The effect of blue light on energy levels in epidermal strips

Red light applied together with blue enhanced stomatal opening in epidermal strips of Commelina communis L. more than red light alone. In red light, stomatal opening was enhanced by exogenously applied ATP and was inhibited by 3-(3,4-dichlorophe-nyl)-l,l-dimethylurea (DCMU), while in the presence of blue light external ATP was almost without effect, and DCMU stimulated stomatal opening. Blue light increased the ATP levels in the epidermal strips. DCMU diminished the amount of ATP in both red light and red + blue light treatments, but did not abolish the stimulatory effect of blue light. Blue light also stimulated the respiration rate of the epidermal strips. Rotenone, which inhibited stomatal opening and respiration rate, abolished the effect of blue light in both processes. These results imply that blue light increases the ATP levels by stimulation of oxidative phosphorylation.     

Further support for the involvement of phytoehrome in stomatal movement

The involvement of phytochrome in stomatal movement in Commelina communis L. is indicated by the following observations: 1) Short irradiation with red or blue light causes opening, of isolated stomata and swelling of guard cell protoplasts. This is reversed by subsequent far red irradiation. 2) In a similar way, stomatal response to prolonged irradiation with red or blue light is decreased by concomitant far red irradiation. 3) Pretreatment with filipin, which interferes with phytochrome binding to membranes, decreases stomatal opening in red and blue light. The stomatal responses to blue and red light are modified by DCMU, N₂, CO_2-enriched atmosphere, and CO_2-free air, which are known to affect, among other processes, chlorophyll fluorescence. Increased chlorophyll fluorescence by DCMU, N₂ and CO₂-enriched atmosphere enhanced stomatal opening in blue light and inhibited it in red light. CO₂-free air, which decreases chlorophyll fluorescence, had the opposite effect.     

Metabolic energy for stomatal opening. Roles of photophosphorylation and oxidative phosphorylation

The supply of energy for stomatal opening was investigated with epidermal peels of Commelina communis L. and Vicia faba L., under white, blue and red irradiation or in darkness. Fluencerate response curves of stomatal opening under blue and red light were consistent with the operation of two photosystems, one dependent on photosynthetic active radiation (PAR) and the other on blue light, in the guard cells. The PAR-dependent system was 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-sensitive and KCN-resistant and showed a relatively high threshold irradiance for its activation; its activity was most prominent at moderate to high irradiances. The blue-light-dependent photosystem was KCN-sensitive, was active at low irradiances, and interacted with the PAR-dependent photosystem at high blue irradiances. Stomatal opening in darkness, caused by CO₂-free air, fusicoccin or high KCl concentrations, was KCN-sensitive and DCMU-resistant. These data indicate that stomatal opening in darkness depends on oxidative phosphorylation for the supply of high-energy equivalents driving proton extrusion. Light-dependent stomatal opening appears to require photophosphorylation from guard-cell chloroplasts and the activation of the blue-light photosystem which could rely either on oxidative phosphorylation or a specific, membrane-bound electron-transport carrier.Abbreviations DCMU 3(3,4-dichlorophenyl)-1-1-dimethylurea - FC fusicoccin - KCN potassium cyanide - PAR photosynthetic active radiation - WL white light     

Different stomatal responses of maize leaves after blue or red illumination under anoxia

Abstract In normal air, illumination with a low level of blue or red light (40 μmol m^?2 s^?1) did not induce stomatal opening in maize plantlets. In CO₂-free air, 40 μmol m^?2 s^?1 of blue or red light promoted an enhancement in stomatal opening. At the same quantum flux, blue light was more efficient than red light and stomatal closure occurred more rapidly with a significantly shorter lag phase after blue light. Anoxia inhibited light-dependent stomatal opening, even under 320 μmol m^?2 s^?1 illumination. However, after 60 min of illumination with 40 μmol m^?2 s^?1 of blue light in anoxia, transient stomatal opening was observed when the plant was returned to darkness and normal air. This transient stomatal opening was weaker after pretreatment with red light. We conclude that a blue-light-dependent process induced under anoxia leads to stomatal opening provided oxygen is present. Possible mechanisms associated with blue-light-effect and the nature of the oxygen-consuming processes are discussed.     

Evidence for phytochrome involvement in light-mediated stomatal movement in Phaseolus vulgaris L.

Observations made with primary leaves of Phaseolus vulgaris L. demonstrated that phytochrome modulates light-induced stomatal movement. Removal of the far-red-absorbing form of the pigment (Pfr) with far-red (FR) radiation decreased the time required by the stomata to reach maximal opening following a dark-to-light transition; this effect of FR was fully reversible with red. Removal of Pfr with FR also decreased the time required to reach maximal closure following a light-to-dark transition, and the rate of closure was dependent on the final irradiation treatment before darkness. No evidence was found for phytochrome involvement in determining stomatal aperture under constant conditions of either darkness of light.Abbreviations and symbols Chl chlorophyll - D darkness - FR far-red - phytochrome photostationary state - Pfr, Pr FR- and R-absorbing forms of phytochrome, respectively - R red     

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.     

Energy Supply for Stomatal Opening in Epidermal Strips of Commelina benghalensis

The influence of light or darkness on stomatal opening in epidermal strips of Commelina benghalensis was evaluated in the presence or absence of O₂ and/or metabolic inhibitors. Opening was restricted in nitrogen and was promoted by NADH and acids of the tricarboxylic acid cycle (succinate and α-ketoglutarate) in CO₂-free air in light as well as in darkness. The enhancement by light of stomatal opening was prevalent under nitrogen or in the presence of the respiratory inhibitors (sodium azide and oligomycin). Respiratory inhibitors decreased the opening in light or darkness under CO₂-free air but exhibited no effect under nitrogen, whereas phosphorylation uncouplers were inhibitory in light or darkness under both CO₂-free air and nitrogen. The results suggest that oxidative phosphorylation is a basic source of energy for stomatal opening, although photophosphorylation could be an energy source.     

A reassessment of the intervention of calmodulin in the regulation of stomatal movement

Commelina cammunis L., a monocotyledonous plant whose stomata are highly sensitive to calcium ions, was used to study calmodulin (CaM) involvement in stomatal movements. CaM was detected and quantified in guard cell and mesophyll cell protoplasts by western blot and by ⁴⁵Ca²⁺-overlays. CaM was found to be 3- to 7-fold more abundant on a per protein basis in guard cell than in mesophyll cell protoplasts. Numerous guard cell proteins that bind CaM in a Ca²⁺-dependent manner were detected by gold-labelled CaM overlays. Using bioassays with epidermal strips, different CaM-antagonists were found to induce a net stimulation of stomatal opening in darkness or under illumination (trifluoperazine > compound 48/80 ∼ fluphenazine > W7 > W5). As CaM is frequently involved in the regulation of phosphorylation processes, the effects of different inhibitors of protein kinases on stomatal movements were studied. In red plus blue light, a promotion of the stomatal aperture was observed in the nanomolar range with K252a and KT5926 and in the micromolar range with KT5720 ≫ ML7 ∼ ML9 ≫ H7 > KN62. Only the inhibitors with a high specificity for Ca²⁺-CaM dependent protein kinases (K252a, KT5926, ML7, ML9) triggered a stomatal opening in darkness and increased stomatal aperture in red plus blue light. Taken together, these data strongly suggest that a Ca²⁺- or a Ca²⁺-CaM-dependent protein kinase plays a central role in the calcium transduction pathway leading to the maintaining of stomatal closure.     

A reassessment of the intervention of calmodulin in the regulation of stomatal movement

Commelina cammunis L., a monocotyledonous plant whose stomata are highly sensitive to calcium ions, was used to study calmodulin (CaM) involvement in stomatal movements. CaM was detected and quantified in guard cell and mesophyll cell protoplasts by western blot and by ⁴⁵Ca²⁺-overlays. CaM was found to be 3- to 7-fold more abundant on a per protein basis in guard cell than in mesophyll cell protoplasts. Numerous guard cell proteins that bind CaM in a Ca²⁺-dependent manner were detected by gold-labelled CaM overlays. Using bioassays with epidermal strips, different CaM-antagonists were found to induce a net stimulation of stomatal opening in darkness or under illumination (trifluoperazine > compound 48/80 ≅ fluphenazine > W7 > W5). As CaM is frequently involved in the regulation of phosphorylation processes, the effects of different inhibitors of protein kinases on stomatal movements were studied. In red plus blue light, a promotion of the stomatal aperture was observed in the nanomolar range with K252a and KT5926 and in the micromolar range with KT5720 ≫ ML7 ≅ ML9 ≫ H7 > KN62. Only the inhibitors with a high specificity for Ca²⁺-CaM dependent protein kinases (K252a, KT5926, ML7, ML9) triggered a stomatal opening in darkness and increased stomatal aperture in red plus blue light. Taken together, these data strongly suggest that a Ca²⁺- or a Ca²⁺-CaM-dependent protein kinase plays a central role in the calcium transduction pathway leading to the maintaining of stomatal closure.     

Inhibition of stomatal opening in sunflower leaves by carbon monoxide,and reversal of inhibition by light

When leaves of Helianthus annuus, whose stomates had been opened in the dark in the absence of CO₂, were exposed to 25% carbon monoxide (CO), stomatal conductivity for water vapor decreased from about 0.4 to 0.2 cm·s^-1. The CO effect on stomatal aperture required a CO/O₂ ratio of about 25. As this ratio was decreased the stomata opened, indicating that inhibitio of cytochrome-c oxidase by CO is competitive in respect to O₂. Photosynthetically active red light was unable to reverse CO-induced stomatal closure even at high irradiances, when CO₂ was absent. When it was present, stomatal opening was occasionally, but not consistently observed. Carbon monoxide did not inhibit photosynthetic carbon reduction in leaves of Helianthus.In contrast to red light, very weak blue light (405 nm) increased the stomatal aperture in the presence of CO. It also increased leaf ATP/ADP ratios which had been decreased in the presence of CO. The blue-light effect was not related to photosynthesis. Neither could it be explained by photodissociation of the cytochrome a ₃-CO complex which has an absorption maximum at 430 nm. The data indicate that ATP derived from mitochondrial oxidative phosphorylation provides energy for stomatal opening in sunflower leaves in the dark as well as in the light. Indirect transfer of ATP from chloroplasts to the cytosol via the triose phosphate/phosphoglycerate exchange which is mediated by the phosphate translocator of the chloroplast envelope can support stomatal opening only if metabolite concentrations are high enough for efficient shuttle transfer of ATP. Blue light causes stomatal opening in the presence of CO by stimulating ATP synthesis.     

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.     

Metabolomics of red‐light‐induced stomatal opening in Arabidopsis thaliana: Coupling with abscisic acid and jasmonic acid metabolism

Environmental stimuli‐triggered stomatal movement is a key physiological process that regulates CO₂ uptake and water loss in plants. Stomata are defined by pairs of guard cells that perceive and transduce external signals, leading to cellular volume changes and consequent stomatal aperture change. Within the visible light spectrum, red light induces stomatal opening in intact leaves. However, there has been debate regarding the extent to which red‐light‐induced stomatal opening arises from direct guard cell sensing of red light versus indirect responses as a result of red light influences on mesophyll photosynthesis. Here we identify conditions that result in red‐light‐stimulated stomatal opening in isolated epidermal peels and enlargement of protoplasts, firmly establishing a direct guard cell response to red light. We then employ metabolomics workflows utilizing gas chromatography mass spectrometry and liquid chromatography mass spectrometry for metabolite profiling and identification of Arabidopsis guard cell metabolic signatures in response to red light in the absence of the mesophyll. We quantified 223 metabolites in Arabidopsis guard cells, with 104 found to be red light responsive. These red‐light‐modulated metabolites participate in the tricarboxylic acid cycle, carbon balance, phytohormone biosynthesis and redox homeostasis. We next analyzed selected Arabidopsis mutants, and discovered that stomatal opening response to red light is correlated with a decrease in guard cell abscisic acid content and an increase in jasmonic acid content. The red‐light‐modulated guard cell metabolome reported here provides fundamental information concerning autonomous red light signaling pathways in guard cells.     

Phytochrome involvement in stomatal movement in Pisum sativum, Vicia faba and Pelargonium sp.

Red and blue light triggered the opening of isolated stomata of Pisum sativum L. cv. Peleg Alvador, Vicia faba L. (unknown cultivar) and Pelargonium sp. The stimulatory effect of short irradiation with red or blue light was reversed by a subsequent short irradiation with far-red light. In Pisum the stimulatory effect of a continuous irradiation with red or blue light was also abolished by a concomitant far-red light. In leaf pieces of P. sativum blue light was more effective than red, but not in isolated guard cells. In the presence of mesophyll, DCMU inhibited stomatal opening in red light more than in blue, and thus increased the relative response to blue light. This was less evident in isolated guard cells.     

Stomatal Opening and Cell Enlargement in Response to Light and Phytohormone Treatments in Primary Leaves of Red-Light-Grown Seedlings of Phaseolus vulgaris L.

Cell enlargement in primary leaves of bean seedlings grown for10 days in dim red light in response to different light andphytohormone treatments was studied. On day 10, bean leaf discswere floated on 1% sucrose with, or without, phytohormones fordifferent periods (up to 24 h) under dim red light, or discswere floated in sucrose solution and irradiated with white orblue light. Cell enlargement was enhanced by continuous whiteand blue light and by benzyladenine, kinetin and gibberellicacid. When seedlings were grown for 8 days under dim red light afterwhich a 2-day dark period was interposed (for the accumulationof inactive phytochrome), cell enlargement was enhanced by a5-min irradiation with red light. This growth induction wasfar-red reversible. The conditions under which cell enlargement was promoted, alsoinduced the opening of the stomata. Red light induced a far-redreversible transient stomatal opening. Based on the kineticsof stomatal opening and cell enlargement we formulated the hypothesisthat cell enlargement in leaves in response to light and phytohormonesis mediated by the stomatal response to these factors. (Received September 30, 1983; Accepted February 27, 1984)     

Sugar and Organic Acid Accumulation in Guard Cells of Vicia faba in Response to Red and Blue Light

Changes in neutral sugar and organic acid content of guard cells were quantitated by high-performance liquid chromatography during stomatal opening in different light qualities. Sonicated Vicia faba epidermal peels were irradiated with 10 [mu]mol m-2 s-1 of blue light, a fluence rate insufficient for the activation of guard cell photosynthesis, or 125 [mu]mol m-2 s-1 of red light, in the presence of 1 mM KCl, 0.1 mM CaCl2. The low-fluence-rate blue light stimulated an average net stomatal opening of 4.7 [mu]m in 2 h, whereas the saturating fluence rate of red light stimulated an average net opening of 3.8 [mu]m in 2 h. Under blue light, the malate content of guard cells increased to 173% of the initial level during the first 30 min of opening and declined as opening continued. Sucrose levels continuously rose throughout the blue light-stimulated opening, reaching 215% of the initial level after 2 h. The starch hydrolysis products maltose and maltotriose remained elevated at all times. Under red light, guard cells showed very little increase in organic acid or maltose levels, whereas sucrose levels increased to 208% of the initial level after 2 h. Total measured organic metabolite concentrations were correlated with stomatal apertures in all cases except where substantial malate increases occurred. These results support the hypothesis that light quality modulates alternative mechanisms of osmotic accumulation in guard cells, including potassium uptake, photosynthetic sugar production, and starch breakdown.     

Guard-cell chloroplasts provide ATP required for H(+) pumping in the plasma membrane and stomatal opening

To elucidate the role of guard-cell chloroplasts (GCCs) in stomatal movement, we investigated the effects of oligomycin, an inhibitor of oxidative phosphorylation, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II, on fusicoccin (FC)-induced H(+) pumping and stomatal opening. FC was found to induce H(+ )pumping in guard-cell protoplasts (GCPs) from Vicia faba and stomatal opening in the epidermis of Commelina benghalensis; and, red light (RL) slightly stimulated these responses. Oligomycin strongly inhibited the pumping and stomatal opening in the dark. RL partially reversed the inhibitions, and DCMU decreased the effect of RL. FC activated the plasma membrane H(+)-ATPase (EC 3.6.1.35) in GCPs similarly irrespective of these treatments, indicating that the H(+)-ATPase activity was not the limiting step in H(+) pumping. Oligomycin significantly decreased the ATP content in GCPs in the dark. RL partially reversed this effect, and DCMU eliminated the effect of RL. A significant part of the ATP produced by photophosphorylation to H(+) pumping was indicated under RL. These results suggest that GCCs supply ATP to the cytosol under RL, and that the ATP is utilized by the plasma membrane H(+)-ATPase for H(+) pumping.     

Light-Induced Stomatal Opening Is Affected by the Guard Cell Protein Kinase APK1b

Guard cells allow land plants to survive under restricted or fluctuating water availability. They control the exchange of gases between the external environment and the interior of the plant by regulating the aperture of stomatal pores in response to environmental stimuli such as light intensity, and are important regulators of plant productivity. Their turgor driven movements are under the control of a signalling network that is not yet fully characterised. A reporter gene fusion confirmed that the Arabidopsis APK1b protein kinase gene is predominantly expressed in guard cells. Infrared gas analysis and stomatal aperture measurements indicated that plants lacking APK1b are impaired in their ability to open their stomata on exposure to light, but retain the ability to adjust their stomatal apertures in response to darkness, abscisic acid or lack of carbon dioxide. Stomatal opening was not specifically impaired in response to either red or blue light as both of these stimuli caused some increase in stomatal conductance. Consistent with the reduction in maximum stomatal conductance, the relative water content of plants lacking APK1b was significantly increased under both well-watered and drought conditions. We conclude that APK1b is required for full stomatal opening in the light but is not required for stomatal closure.     

Photomorphogenesis of the Gynophore, Pod and Embryo in Peanut, Arachis hypogaea L.

Darkened excized gynophores ceased to elongate after 8–10days in vitro and started to form a pod. Gynophore elongationwas inhibited to a greater extent in total darkness than underlow irradiance, while pod and embryo growth was stimulated indarkness only. Intact gynophores, enclosed in transparent vials containingglass beads, continued to elongate in both light and darkness.In light the elongating gynophores thickened as they penetratedbetween the glass beads, forming a seedless pod at the bottomof the vials. In the dark the elongating gynophores producedsmall pods in which the seeds had started to grow. Excized gynophores elongated in vitro under continuous whitelight at a rate similar to that of intact exposed gynophores.The rate of elongation in vitro, was lower under continuousblue or red-enriched light, than under white light, and wasfurther reduced under continuous far-red irradiation. Pods didnot form during any of the continuous irradiation treatmentsbut only after transfer to darkness, the largest pods formingafter continuous far-red irradiation. As little as 10 min daily exposure to red or far-red irradiancehad the same effect on gynophore elongation as continuous irradiation.Pods formed only when the daily periods of far-red irradiationwere 30 min or less. Reducing the daily exposures to 2 min decreasedthe time to onset of pod formation from 30 to 16 days. Far-redfollowing red irradiation was effective in inhibiting gynophoreelongation stimulated by red irradiation. Pod formation in red/far-redirradiation was only 50 per cent of that observed in far-redirradiation. The involvement of light in continual gynophoreelongation and in the concomitant inhibition of proembryo growthis discussed. Arachis hypogaea L., peanut, gynophore, photomorphogenesis, embryo development, pod development, proembryo     

Ion movements during stomatal opening in darkness after illumination under nitrogen

In Zea mays L., Bryophylum laxiflorum Bak., Gossypium hirsutum L., Helianthus annuus L., Oryza sativa L. and Vigna radiata L., a pre-illumination in nitrogen causes transient stomatal opening upon returning the plant to darkness and normal air. In Zea mays L. hybrid INRA 508, K⁺ and Cl⁻ fluctuations in the stomatal complex during this stomatal opening in darkness were similar to those observed during a light-induced opening in normal air. These results are consistent with a two-phase mechanism for stomatal opening: a light phase that may occur in the absence of oxygen and carbon dioxide, and a dark phase during which oxygen is necessary for ion accumulation and other mechanisms of osmotic adjustment.