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.     

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.     

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.     

Interactions between a blue-green reversible photoreceptor and a separate UV-B receptor in stomatal guard cells

Stomatal opening exhibits two main peaks of activity in the visible range-a red peak, mediated by photosynthesis, and a blue peak, mediated by one or more blue light (BL) photoreceptors. In addition, a pronounced peak in the UV-B region has been characterized, as has a smaller UV-A peak. The BL-induced stomatal opening can be reversed by green light (GL). Here we report that UV-B-induced opening is also antagonized by GL. To determine whether UV-B is being absorbed by the BL photoreceptor or by a separate UV-B receptor, the UV-B responses of two different Arabidopsis mutants, npq1 and phot1/phot2, were tested. Both putative BL-photoreceptor mutants exhibited normal stomatal opening in response to UV-B, consistent with the existence of a separate UV-B photoreceptor. Moreover, GL failed to antagonize UV-B-induced stomatal opening in the phot1/phot2 double mutant and only partially antagonized UV-B opening in npq1. Thus, both phot1 and phot 2, as well as zeaxanthin, are required for the normal GL inhibition of UV-B. A model for a photoreceptor network that regulates stomatal opening is presented. Unlike the situation in guard cells, the UV-B bending response of Arabidopsis hypocotyls during phototropism appears to be mediated by phototropins.     

Does photorespiration protect the photosynthetic apparatus in french bean leaves from photoinhibition during drought stress?

Dithiothreitol (DTT), an inhibitor of violaxanthin de-epoxidation and zeaxanthin formation in chloroplasts, inhibited blue-light-stimulated stomatal opening in epidermal peels of Vicia faba L. in a concentration-dependent fashion. Complete inhibition was observed at 3 mM DTT. The DTT effect was specific for the stomatal response to blue light, and the red-light-stimulated opening, which depends on photosynthetic reactions in the guard cells, was unaffected. Preirradiation of stomata in epidermal peels with increasing photon fluence rates of red light, prior to an incubation in 10 mol·m^-2·s^-1 of blue light and 100 mol·m^-2·s^-1 red light, resulted in a DTT-sensitive, blue-light-stimulated opening that was proportional to the fluence rate of the red light pre-treatment. Guard cells in epidermal peels and guard-cell protoplasts irradiated with red light showed increases in their zeaxanthin content that depended on the fluence rate of red light, or on the incubation time. The increases in zeaxanthin concentration were inhibited by DTT. The obtained results indicate that zeaxanthin could function as a photoreceptor mediating the stomatal responses to blue light.Abbreviation DTT dithiothreitol This work was supported by grants from the National Science Foundation and the US Department of Energy to E.Z.     

Induction of CAM in Mesembryanthemum crystallinum Abolishes the Stomatal Response to Blue Light and Light-Dependent Zeaxanthin Formation in Guard Cell Chloroplasts

Facultative CAM plants such as Mesembryanthemum crystallinum(ice plant) possess C3 metabolism when unstressed but developCAM under water or salt stress. When ice plants shift from C3metabolism to CAM, their stomata remain closed during the dayand open at night. Recent studies have shown that the stomatalresponse of ice plants in the C3 mode depends solely on theguard cell response to blue light. Recent evidence for a possiblerole of the xanthophyll, zeaxanthin in blue light photoreceptionof guard cells led to the question of whether changes in theregulation of the xanthophyll cycle in guard cells parallelthe shift from diurnal to nocturnal stomatal opening associatedwith CAM induction. In the present study, light-dependent stomatalopening and the operation of the xanthophyll cycle were characterizedin guard cells isolated from ice plants shifting from C3 metabolismto CAM. Stomata in epidermis detached from leaves with C3 metabolismopened in response to white light and blue light, but they didnot open in response to red light. Guard cells from these leavesshowed light-dependent conversion of violaxan-thin to zeaxanthin.Induction of CAM by NaCI abolished both white light- and bluelight-stimulated stomatal opening and light-dependent zeaxanthinformation. When guard cells isolated from leaves with CAM weretreated with 100 mM ascorbate, pH 5.0 for 1 h in darkness, guardcell zeaxanthin content increased at rates equal to or higherthan those stimulated by light in guard cells from leaves inthe C3 mode. The ascorbate effect indicates that chloroplastsin guard cells from leaves with CAM retain their competenceto operate the xanthophyll cycle, but that zeaxanthin formationdoes not take place in the light. The data suggest that inhibitionof light-dependent zeaxanthin formation in guard cells mightbe one of the regulatory steps mediating the shift from diurnalto nocturnal stomatal opening typical of plants with CAM. (Received July 5, 1996; Accepted December 12, 1996)     

FLOWERING LOCUS T regulates stomatal opening

Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange for photosynthesis in response to light, CO(2), and phytohormone abscisic acid. Phototropins (phot1 and phot2) are plant blue-light receptor kinases and mediate stomatal opening via activation of the plasma membrane H(+)-ATPase. However, the signaling mechanism from phototropins to the H(+)-ATPase has yet to be determined. Here, we show that FLOWERING LOCUS T (FT) is expressed in guard cells and regulates stomatal opening. We isolated an scs (suppressor of closed-stomata phenotype in phot1 phot2) 1-1 mutant of Arabidopsis thaliana and showed that scs1-1 carries a novel null early flowering 3 (elf3) allele in a phot1 phot2 background. scs1-1 (elf3 phot1 phot2 triple mutant) had an open-stomata phenotype with high H(+)-ATPase activity and showed increased levels of FT mRNA in guard cells. Transgenic plants overexpressing FT in guard cells showed open stomata, whereas a loss-of-function FT allele, ft-1, exhibited closed stomata and failed to activate the H(+)-ATPase in response to blue light. Our results define a new cell-autonomous role for FT and demonstrate that the flowering time genes ELF3 and FT are involved in the regulation of H(+)-ATPase by blue light in guard cells.     

Genetic variation of stomatal conductance, blue light sensitivity and zeaxanthin content in guard cells of Pima cotton (Gossypium barbadense)

Pima S‐6 ( Gossypium barbadense L.) is a modern line with high stomatal conductance, while B368 is a primitive cotton with low conductance. The blue light sensitivity of adaxial guard cells, probed as the blue light‐dependent enhancement of the red light‐induced chlorophyll a fluorescence quenching, was investigated in these two cotton lines with contrasting stomatal conductance. Adaxial guard cells isolated from Pima S‐6 cotton plants had a significantly higher carotenoid content and a higher blue light sensitivity than those isolated from B368 plants. In a growth chamber‐grown F₂ population of a cross between these two lines, adaxial stomatal conductances of individual plants segregated over a range exceeding the average conductances of the parents. Carotenoid content and the blue light sensitivity of adaxial guard cells also segregated. The concentrations of xanthophylls and β‐carotene in the adaxial guard cells were poorly correlated with the blue light response, except for zeaxanthin. The co‐segregation of stomatal conductance and blue light sensitivity suggested that the stomatal response to blue light may play a role in the regulation of stomatal conductance in the intact leaf. Zeaxanthin content and blue light sensitivity also co‐segregated, suggesting that both parameters are under genetic control. The co‐segregation of zeaxanthin content, blue light sensitivity and stomatal conductance provides further evidence for a role of zeaxanthin in the blue light photoreception of guard cells.     

The fern Adiantum capillus-veneris lacks stomatal responses to blue light

We investigated the responses of stomata to light in the fern Adiantum capillus-veneris, a typical species of Leptosporangiopsida. Stomata in the intact leaves of the sporophytes opened in response to red light, but they did not open when blue light was superimposed on the red light. The results were confirmed in the isolated Adiantum epidermis. The red light-induced stomatal response was not affected by the mutation of phy3, a chimeric protein of phytochrome and phototropin in this fern. The lack of a blue light-specific stomatal response was observed in three other fern species of Leptosporangiopsida, i.e. Pteris cretica, Asplenium scolopendrium and Nephrolepis auriculata. Fusicoccin, an activator of the plasma membrane H(+)-ATPase, induced both stomatal opening and H(+) release in the Adiantum epidermis. Adiantum phototropin genes AcPHOT1 and AcPHOT2 were expressed in the fern guard cells. The transformation of an Arabidopsis phot1 phot2 double mutant, which lost blue light-specific stomatal opening, with AcPHOT1 restored the stomatal response to blue light. Taken together, these results suggest that ferns of Leptosporangiopsida lack a blue light-specific stomatal response, although the functional phototropin and plasma membrane H(+)-ATPase are present in this species.     

The stomata of the fern Adiantum capillus-veneris do not respond to CO2 in the dark and open by photosynthesis in guard cells

The stomata of the fern Adiantum capillus-veneris lack a blue light-specific opening response but open in response to red light. We investigated this light response of Adiantum stomata and found that the light wavelength dependence of stomatal opening matched that of photosynthesis. The simultaneous application of red (2 micromol m(-2) s(-1)) and far-red (50 micromol m(-2) s(-1)) light synergistically induced stomatal opening, but application of only one of these wavelengths was ineffective. Adiantum stomata did not respond to CO2 in the dark; the stomata neither opened under a low intercellular CO2 concentration nor closed under high intercellular CO2 concentration. Stomata in Arabidopsis (Arabidopsis thaliana), which were used as a control, showed clear sensitivity to CO2. In Adiantum, stomatal conductance showed much higher light sensitivity when the light was applied to the lower leaf surface, where stomata exist, than when it was applied to the upper surface. This suggests that guard cells likely sensed the light required for stomatal opening. In the epidermal fragments, red light induced both stomatal opening and K+ accumulation in guard cells, and both of these responses were inhibited by a photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The stomatal opening was completely inhibited by CsCl, a K+ channel blocker. In intact fern leaves, red light-induced stomatal opening was also suppressed by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that Adiantum stomata lack sensitivity to CO2 in the dark and that stomatal opening is driven by photosynthetic electron transport in guard cell chloroplasts, probably via K+ uptake.     

Blue light regulation of stomata in wheat seedlings. II. Action spectrum and search for action dichroism

The stomatal conductance response to low intensity blue light was studied in wheat seedlings ( Triticum aestivum L. cv. Starke II, Weibull) under red background illumination. Reciprocity was shown to be valid for illumination times from 10 s up to about 2 min. The action spectrum, constructed from fluence rate response curves, showed a maximum peak at 445–450 nm, another peak at 470 nm, a slight shoulder at 420 nm and a plateau between 370–400 nm. The relationship with action spectra for other blue light responses is discussed. The blue light response of wheat stomata did not exhibit action dichroism (the direction of the electrical vector of polarized blue light did not influence the response of the guard cells).     

Biochemical characterization of plasma membrane H+-ATPase activation in guard cell protoplasts of Arabidopsis thaliana in response to blue light

Recent genetic analysis showed that phototropins (phot1 and phot2) function as blue light receptors in stomatal opening of Arabidopsis thaliana, but no biochemical evidence was provided for this. We prepared a large quantity of guard cell protoplasts from Arabidopsis. The immunological method indicated that phot1 was present in guard cell protoplasts from the wild-type plant and the phot2 mutant, that phot2 was present in those from the wild-type plant and the phot1 mutant, and that neither phot1 nor phot2 was present in those from the phot1 phot2 double mutant. However, the same amounts of plasma membrane H+-ATPase were found in all of these plants. H+ pumping was induced by blue light in isolated guard cell protoplasts from the wild type, from the single mutants of phototropins (phot1-5 and phot2-1), and from the zeaxanthin-less mutant (npq1-2), but not from the phot1 phot2 double mutant. Moreover, increased ATP hydrolysis and the binding of 14-3-3 protein to the H+-ATPase were found in response to blue light in guard cell protoplasts from the wild type, but not from the phot1 phot2 double mutant. These results indicate that phot1 and phot2 mediate blue light-dependent activation of the plasma membrane H+-ATPase and illustrate that Arabidopsis guard cell protoplasts can be useful for biochemical analysis of stomatal functions. We determined isogenes of the plasma membrane H+-ATPase and found the expression of all isogenes of functional plasma membrane H+-ATPases (AHA1-11) in guard cell protoplasts.     

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.     

The Arabidopsis small G protein ROP2 is activated by light in guard cells and inhibits light-induced stomatal opening

ROP small G proteins function as molecular switches in diverse signaling processes. Here, we investigated signals that activate ROP2 in guard cells. In guard cells of Vicia faba expressing Arabidopsis thaliana constitutively active (CA) ROP2 fused to red fluorescent protein (RFP-CA-ROP2), fluorescence localized exclusively at the plasma membrane, whereas a dominant negative version of RFP-ROP2 (DN-ROP2) localized in the cytoplasm. In guard cells expressing green fluorescent protein-ROP2, the relative fluorescence intensity at the plasma membrane increased upon illumination, suggesting that light activates ROP2. Unlike previously reported light-activated factors, light-activated ROP2 inhibits rather than accelerates light-induced stomatal opening; stomata bordered by guard cells transformed with CA-rop2 opened less than controls upon light irradiation. When introduced into guard cells together with CA-ROP2, At RhoGDI1, which encodes a guanine nucleotide dissociation inhibitor, inhibited plasma membrane localization of CA-ROP2 and abolished the inhibitory effect of CA-ROP2 on light-induced stomatal opening, supporting the negative effect of active ROP2 on stomatal opening. Mutant rop2 Arabidopsis guard cells showed phenotypes similar to those of transformed V. faba guard cells; CA-rop2 stomata opened more slowly and to a lesser extent, and DN-rop2 stomata opened faster than wild-type stomata in response to light. Moreover, in rop2 knockout plants, stomata opened faster and to a greater extent than wild-type stomata in response to light. Thus, ROP2 is a light-activated negative factor that attenuates the extent of light-induced changes in stomatal aperture. The inhibition of light-induced stomatal opening by light-activated ROP2 suggests the existence of feedback regulatory mechanisms through which stomatal apertures may be finely controlled.     

Light-induced stomatal movement of selected Arabidopsis thaliana mutants

Various Arabidopsis thaliana mutants with defects in phytohormone signal transduction or the reception of light were analysed with regard to their stomatal response in a red, red/blue light irradiation programme. Stomatal response to light was detected with a customized gas exchange measurement device, optimized for the small model plant. Small transpiration-kinetic variations of the two wild-type lines Columbia (Col) and Landsberg erecta (Ler) were observed. A comparison of the mutant lines to the respective wild type revealed significant differences for the phytochrome A (phyA-103), the abscisic acid insensitive (aba3-2) and the auxin resistant (axr1-3) mutant. Furthermore, the zeaxanthin-less mutant line npq1-2 showed no alterations in stomatal response to light.     

Abaxial and adaxial stomata from Pima cotton (Gossypium barbadense L.) differ in their pigment content and sensitivity to light quality

Sensitivity to light quality and pigment composition were analysed and compared in abaxial and adaxial stomata of Gossypium barbadense L. (Pima cotton). In most plants, abaxial (lower) stomatal conductances are higher than adaxial (upper) ones, and stomatal opening is more sensitive to blue light than to red. In greenhouse-grown Pima cotton, abaxial stomatal conductances were two to three times higher than adaxial ones. In contrast, adaxial stomatal conductances were 1·5 to two times higher than abaxial ones in leaves from growth chamber-grown plants. To establish whether light quality was a factor in the regulation of the relationship between abaxial and adaxial stomatal conductances, growth-chamber-grown plants were exposed to solar radiation outdoors and to increased red light in the growth chamber. In both cases, the ratios of adaxial to abaxial stomatal conductance reverted to those typical of greenhouse plants. We investigated the hypothesis that adaxial stomata are more sensitive to blue light and abaxial stomata are more sensitive to red light. Measurements of stomatal apertures in mechanically isolated epidermal peels from growth chamber and greenhouse plants showed that adaxial stomata opened more under blue light than under red light, while abaxial stomata had the opposite response. Using HPLC, we quantified the chlorophylls and carotenoids extracted from isolated adaxial and abaxial guard cells. All pigments analysed were more abundant in the adaxial than in the abaxial guard cells. Antheraxanthin and β-carotene contents were 2·3 times higher in adaxial than in abaxial guard cells, comparing with ad/ab ratios of 1·5–1·9 for the other pigments. We conclude that adaxial and abaxial stomata from Pima cotton have a differential sensitivity to light quality and their distinct responses are correlated with different pigment content.     

Structural and functional properties of the coleoptile chloroplast: Photosynthesis and photosensory transduction

Recent studies have shown that guard cell and coleoptile chloroplasts appear to be involved in blue light photoreception during blue light-dependent stomatal opening and phototropic bending. The guard cell chloroplast has been studied in detail but the coleoptile chloroplast is poorly understood. The present study was aimed at the characterization of the corn coleoptile chloroplast, and its comparison with mesophyll and guard cell chloroplasts. Coleoptile chloroplasts operated the xanthophyll cycle, and their zeaxanthin content tracked incident rates of solar radiation throughout the day. Zeaxanthin formation was very sensitive to low incident fluence rates, and saturated at around 800–1000 mol m^–2 s^–1. Zeaxanthin formation in corn mesophyll chloroplasts was insensitive to low fluence rates and saturated at around 1800 mol m^–2 s^–1. Quenching rates of chlorophyll a fluorescence transients from coleoptile chloroplasts induced by saturating fluence rates of actinic red light increased as a function of zeaxanthin content. This implies that zeaxanthin plays a photoprotective role in the coleoptile chloroplast. Addition of low fluence rates of blue light to saturating red light also increased quenching rates in a zeaxanthin-dependent fashion. This blue light response of the coleoptile chloroplast is analogous to that of the guard cell chloroplast, and implicates these organelles in the sensory transduction of blue light. On a chlorophyll basis, coleoptile chloroplasts had high rates of photosynthetic oxygen evolution and low rates of photosynthetic carbon fixation, as compared with mesophyll chloroplasts. In contrast with the uniform chloroplast distribution in the leaf, coleoptile chloroplasts were predominately found in the outer cell layers of the coleoptile cortex, and had large starch grains and a moderate amount of stacked grana and stroma lamellae. Several key properties of the coleoptile chloroplast were different from those of mesophyll chloroplasts and resembled those of guard cell chloroplasts. We propose that the common properties of guard cell and coleoptile chloroplasts define a functional pattern characteristic of chloroplasts specialized in photosensory transduction.Abbreviations Ant or A antheraxanthin - dv/dt fluorescence quenching rate - Fm maximum yield of fluorescence with all PS II reaction centers closed - Fo yield of instantaneous fluorescence with all PS II reaction centers open - Vio or V violaxanthin - Zea or Z zeaxanthin     

Enhancement of the Stomatal Response to Blue Light by Red Light, Reduced Intercellular Concentrations of CO(2), and Low Vapor Pressure Differences

The effects of environmental parameters on the blue light response of stomata were studied by quantifying transient increases in stomatal conductance in Commelina communis following 15 seconds by 0.100 millimole per square meter per second pulses of blue light. Because conductance increases were not observed following red light pulses of the same or greater (30 seconds by 0.200 millimole per square meter per second) fluences, the responses observed could be reliably attributed to the specific blue light response of the guard cells, rather than to guard cell chlorophyll. In both Paphiopedilum harrisianum, which lacks guard cell chloroplasts, and Commelina, the blue light response was enhanced by 0.263 millimole per square meter per second continuous background red light. Thus, the blue light response and its enhancement do not require energy derived from red-light-driven photophosphorylation by the guard cell chloroplasts. In Commelina, reduction of the intercellular concentration of CO₂ by manipulation of ambient CO₂ concentrations resulted in an enhanced blue light response. In both Commelina and Paphiopedilum, the blue light response was decreased by an increased vapor pressure difference. The magnitude of blue-light-specific stomatal opening thus appears to be sensitive to environmental conditions that affect the carbon and water status of the plant.     

Reversal of blue light-stimulated stomatal opening by green light

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.     

Stomatal responses to light in the facultative Crassulacean acid metabolism species, Portulacaria afra

Guard cell responses to light are mediated by guard cell chlorophyll and by a specific blue light photoreceptor. Gas exchange and epidermal peel techniques were employed to investigate these responses in the facultative Crassulacean acid metabolism (CAM) species, Portulacaria afra (L.) Jacq. In P. afra individuals performing C₃ metabolism, red light stimulated an increase in leaf conductance in intact leaves and stomatal opening in isolated epidermal peels, indicating the presence in guard cells of the chlorophyll-mediated response to light. Under a background of continuous red illumination, conductance exhibited transient increases following pulses of blue but not red light, indicating that the specific stomatal response to blue light was also operative. In contrast, in CAM individuals, conductance in gas exchange experiments and stomatal opening in epidermal peel experiments were not stimulated by red light. In CAM plants, conductance did not increase following blue light pulses administered over a range of temperatures, vapor pressure differences (VPD), ambient CO₂ concentrations and background red light intensities. These results indicate that P. afra does possess typical guard cell responses to light when performing C₃ metabolism. The metabolic pathways mediating these responses are either lost or inhibited when CAM is induced.