 2008; Vahisalu et al., 2008). In other words, the deactivation of S-type anion channels would lead to fast stomatal opening in the light. Mesophyll signals at low CO2 in the light may deactivate S-type anion channels, and speed up stomatal opening.
Although stomata in the leaf segment closed rapidly in white light and at high CO2, stomata in the epidermal strip barely closed within 1 h under these conditions (Fig. 7d,e). Previous studies have indicated clearly that stomata in the epidermal strip close at high CO2 (Schwartz et al., 1988; Webb et al., 1996; Webb & Hetherington, 1997; Hu et al., 2010). To check whether this phenomenon occurred in our system, the epidermal strip was maintained at high CO2 for > 1 h. The stomata on the epidermal strip closed slowly from an aperture of 4.6 to 2.8 μm over a 3-h period (Fig. S1). The degree of stomatal closure was comparable with that observed in previous studies. In addition, when the epidermal strip was maintained in the dark, stomata closed rapidly within 1 h (Figs 7g, S1, Table 1). Therefore, the stomata in the epidermal strip had the potential to close; the closure under high CO2 conditions was not an experimental artefact. By contrast, when the epidermal strip was placed on the mesophyll segment, stomata closed rapidly (Fig. 7f ). These data indicate that the mesophyll contributes to the rapid induction of stomatal closure at high CO2. Stomata in the leaf segment treated with DCMU closed rapidly in white light at high CO2 in a manner similar to that of the control (Fig. 10c,d). Hence, we questioned what was responsible for the rapid stomatal closure in the DCMU-treated leaf segment. As photophosphorylation was inhibited in the DCMU-treated leaf segment, the ATP required for stomatal opening would gradually decline. However, stomata in the DCMU-treated leaf segment continued to open gradually for at least 3 h when the CO2 concentration was maintained at 100 ppm (Fig. 10e). On the basis of this information, it is unlikely that stomatal closure in the DCMU-treated leaf segment at high CO2 is caused by a shortage of ATP. It is also unlikely that stomatal closure at high CO2 is dependent on mesophyll photosynthesis.
In contrast with the effects of low CO2, S-type anion channels were activated at high CO2 (Roelfsema et al., 2002). Mesophyll signals at high CO2 may cause the activation of S-type anion channels for fast stomatal closure.
Abscisic acid (ABA) is one of the candidates of the mesophyll signal that might induce stomatal closure. CA catalyses the reversible reaction of CO2 + H2O H+ + , with this enzyme also being present in mesophyll cells (Badger & Price, 1994). When ambient CO2 concentrations are high, CO2 concentrations in mesophyll cells inevitably increase. Then, the equilibrium favours the production of H+ and . Thus, the pH inside the mesophyll cells becomes more acidic. Under these acidic conditions, ABA would exist in its uncharged form (ABAH), which is able to diffuse across the cell membrane (Slovik et al., 1992). Therefore, when CO2 concentrations are high, ABA would be released from the mesophyll. Thus, ABA moves from the mesophyll to the guard cells, in turn inducing stomatal closure. Further studies are required to test this hypothesis.
Mesophyll signals move from the mesophyll to the epidermis via the apoplast
Our data demonstrate that the mesophyll plays a critical role in controlling stomatal aperture. Like previous studies, we also propose the existence of ‘mesophyll signals’ (substances controlling stomatal aperture), which are released from the mesophyll and move towards the epidermis (Lee & Bowling, 1992, 1993, 1995; Mott et al., 2008). Sibbernsen & Mott (2010) found that stomata close rapidly when liquids are microinjected into the intercellular spaces of leaves. As the transfer of gaseous substances from the mesophyll to the epidermis was blocked by microinjection, the authors suggested that mesophyll signals are gaseous. However, the results of Sibbernsen & Mott (2010) would not eliminate the involvement of aqueous mesophyll signals. Our study showed that mesophyll signals inducing stomatal opening are probably produced during the course of photosynthesis and are released from the mesophyll. For photosynthesis, a CO2 supply to the mesophyll is needed (Fig. 10a,b). When the intercellular space floods, the CO2 supply to the mesophyll is blocked, which would consequently suppress the production of both gaseous and aqueous mesophyll signals. It is also possible that the liquid occupying the substomatal cavities suppresses respiration in guard cells, inhibiting the O2 supply from the intercellular space to the guard cell.
We examined whether the mesophyll signals are gaseous. Small molecules in the liquid may diffuse across a cellophane film, but not across a polyethylene one. We inserted spacers made of these films between the epidermis and the mesophyll, and investigated whether the mesophyll signals regulating stomatal responses were aqueous. In our system, the mesophyll segment was sandwiched by the gel and the doughnut-shaped spacer (Fig. 4, right). As described in the section on ‘Stomatal opening’, stomatal opening was strongly dependent on photosynthesis. With the spacer having no holes, CO2 for photosynthesis cannot be supplied to the mesophyll, and thereby the release of the mesophyll signals would be inhibited. Indeed, when we inserted the spacer without a hole, we could not observe stomatal opening in red light (data not shown). Therefore, we used the ‘doughnut-shaped’ spacers to deliver CO2 through the stomata in the epidermis to the mesophyll (Fig. 4).
When the doughnut-shaped polyethylene spacer (50 μm thick) was inserted between the epidermis and the mesophyll, the stomata could not respond to CO2 (Fig. 8b,e). However, when the doughnut-shaped cellophane spacer (50 μm thick) was inserted between the epidermis and the mesophyll segment, the stomata opened at low CO2 (Fig. 8c) and closed at high CO2 (Fig. 8f). This indicates that the stomata in the cellophane spacer-inserted samples respond rapidly to CO2 in a manner similar to that of the stomata in the leaf segment (Fig. 7a,d). On the basis of these results, the mesophyll signals inducing both stomatal opening and closure appear to move from the mesophyll to the epidermis via the aqueous phase in the apoplast. If gaseous mesophyll signals were present, the effects on the regulation of stomatal aperture would be less important relative to those of the aqueous mesophyll signals.
In summary, the data presented in this study indicate that both stomatal opening and closure are strongly regulated by aqueous signals from the mesophyll. Stomatal opening is dependent on mesophyll photosynthesis, whereas stomatal closure is less dependent on mesophyll photosynthesis. In an effort to identify such mesophyll signals, we are currently devising a method to collect the mesophyll apoplastic solution.