Green light reversal of blue light-stimulated stomatal opening was discovered in isolated stomata. The present study shows that the response also occurs in stomata from intact leaves. Arabidopsis thaliana plants were grown in a growth chamber under blue, red and green light. Removal of the green light opened the stomata and restoration of green light closed them to baseline values under experimental conditions that rule out a mesophyll-mediated effect. Assessment of the response to green light over a daily time course showed that the stomatal sensitivity to green light was observed only in the morning, which coincided with the use of potassium as a guard cell osmoticum. Sensitivity to green light was absent during the afternoon phase of stomatal movement, which was previously shown to be dominated by sucrose osmoregulation in Vicia faba. Hence, the shift away from potassium-based osmoregulation in guard cells is further postulated to entail a shift from blue light to photosynthesis as the primary component of the stomatal response to light. Stomata from intact leaves of the zeaxanthin-less, npq1 mutant of Arabidopsis failed to respond to the removal or restoration of green light in the growth chamber, or to short, high fluence pulses of blue or green light. These data confirm previous studies showing that npq1 stomata are devoid of a specific blue light response. In contrast, stomata from intact leaves of phot1 phot2 double mutant plants had a reduced but readily detectable response to the removal of green light and to blue and green pulses.

Stomata in epidermal strips from growth chamber-grown Vicia faba leaves opened less in response to white light than did stomata from greenhouse-grown leaves. Chlorophyll-mediated, red light-stimulated opening was similar in stomata from the two growth conditions, but stomata from the growth chamber environment had a severely reduced response to blue light. Transfer of plants between the two growth conditions resulted in an acclimation of the stomatal blue light response. Stomata lost blue light sensitivity within 1 d of transfer to growth chamber conditions and gained sensitivity to blue light over an 8 d period after transfer to a greenhouse. Short-term transfer experiments confirmed that the rapid loss of blue light sensitivity was an acclimation response, requiring between 12 and 20 h exposure to growth chamber conditions. The acclimation of the stomatal response to blue light was inversely related to a previously reported acclimation response in which stomata change between high CO2 sensitivity under growth chamber conditions and low CO2 sensitivity under greenhouse conditions. The time courses of the blue light and CO2 acclimation responses were virtually identical, suggesting the possibility of a common acclimation mechanism.

Recent studies have shown that blue light-specific stomatal opening is reversed by green light and that far-red light can be used to probe phytochrome-dependent stomatal movements. Here, blue-green reversibility and far-red light were used to probe the stomatal responses of the npq1 mutant and the phot1 phot2 double mutant of Arabidopsis. In plants grown at 50 μmol m-2 s-1, red light (photosynthetic)-mediated opening in isolated stomata from wild type (WT) and both mutants saturated at 100 μmol m-2 s-1. Higher fluence rates caused stomatal closing, most likely due to photo-inhibition. Blue light-specific opening, probed by adding blue light (10 μmol m-2 s-1) to a 100 μmol m-2 s-1 red background, was found in WT, but not in npq1 or phot1 phot2 double mutant stomata. Under 50 μmol m-2 s-1 red light, 10 μmol m-2 s-1 blue light opened stomata in both WT and npq1 mutant stomata but not in the phot1 phot2 double mutant. In npq1, blue light-stimulated opening was reversed by far-red but not green light, indicating that npq1 has a phytochrome-mediated response and lacks a blue light-specific response. Stomata of the phot1 phot2 double mutant opened in response to 20 to 50 μmol m-2 s-1 blue light. This opening was green light reversible and far-red light insensitive, indicating that stomata of the phot1 phot2 double mutant have a detectable blue light-specific response.

Recent studies have shown that blue light-specific stomatal opening is reversed by green light and that far-red light can be used to probe phytochrome-dependent stomatal movements. Here, blue-green reversibility and far-red light were used to probe the stomatal responses of the npq1 mutant and the phot1 phot2 double mutant of Arabidopsis. In plants grown at 50 mumol m(-2) s(-1), red light (photosynthetic)-mediated opening in isolated stomata from wild type (WT) and both mutants saturated at 100 mumol m(-2) s(-1). Higher fluence rates caused stomatal closing, most likely due to photo-inhibition. Blue light-specific opening, probed by adding blue light (10 mumol m(-2) s(-1)) to a 100 mumol m(-2) s(-1) red background, was found in WT, but not in npq1 or phot1 phot2 double mutant stomata. Under 50 mumol m(-2) s(-1) red light, 10 mumol m(-2) s(-1) blue light opened stomata in both WT and npql mutant stomata but not in the phot1 phot2 double mutant. In npql, blue light-stimulated opening was reversed by far-red but not green light, indicating that npql has a phytochrome-mediated response and lacks a blue light-specific response. Stomata of the phot1 phot2 double mutant opened in response to 20 to 50 mumol m(-2) s(-1) blue light. This opening was green light reversible and far-red light insensitive, indicating that stomata of the phot1 phot2 double mutant have a detectable blue light-specific response.

Previous work has shown that stomata of growth chamber-grown Vicia faba leaves have an enhanced CO2 response when compared with stomata of greenhouse-grown plants. This guard cell response to CO2 acclimatizes to the environmental conditions on the transfer of plants between the two environments. In the present study, air relative humidity is identified as a key environmental factor mediating the changes in stomatal sensitivity to CO2. In the greenhouse environment, elevation of relative humidity to growth chamber levels resulted in an enhanced CO2 response, whereas a reduction in the light level to that comparable to growth chamber conditions had no effect on stomatal CO2 sensitivity. The transfer of plants between humidified and normal greenhouse conditions resulted in an acclimation response with a time-course matching that previously obtained in transfers of plants between greenhouse and growth chamber environments. The high stomatal sensitivity to CO2 of growth chamber-grown plants could be reduced by lowering growth chamber relative humidity and then restored with its characteristic acclimation time-course by an elevation of relative humidity. Leaf temperature was unchanged during this restoration, eliminating it as a primary factor in the acclimation response. Humidity regulation of stomatal CO2 sensitivity could function as a signal for leaves inside dense foliage canopies, promoting stomatal opening under low light, low CO2 conditions.

Reversal by green light of blue-light-stimulated stomatal opening was found across a number of plant species, including leguminous and nonleguminous dicots and grass and nongrass monocots. Simultaneous exposure to equal fluence rates of blue and green light resulted in ∼50% reversal of normal blue light opening. Complete reversal occurred when the fluence rate of green light was approximately twice that of blue light. These results suggest that blue-green reversibility of stomatal opening is a basic photobiological property of guard cells. The blue-green reversibility of stomatal opening has been hypothesized to ensue from the cycling of two interconvertible, isomeric forms of the blue-light photoreceptor, zeaxanthin. Testing of blue-green reversibility could provide a valuable diagnostic tool for zeaxanthin-mediated blue-light photoperception.

Stomata of growth chamber-grown Vicia faba leaves have an enhanced CO2 response, measured as change in stomatal aperture, compared to stomata of greenhouse-grown leaves. Reciprocal transfer experiments showed that the stomatal response to CO2 acclimated to the growing environment. Stomata of growth chamber-grown leaves transferred to a greenhouse lost their high CO2 sensitivity within 2-3 d while stomata of greenhouse-grown leaves transferred to a growth chamber acquired a high CO2 sensitivity within 5-7 d. Experiments measuring the CO2 responses of stomata in detached epidermis showed that growth chamber and greenhouse-grown stomata have the same contrasting CO2 sensitivity observed in the intact leaf, indicating that the responses reflect intrinsic guard cell properties. The acclimation properties of the CO2 response of guard cells have implications for the understanding of stomatal function under the predicted increases in atmospheric CO2.

Guard cells of the orchid genus, Paphiopedilum have been reported to lack developed chloroplasts and detectable chlorophyll a autofluorescence. Paphiopedilum stomata lack a photosynthesis-dependent opening response but have a blue light-specific opening. The present study found that low fluence rate green and red light elicited stomatal opening in Paphiopedilum and this opening was reversed by far red light, indicating the presence of a phytochrome-mediated opening response. Phytochrome-dependent, red light-stimulated opening was largest under low fluence rates and decreased to near zero as fluence rate increased. A recently discovered green light reversibility of blue light-specific stomatal opening was used to probe the properties of the blue light response in Paphiopedilum stomata. Blue light-stimulated opening was completely reversed by green light in the presence of far red light. Red light enhanced the blue light response of Paphiopedilum guard cells when given as a pretreatment or together with blue light. Analysis of guard cell pigments showed that guard cells have small amounts of chlorophyll a and b, zeaxanthin, violaxanthin, antheraxanthin and lutein. Zeaxanthin content increased in response to blue light or ascorbate and declined in the dark or under illumination in the presence of dithiothreitol, indicating the presence of an active xanthophyll cycle. Thus Paphiopedilum stomata possess both a blue light-mediated opening response with characteristics similar to species with normal chloroplast development and a novel phytochrome-mediated opening response.

Summary The guard cell chloroplast is the site of perception of blue light and of photosynthetically active radiation, and of at least one of the mechanisms sensing CO 2 in the guard cell. The guard cell chloroplast has been the focus of intense controversy over its capacity for light sensing and photosynthetic carbon fixation, and the osmoregulatory mechanisms mediating stomatal movements. It is argued here that a primary reason behind these long‐lived controversies is the remarkable plasticity of the guard cell, which has resulted in responses being generalized as basic properties when opposite responses appear to be the norm under different environmental or experimental conditions. Examples of guard cell plasticity are described, including variation of chlorophyll fluorescence transients over a daily course, acclimation of the guard cell responses to blue light and CO 2 , the shift from potassium to sucrose in daily courses of osmoregulation and the transduction of red light into different osmoregulatory pathways. Recent findings on the properties of the guard cell chloroplast are also presented, including the role of the chloroplastic carotenoid, zeaxanthin, in blue light photoreception, the blue‐green reversibility of stomatal movements, and the involvement of phytochrome in the stomatal response to light in the orchid, Paphiopedilum .

Blue light-stimulated stomatal opening in detached epidermis of Vicia faba is reversed by green light. A 30 s green light pulse eliminated the transient opening stimulated by an immediately preceding blue light pulse. Opening was restored by a subsequent blue light pulse. An initial green light pulse did not alter the response to a subsequent blue light pulse. Reversal also occurred under continuous illumination, with or without a saturating red light background. The magnitude of the green light reversal depended on fluence rate, with full reversal observed at a green light fluence rate twice that of the blue light. Continuous green light given alone stimulated a slight stomatal opening, and had no effect on red light-stimulated opening. An action spectrum for the green light effect showed a maximum at 540 nm and minor peaks at 490 and 580 nm. This spectrum is similar to the action spectrum for blue light-stimulated stomatal opening, red-shifted by about 90 nm. The carotenoid zeaxanthin has been implicated as a photoreceptor for the stomatal blue light response. Blue/green reversibility might be explained by a pair of interconvertible zeaxanthin isomers, one absorbing in the blue and the other in the green, with the green absorbing form being the physiologically active one.

The Arabidopsis mutant npq1, which cannot accumulate zeaxanthin because of a defective violaxanthin deepoxidase, was used to investigate the role of zeaxanthin in the stomatal response to blue light. Neither dark-adapted nor light-treated guard cells or mesophyll cells of the npq1 mutant contained detectable zeaxanthin. In contrast, wild-type guard cells had a significant zeaxanthin content in the dark and accumulated large amounts of zeaxanthin when illuminated. The well-documented red light enhancement of blue light-stimulated stomatal opening, in which increasing fluence rates of background red light result in increased response to blue light, was used to probe the specific blue light response of Arabidopsis stomata. Stomata from the npq1 mutant did not have a specific blue light response under all fluence rates of background red light tested. On the other hand, stomata from leaves of hy4 (cry 1), an Arabidopsis mutant lacking blue light-dependent inhibition of hypocotyl elongation, had a typical enhancement of the blue light response by background red light. The lack of a specific blue light response in the zeaxanthinless npq1 mutant provides genetic evidence for the role of zeaxanthin as a blue light photoreceptor in guard cells.

The mechanisms mediating CO2 sensing and light–CO2 interactions in guard cells are unknown. In growth chamber‐grown Vicia faba leaves kept under constant light (500 μmol m–2 s–1) and temperature, guard cell zeaxanthin content tracked ambient [CO2] and stomatal apertures. Increases in [CO2] from 400 to 1200 cm3 m–3 decreased zeaxanthin content from 180 to 80 mmol mol–1 Chl and decreased stomatal apertures by 7·0 μm. Changes in zeaxanthin and aperture were reversed when [CO2] was lowered. Guard cell zeaxanthin content was linearly correlated with stomatal apertures. In the dark, the CO2‐induced changes in stomatal aperture were much smaller, and guard cell zeaxanthin content did not change with chamber [CO2]. Guard cell zeaxanthin also tracked [CO2] and stomatal aperture in illuminated stomata from epidermal peels. Dithiothreitol (DTT), an inhibitor of zeaxanthin formation, eliminated CO2‐induced zeaxanthin changes in guard cells from illuminated epidermal peels and reduced the stomatal CO2 response to the level observed in the dark. These data suggest that CO2‐dependent changes in the zeaxanthin content of guard cells could modulate CO2‐dependent changes of stomatal apertures in the light while a zeaxanthin‐independent CO2 sensing mechanism would modulate the CO2 response in the dark.

Stomatal apertures are regulated by changes in the solute content of guard cells. The identity of the solutes involved in guard cell osmoregulation has been the subject of much study. Early consensus in the field held that carbohydrates derived from starch constitute the principal osmoticum. This starch-sugar hypothesis has been replaced by the present paradigm of guard cell osmoregulation by K+ and its counterions. Recent studies, however, show that both K+ and sucrose are primary guard cell osmotica, and that the use of these two solutes is separated into two distinct phases in which one or the other constitutes the dominant osmoticum. In the intact leaf, opening at the beginning of a daily cycle is supported by K+ and its counterions, malate(2-) and Cl-. Malate(2-) is the dominant counterion in growth chamber-grown leaves, whereas Cl- predominates in a greenhouse environment. In the second half of the daily cycle, K+ content in guard cells decreases drastically and sucrose becomes the dominant solute. Manipulation of stomatal apertures by altering ambient CO2 concentration shows that either K+ or sucrose accumulation can sustain rapid opening. The functional implications of two distinct osmoregulatory phases of stomatal movements remains to be elucidated. The guard cell content of K+, its counterions, and sucrose can be modulated by at least three osmoregulatory pathways in guard cells. Experimental conditions favouring three distinct pathways have been established, but major uncertainties remain about the control of guard cell solute content in the intact leaf.

Abaxial stomata from Vicia faba leaves grown in a growth chamber under constant light, temperature and humidity showed an elaborate pattern of aperture changes over the course of a light cycle. These aperture changes were tightly correlated with changes in chamber CO2 concentration (r2=0.83). Changes in chamber [CO2] resulted, in turn, from substantial daily fluctuations in ambient [CO2], typical of the Los Angeles environment, with a constant offset caused by photosynthesis and respiration of the plants within the chamber. The dominant role of the stomatal response to CO2 in the control of aperture was confirmed by manipulation of chamber [CO2]. Fast (15 min) increases and decreases in [CO2] caused rapid decreases and increases in aperture, while constant [CO2] resulted in constant aperture. In contrast, aperture changes in comparable plants grown under greenhouse conditions were tightly correlated with changes in incident solar radiation (r2=0.80), and poorly correlated with changes in [CO2] (r2=0.09). Greenhouse-grown plants transferred to growth chamber conditions showed no apparent response to CO2. These data indicate that growth-chamber-grown V. faba leaves provide an experimental system optimally suited for the study of the stomatal response to CO2, and suggest that acclimation to environmental conditions alters the sensitivity of stomata to CO2.

Solute content of stomata from intact onion cotyledons grown under either greenhouse or growth chamber conditions was followed over the course of a daily light cycle to determine patterns of osmoregulation. Initial opening of stomata was well correlated with guard cell potassium accumulation under both growth conditions. Subsequently, however, there was a consistent decrease in guard cell potassium content despite constant or increasing aperture. Although a secondary increase in potassium was sometimes observed during the second half of the light cycle, guard cell potassium content was poorly correlated with aperture, Sucrose levels in guard cells increased 60% during the period of declining potassium content, suggesting its use as an alternate osmoticum. Guard cells are postulated to use multiple pathways for the production of osmotica over the course of a complete daily cycle of stomatal movements.

Osmoregulation in guard cells of intact, attached Vicia faba leaves grown under growth chamber and greenhouse conditions was studied over a daily light cycle of stomatal movements. Under both growth conditions guard cells had two distinct osmoregulatory phases. In the first (morning) phase, opening was correlated with K+ uptake and, to a lesser extent, sucrose accumulation. In the second (afternoon) phase, in which apertures were maximal, K+ content declined and sucrose became the dominant osmoticum. Reopening of the stomata after a CO2-induced closure was accompanied by accumulation of either K+ or sucrose, depending on the time of day, indicating that a single environmental signal may use multiple osmoregulatory pathways. Malate accumulation, correlated with K+ uptake, was detected under growth chamber but not greenhouse conditions, whereas Cl- was the main K+ counterion in the greenhouse. These results indicate that guard-cell osmoregulation in the intact leaf depends on at least two different osmoregulatory pathways, K+ transport and sucrose metabolism. Furthermore, the relative importance of the K+ counterions malate and Cl- appears to be environment-dependent.