The guard cell chloroplast: a perspective for the twenty-first century.

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 CO2 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 CO2 , 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.

Reversal by green light of blue light-stimulated stomatal opening in intact, attached leaves of Arabidopsis operates only in the potassium-dependent, morning phase of movement.

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.

The blue light-specific response of Vicia faba stomata acclimates to growth environment.

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.

Relative humidity is a key factor in the acclimation of the stomatal response to CO(2).

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.

The CO(2) response of Vicia guard cells acclimates to growth environment.

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

Blue light and phytochrome-mediated stomatal opening in the npq1 and phot1 phot2 mutants of Arabidopsis.

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 micromol m-2 s-1, red light (photosynthetic)-mediated opening in isolated stomata from wild type (WT) and both mutants saturated at 100 micromol 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 micromol m-2 s-1) to a 100 micromol m-2 s-1 red background, was found in WT, but not in npq1 or phot1 phot2 double mutant stomata. Under 50 micromol m-2 s-1 red light, 10 micromol 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 micromol 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.

Green light reversal of blue-light-stimulated stomatal opening is found in a diversity of plant species.

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.

Phytochrome and blue light-mediated stomatal opening in the orchid, paphiopedilum.

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.