Stomatal responses to light and CO2 were investigated using isolated epidermes of Tradescantia pallida, Vicia faba and Pisum sativum. Stomata in leaves of T. pallida and P. sativum responded to light and CO2, but those from V. faba did not. Stomata in isolated epidermes of all three species could be opened on KCl solutions, but they showed no response to light or CO2. However, when isolated epidermes of T. pallida and P. sativum were placed on an exposed mesophyll from a leaf of the same species or a different species, they regained responsiveness to light and CO2. Stomatal responses in these epidermes were similar to those in leaves in that they responded rapidly and reversibly to changes in light and CO2. Epidermes from V. faba did not respond to light or CO2 when placed on mesophyll from any of the three species. Experiments with single optic fibres suggest that stomata were being regulated via signals from the mesophyll produced in response to light and CO2 rather than being sensitized to light and CO2 by the mesophyll. The data suggest that most of the stomatal response to CO2 and light occurs in response to a signal generated by the mesophyll.
Plants must balance the demand of the mesophyll for CO2 with the supply of CO2 through the stomata. This balance prevents unnecessary water loss on the one hand, or excessive limitation of photosynthesis on the other, and is maintained in large part by stomatal responses to light and intercellular CO2 concentration (ci). Under most circumstances, the degree of coordination between the stomatal supply of CO2 and the mesophyll demand for CO2 is very high; so high that it has prompted speculation that stomatal conductance is linked to mesophyll photosynthesis rate (Wong, Cowan & Farquhar 1979; Buckley, Mott & Farquhar 2003). Although there are indications that this might be the case (Lee & Bowling 1992; Messinger, Buckley & Mott 2006), plants with reduced rates of photosynthesis because of antisense reductions in the photosynthetic system seem to have normal stomatal responses (Price et al. 1998; Caemmerer et al. 2004), a result that seems inconsistent with an effect of the mesophyll. Most current literature assumes that the mesophyll has little or no effect on stomatal conductance, and that stomatal responses to light and CO2 reside primarily in the guard cells. Yet, the complete mechanisms for these responses remain elusive.
It is clear that in a leaf, stomata respond to both red and blue light. The blue-light response saturates at low fluences (around 50 µmol m−2 s−1), and is most pronounced in the presence of high-fluence red light (Shimazaki et al. 2007). The receptor for blue light is controversial. At least part of the blue-light response is caused by phototropins (Kinoshita et al. 2001; Marten, Hedrich & Roelfsema 2007), but there is also evidence that zeaxanthin also plays a role (Zeiger & Zhu 1998). It has been shown that blue light activates a plasma membrane ATPase in guard cells (Kinoshita & Shimazaki 1999), but little else is known about this pathway. The red-light response, which is actually a response to both red and blue light, is clearly mediated by the absorption of light by chlorophyll, and this response saturates at much higher fluences than the blue-light response (Shimazaki et al. 2007). In a leaf, some of the response to high fluence red light is undoubtedly due to reductions in ci. However, stomata respond to red light even when ci is maintained constant by adjusting the ambient CO2 concentration (Messinger et al. 2006), so other mechanisms must operate as well. Most current hypotheses for the red-light response centre on a role for guard cell chloroplasts (Tominaga, Kinoshita & Shimazaki 2001; Shimazaki et al. 2007).
Less is known about the response of stomata to CO2. The response is clearly to ci rather than to the ambient CO2 concentration (Mott 1988), but the sensor for this response is unknown (Vavasseur & Raghavendra 2005). It has been suggested several times that stomatal responses to light and CO2 could be accounted for by one mechanism if stomata responded to zeaxanthin (Zhu et al. 1998) or the balance between ribulose 1, 5 bisphosphate (RuBP) carboxylation and RuBP regeneration (Farquhar & Wong 1984; Buckley et al. 2003). Unequivocal evidence for either mechanism is lacking, however.
Much of the recent work on stomatal responses to CO2 and light has been performed using stomata in isolated epidermes. Although there are reports of stomatal responses to light or CO2 in isolated epidermes, these responses are quite different from the responses in leaves. For example, light-induced opening in isolated epidermes is typically investigated by incubating them in a buffer containing KCl, and then measuring apertures for an average of many stomata over time. In these studies, opening typically occurs over a time frame of several hours (e.g. Fischer 1968; Tallman & Zeiger 1988; Olsen et al. 2002), and although illuminated stomata open more rapidly, stomata in darkness often show some opening as well (Olsen et al. 2002). In contrast, stomatal responses to light in leaves are typically complete within 1 h. Stomata in leaves also close very rapidly in response to darkness, yet there are no published reports of stomata in isolated epidermes closing in response to darkness. Stomatal responses to CO2 in isolated epidermes are often small (Young et al. 2006) or nonexistent (Willmer & Mansfield 1970; Travis & Mansfield 1979; Lee & Bowling 1992), although there are some studies showing larger responses (e.g. Snaith & Mansfield 1982; Brearley, Venis & Blatt 1997).
The differences between stomatal responses to light and CO2 in leaves and those in isolated epidermes have been recognized in the literature (Travis & Mansfield 1979; Lee & Bowling 1992, 1995), and the validity of studying stomatal responses in isolated epidermes has been questioned (Willmer & Mansfield 1969). In addition to these inconsistencies, there are some studies suggesting that guard cells have little or no response to red light. For example, isolated guard cell protoplasts or guard cells in isolated epidermes respond to blue light but not to red light (Zeiger & Hepler 1977; Lee & Bowling 1993; Roelfsema et al. 2002). In addition, although individual guard cells do not appear to respond to red light, illuminating a wider area of the leaf around the stoma caused hyperpolarization of guard cells (Roelfsema et al. 2002).
In this study, we investigated the role of the mesophyll in stomatal responses to CO2 and light using leaves, isolated epidermes and isolated epidermes that had been placed over an exposed mesophyll from another leaf. We also used individual strands of optic fibre to illuminate small areas of a leaf. Our data suggest that for the plants used in this study, much of the stomatal response to light and CO2 depends on a signal from the mesophyll.
METHODS AND MATERIALS
Tradescantia pallida, Vicia faba and Pisum sativum were grown in a controlled environment greenhouse with day and night temperatures of 30 and 20 °C, respectively. Day length was extended to 16 h with high-pressure sodium lamps [photon flux density (PFD) approximately 1000 µmol m−2 s−1 at the top of the plant] when necessary. Plants were grown in 1 L pots containing peat:perlite: vermiculite (1:1:1 by volume), and were watered to excess daily with a dilute nutrient solution containing 9.1 mm N, 1.8 mm P, 2.7 mm K (Peter's 20-10-20; Grace Sierra Horticultural Products Co., Milpitas, CA, USA) and 11 µm chelated Fe. Plants used for experiments were at least 4 weeks old, and the leaves chosen for experiments were fully mature but not senescing. Isolated epidermes were prepared from the lower surface of all species by removing the upper epidermis and the mesophyll with a razor blade. Visual examination of these epidermes revealed that (1) all stomata were closed, (2) essentially no mesophyll cells remained, and (3) most epidermal cells were alive. The isolated epidermis was washed in distilled water and either (1) placed in a petri dish on filter paper saturated with distilled water or 50 mm KCl and 1 mm CaCl2, or (2) grafted to a section of exposed mesophyll from another leaf. In the latter case, we placed the isolated epidermes on top of and in contact with the exposed mesophyll. The petri dish was covered and placed in darkness for approximately 12 h, and then illuminated for approximately 3 h before use. Exposed mesophyll was produced by peeling away the lower epidermis of V. faba and P. sativum, and carefully removing the lower epidermis from a leaf with a razor blade for T. pallida.
Epidermes on wet filter paper or on exposed mesophyll were then mounted in a treatment chamber. The chamber and the experimental setup have been described previously (Shope, Peak & Mott 2008). Briefly, it consisted of two identical aluminum compartments separated by the epidermis or leaf (depending on the experiment). The top of the upper compartment and the bottom of the lower compartment were made of optical quality glass, and the temperature of the two compartments was controlled to be 23 °C by circulating water from a temperature-controlled bath through water ports in the aluminum body. The lower chamber (next to the filter paper or adaxial epidermis of the exposed mesophyll, depending on the experiment) was sealed from the outside air and was maintained nearly saturated with water vapour by placing wet tissue paper in the chamber. The air in the upper chamber was provided from compressed-gas tanks of CO2-free air and 1% CO2 in air. These two gases were mixed using mass flow controllers, and the CO2 concentration of the resulting mixture was determined with a CO2 infrared gas analyser. The gas mixture was humidified by bubbling it through distilled water at 35 °C, and then passing it through a helical condenser at 22.9 °C. The flow rate through the chamber was approximately 1 L min−1. For experiments using leaves, the leaf was placed upside down (abaxial surface up) in the chamber described previously. The leaf, epidermis, or grafted epidermis was illuminated from above with a fibre-optic illuminator that utilized a xenon bulb (Schott, Elmsford, NY, USA). The PFD was 340 µmol m−2 s−1.
For experiments in which a single stoma was illuminated with an individual strand of optic fibre, the leaf was placed upside down on the bottom compartment of the chamber, and was held down with an aluminum ring. Air from the gas-mixing system was then passed over the abaxial surface of the leaf by placing a fan-shaped manifold on the end of the gas tube and positioning the manifold next to the leaf surface. Optic fibres (Polymicro Technologies, Phoenix, AZ, USA) were positioned near the leaf surface using a micromanipulator. Light was provided to the optic fibres by focusing a halogen light source on the end of the fibre using a convex lens. When the entire area of the leaf in the chamber was to be illuminated, the fibre-optic illuminator described previously was used.
To match the intensity of the single optic fibre to the PFD provided by the fibre-optic illuminator (340 µmol m−2 s−1), we used a peltier-cooled digital camera (CoolSnap HQ; Roper Scientific, Photometrics, Tucson, AZ, USA) to acquire a greyscale image of white paper while it was illuminated with the fibre-optic illuminator. Integration time was adjusted to give a greyscale value midway between black and white. Using the same setup, we acquired images of the bright spot produced by the single fibre optic, and adjusted the light source until the greyscale intensity matched that produced by the fibre-optic illuminator.
In all experiments, the CCD camera mentioned previously was used to capture images of the stomata, and apertures were measured using a digital image processing software (ImagePro; Media Cybernetics, Bethesda, MD, USA). Two protocols were used for experiments in dark. In some experiments, a low intensity of light above 700 nm was provided to the leaf using a 700 nm high-pass filter (no. 7-69; Kopp, Pittsburgh, PA, USA). The peltier-cooled CCD camera was sensitive to wavelengths above 700 nm and could acquire images in the absence of light below 700 nm. In other experiments, white light was applied to the leaf for approximately 15 s while images were acquired. No difference in stomatal behavior was noted for the experiments using these two protocols.
In the experiments shown in Figs 1 through 9, PFD was changed sequentially from 340 to 0, and then back to 340 µmol m−2 s−1. CO2 concentration was then changed sequentially from 120 to 540, and back to 120 µmol mol−1. One hour was allowed for stomata to respond to darkness or to 540 µmol mol−1 CO2. If there was no response to darkness, only 30 min were allowed for a stomatal response to the increase in PFD to 340 µmol m−2 s−1. Each figure shows the results from one experiment, but all experiments were repeated at least three times using different plant material with similar results. Variation in the individual experiments is discussed below.
Stomata in leaves of T. pallida were found to respond rapidly and reversibly in response to light and CO2 (Fig. 1). In all experiments, stomata closed by at least 70% (usually 100%) within 30 min when PFD was reduced from 340 µmol m−2 s−1 to darkness, and stomata closed by at least 30% within 1 h when CO2 was increased from 120 to 540 µmol mol−1. Stomata reopened when PFD was returned to 340 µmol m−2 s−1 or when CO2 was returned to 120 µmol mol−1. Although reopening was sometimes not complete – that is, apertures did not return completely to the values before the closing response – there was always a clear response of aperture to each perturbation. These responses were always apparent within a few minutes of a change in conditions and were typically complete within 1 h. The only exception to this was an occasional lag between re-illumination and the beginning of the opening response, but this lag never exceeded 20 min. It is notable that stomata closed completely in darkness but remained slightly open in high CO2.
In contrast to leaves, stomata in isolated epidermes of T. pallida were found to be completely insensitive to darkness and high CO2. The data shown in Fig. 2 are for epidermes that were incubated on 50 mm KCl and 1 mm CaCl2. However, we performed experiments with epidermes that were incubated on distilled water with identical results (data not shown). We also investigated the effect of leaving the epidermis wet with the incubation solution or blotting it dry before placing it in the chamber, but this had no effect on the data. Finally, we tried various humidity treatments for the outer surface, and we tried placing the epidermis on a piece of filter paper that was saturated with either KCl solution or distilled water. No treatment or combination of treatments was found that produced any measurable response to CO2 and light. The stomata in these epidermes were sensitive to changes in humidity as demonstrated in a previous study (Shope, Peak & Mott 2008), and the guard cells were clearly turgid.
To investigate the role of the mesophyll in stomatal responses, we grafted isolated epidermes of T. pallida to mesophyll from another T. pallida leaf. Stomata in these epidermes responded to darkness and CO2 in a manner that was similar to the response of stomata in T. pallida leaves (Fig. 3). Stomata closed completely in response to darkness, reopened in response to light, and closed by at least 30% in response to high CO2. In experiments with grafted epidermes, it was important that good contact was maintained between the exposed mesophyll and the isolated epidermis, and that no liquid water was present on either the mesophyll or the epidermis. Not every grafted epidermis showed open stomata, and not all stomata were open on any given epidermis, but all stomata that opened on any particular graft responded rapidly and reversibly to light and CO2 as shown in Fig. 3.
Remarkably, stomata also responded to light and CO2 in epidermes from T. pallida that were placed on an exposed mesophyll from V. faba (Fig. 4). Responses to light and CO2 in these epidermes were similar to those for leaves or for the T. pallida epidermes that were placed on exposed mesophyll of T. pallida. When these responsive grafted epidermes were separated from the mesophyll and were placed on a wet filter paper, stomata closed immediately and did not open within 3 h despite continuous illumination (data not shown).
Stomata in leaves of V. faba did not show a measurable response to light or CO2 within the first 60 min of a change in conditions (Fig. 5). The same was true for stomata in isolated epidermes (Fig. 6) or epidermes that were grafted to mesophyll from V. faba (data not shown). Even grafting an epidermis of V. faba to mesophyll from T. pallidum did not cause the stomata to respond to either light or CO2 (Fig. 7).
To be sure that the results with the grafted epidermes of T. pallida were not unique to that species, we performed additional experiments with P. sativum. Leaves of P. sativum were found to respond to light and CO2 in a manner similar to that found in T. pallida (Fig. 8). Although stomata did not close completely in response to darkness in all experiments, they always showed at least a 50% reduction in aperture within 30 min, and they always showed at least a 30% reduction in aperture in response to high CO2. In contrast, isolated epidermes showed no measurable response to either light or CO2 (data not shown). Epidermes from P. sativum that were grafted onto mesophyll from T. pallida (Fig. 9) or V. faba (data not shown) showed rapid and reversible responses to light and CO2 that were similar to those of leaves.
To further investigate the role of the mesophyll, we used intact leaves to compare the effect of illuminating a single stoma with the effect of illuminating a single stoma and a small area of the leaf surrounding it (Fig. 10). Before the experiment, the leaf was placed in the light (300 µmol m−2 s−1) at a CO2 concentration of 120 µmol mol−1 and a nearly saturating value of humidity to be sure that the stomata in the field of view would open. As soon as stomata were open more than 10 µm, the light was turned out, and stomata were allowed to close for approximately 1 h, after which all stomata in the field of view were closed. Light was then applied to a single stoma with a 40 µm optic fibre, which produced an illuminated area approximately 100 µm in diameter. Because the guard cells of T. pallida are about 75 µm long, this provided light to both guard cells but minimized the area around the stoma that was illuminated. No opening of the stoma was observed within 6 h of applying the light (data not shown). This experiment was then repeated, but only 1 h was allowed for the illuminated stoma to open, after which the entire portion of the leaf in the chamber was illuminated at 300 µmol m−2 s−1 using the fibre-optic illuminator (Fig. 10). The single illuminated stoma did not respond to the 1 h illumination, but when the entire leaf area was illuminated, all stomata in the field opened rapidly. Next, we turned off the illumination for the leaf, but kept the single stoma illuminated with the optic fibre. All stomata in the field, including the one illuminated with the 40 µm fibre, closed rapidly (Fig. 10). Finally, we illuminated the same single stoma with a 200 µm fibre optic, which produced an illuminated area of approximately 400 µm in diameter. The single illuminated stoma opened, and several neighbouring stomata, which were not illuminated by the optic fibre, also opened (Fig. 10). This experiment was repeated three times with similar results.
Stomata in isolated epidermes of T. pallida, P. sativum and V. faba used in this study did not show measurable responses to light and CO2, but stomata in leaves of T. pallida and P. sativum did respond rapidly and reversibly to these factors. When an isolated epidermis of T. pallida or P. sativum was grafted to the mesophyll of another leaf of the same species, the response of stomata to these factors was restored. Remarkably, functional grafts with stomata that responded rapidly and reversibly to CO2 and light could even be produced between an epidermis of T. pallida or P. sativum and the mesophyll of another species.
Although stomata in leaves of T. pallida and P. sativum showed a clear response to light and CO2, those of V. faba did not. The insensitivity of stomata in leaves of V. faba is unusual, but reports of stomata in leaves that are insensitive to light and CO2 can be found in the literature, particularly for well-watered greenhouse plants (Raschke 1975), and we have found this species to be unresponsive to CO2 and light in previous experiments (data not shown). It is noteworthy that stomata in the epidermes of V. faba did not respond to CO2 or light when grafted to the mesophyll from V. faba or the other two species. Yet, stomata in epidermes from T. pallida and P. sativum did respond to CO2 and light when grafted to the mesophyll of V. faba.
The data for isolated and grafted epidermes suggest that the mesophyll is necessary for stomata to respond to light and CO2. There are at least two plausible explanations for the effect of the mesophyll. Firstly, it is possible that something produced by the mesophyll sensitizes guard cells to CO2 and light. Secondly, it is possible that the mesophyll generates a signal (or signals) in response to light and CO2 that regulates stomatal aperture. These two scenarios, although similar, differ in one important aspect. In the first scenario, the guard cells contain the sensory mechanisms for light and CO2. In the latter, the sensory mechanisms reside in the mesophyll, and one or more signals must be passed from the mesophyll to the guard cells.
The results of the experiments in which a single stoma was illuminated (Fig. 10) support the second hypothesis, that is, that the mesophyll is the sensory mechanism for light and CO2, and that stomata respond via a signal from the mesophyll. If the mesophyll were simply sensitizing the guard cells to CO2 and light, then illuminating a single guard cell pair in a leaf should cause that stoma, and not others, to open. However, our results show that the illuminated stoma opened only if the surrounding mesophyll was also illuminated, and that neighbouring, non-illuminated stomata also opened when the mesophyll was illuminated.
These results are consistent with a similar experiment in which illuminating a single guard cell with red light did not induce a change in membrane potential, but illuminating an area of the mesophyll around the guard cell did cause hyperpolarization of the guard cell membrane (Roelfsema et al. 2002). In that study, the mesophyll effect was attributed to a reduction in ci caused by light-induced photosynthetic CO2 uptake. Several studies, however, argue against this interpretation. Firstly, stomata have been shown to respond to light even when ci is maintained constant by manipulating ambient CO2(Messinger et al. 2006). Secondly, the response of stomata to ci and the response of ci to light are both too small to produce the large changes in stomatal conductance that are observed in response to light (Sharkey & Raschke 1981). Data in this study support this because increasing ambient CO2 from 120 to 540 ppm did not cause stomata to close completely, while darkness did cause them to close completely. Gas exchange experiments on similar leaves showed that ci increased only to about 200 ppm when leaves were darkened (data not shown).
The fact that stomata in isolated epidermes of all three species used in this study were completely insensitive to light and CO2 despite extensive efforts to find conditions that produced a response might seem unusual, but it does not directly contradict any studies of which we are aware. Most studies on light-induced opening of stomata in isolated epidermes show opening rates for epidermes floating on KCl solutions. In these studies, illuminated stomata typically open slowly over the course of several hours (e.g. Fischer 1968; Tallman & Zeiger 1988; Olsen et al. 2002), and control treatments in the dark sometimes show stomatal opening, albeit slower than the illuminated treatment (Olsen et al. 2002). In contrast to these studies, we used epidermes that had been incubated on KCl or distilled water for over 12 h and for which the stomata were already open. We then, unsuccessfully, attempted to close these stomata with darkness or high CO2, and reopen them with light or low CO2. The stomata in leaves of T. pallida and P. sativum responded rapidly and reversibly to these perturbations, but isolated epidermes did not. Indeed, we could find no data in the literature showing rapid responses of stomata in isolated epidermes to light, and this lack of rapid responses to light has been previously noted as a major difference between stomata in leaves and stomata in isolated epidermes (Lee & Bowling 1995). Similarly, there are very few reports of a CO2 response in stomata in isolated epidermes (Snaith & Mansfield 1982; Brearley et al. 1997), and these studies typically show a much smaller response in isolated epidermes than in leaves (Webb et al. 1996; Young et al. 2006). Based on our results, we suggest that the processes responsible for stomatal opening in leaves are different from those causing stomatal opening in isolated epidermes floating on KCl solutions.
Based on the results discussed previously, we speculate that the mesophyll produces an opening signal in proportion to light intensity and inversely proportional to CO2 concentration. This signal appears to be diffusible over short distances because in the experiment shown in Fig. 10, unilluminated stomata near the illuminated area opened in addition to illuminated stoma. The fact that the mesophyll from V. faba can regulate stomata in the epidermes from T. pallida and P. sativum suggests that the signal is common among these three plants. In addition, because T. pallida is a monocot in the family Commelinaceae, and V. faba and P. sativum are dicots in the family Fabaceae, it seems possible that the signal is common across a wide range of taxa. It is noteworthy that epidermes from leaves of V. faba were not sensitive to light and CO2 even when placed on the mesophyll of T. pallida or P. sativum. In contrast, the mesophyll of V. faba was able to produce stomatal responses in epidermes of both P. sativum and T. pallida. This shows that the mesophyll from all three species produced the signal, and that the insensitivity of the stomata in leaves of V. faba to light and CO2 was caused by the inability of V. faba guard cells to respond to the mesophyll signal.
We did not specifically investigate the responses of the stomata to blue and red light in this study. However, we suggest that our results pertain primarily to the red-light response of the stomata for two reasons. Firstly, in contrast to red light, blue light has been shown to have direct effects on the plasma membrane (Roelfsema et al. 2001; Marten et al. 2007) and ATPase activity (Kinoshita & Shimazaki 1999) in isolated guard cells. Secondly, the blue-light response is typically most pronounced in the presence of high-intensity red light and therefore may not have been discernable in our experiments because light was changed from darkness to a relatively high intensity.
In summary, the data presented in this study suggest that most of the stomatal response to CO2 and light in leaves is the result of a signal from the mesophyll rather than a direct response of guard cells to CO2 and light. This signal appears to cause stomata to open, and its presence in leaves may represent a fundamental difference between stomatal functioning in leaves and that in isolated epidermes floated on KCl solutions. The identity of this signal and the mechanism by which it affects guard cells await further research.
We thank Tom Buckley for the original suggestion to try placing epidermes back on mesophyll. We thank Rand Hooper and Jason Jacobs for excellent technical assistance.