Guard Cell Chloroplasts Research Articles

A nitric oxide burst precedes apoptosis in angiosperm and gymnosperm callus cells and foliar tissues

Leaves and callus of Kalanchoë daigremontiana and Taxus brevifolia were used to investigate nitric oxide-induced apoptosis in plant cells. The effect of nitric oxide (NO) was studied by using a NO donor, sodium nitroprusside (SNP), a nitric oxide-synthase (NOS) inhibitor, N:(G)-monomethyl-L-arginine (NMMA), and centrifugation (an apoptosis-inducing treatment in these species). NO production was visualized in cells and tissues with a specific probe, diaminofluorescein diacetate (DAF-2 DA). DNA fragmentation was detected in situ by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) method. In both species, NO was detected diffused in the cytosol of epidermal cells and in chloroplasts of guard cells and leaf parenchyma cells. Centrifugation increased NO production, DNA fragmentation and subsequent cell death by apoptosis. SNP mimicked centrifugation results. NMMA significantly decreased NO production and apoptosis in both species. The inhibitory effect of NMMA on NO production suggests that a putative NOS is present in Kalanchoë and Taxus cells. The present results demonstrated the involvement of NO on DNA damage leading to cell death, and point to a potential role of NO as a signal molecule in these plants.

Characterization of the activity of a plastid-targeted green fluorescent protein in Arabidopsis

In Arabidopsis thaliana the PALE CRESS (PAC) gene product is required for both chloroplast and cell differentiation. Transgenic Arabidopsis plants expressing a translational fusion of the N-terminal part of the PAC protein harboring the complete plastid-targeting sequence and the green fluorescent protein (GFP) exhibit high GFP fluorescence. Detailed analyses based on confocal imaging of various tissues and cell types revealed that the PAC-GFP fusion protein accumulates in chloroplasts of mature stomatal guard cells. The GFP fluorescence within the guard cell chloroplasts is not evenly distributed and appears to be concentrated in suborganellar regions. GFP localization studies demonstrate that thin tubular projections emanating from chloroplasts and etioplasts often connect the organelles with each other. Furthermore, imaging of non-green and etiolated tissue further revealed that GFP fluorescence is present in proplastids, etioplasts, chromoplasts, and amyloplasts. Even photobleaching of carotenoid-free plastids does not affect PAC-GFP accumulation in the organelles of the guard cells indicating that the protein translocation machinery is functional in all types of plastids. The specific accumulation of GFP in guard cell chloroplasts, their tubular connections, the translocation of the precursor polypeptide into the different types of organelles, as well as the use of a plastid-targeted GFP protein as a versatile marker is discussed in the context of previously described observations.

Use of fluorescein diacetate (FDA) to determine ploidy of in vitro watermelon shoots

Ploidy of watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai shoots and plantlets was estimated by painting the lower epidermis of intact in vitro-derived leaves with fluorescein diacetate (FDA) and observing fluorescence of guard cell chloroplasts with a microscope and UV light. Leaves from in vitro shoot-tip cultures of known diploid cultivars and tetraploid breeding lines were used to establish the mean number of chloroplasts per guard cell pair. Leaves from diploid and tetraploid shoot cultures had 9.7 and 17.8 chloroplasts per guard cell pair, respectively. This method then was used to estimate the ploidy of shoots regenerated from cotyledon explants of the diploid cultivar Minilee. Approximately 11% of the 188 regenerated shoots were classified as tetraploid during in vitro culture. Putative tetraploids were transplanted to the field and self-pollinated. About 45% of tetraploids identified in vitro produced fruit and viable seed. Chloroplast counts of R1 progeny were used to confirm their ploidy. All of the putative diploids were confirmed diploid and all putative tetraploids proved to be non-chimeric true breeding tetraploids.

Development of ultrastructural damage to chloroplasts in a plastoquinone-deficient mutant of maize

The ultrastructure of a recently discovered mutant of maize (mutant hcf103-114) that is completely lacking plastoquinone (Cook, W.B., Miles, D., 1992. Nuclear mutations affecting plastoquinone accumulation in maize. Photosyn. Res. 31, 99–111) was investigated. This mutant fails to green and dies at an early age. Tissues along a developmental gradient (from base to tip of a maize leaf) were fixed and prepared for examination via electron microscopy. Initial development was normal in both mesophyll cell (MC) and bundle sheath cell (BSC) chloroplasts. Starch, which was abundant in BSC chloroplasts of wild type maize, did not accumulate in the mutant. As tissue aging progressed, both plastid types exhibited symptoms typical of photooxidative injury. Injury, seen as chloroplast swelling, lipid accumulation and envelope disruption, appeared sooner in BSC chloroplasts than in MC chloroplasts. Chloroplasts in guard cells possessed starch granules and only showed ultrastructural injury after the starch granules disappeared. Stomata developed normally in the hcf103-114 mutant. The results are discussed in terms of the known roles of plastoquinone in chloroplast metabolism.

Ploidy Determination in Musa Germplasm Using Pollen and Chloroplast Characteristics

We attempted to determine ploidy level in the gametophyte and the sporophyte of Musa using pollen and chloroplast characteristics, respectively. In the gametophyte, interploidy differences accounted for 63.8% of the genetic variance for pollen diameter and 87.5% for pollen stainability, the remainder being attributable to intraploidy differences among clones. While pollen count and stainability effectively separated triploid accessions from diploids or tetraploids, they did not discriminate between diploids and tetraploids. In the sporophyte, the relative contributions of interploidy and intraploidy differences to genetic variation in the number of chloroplasts in stomatal guard cells were 70.8% and 29.2%, respectively. Although pollen diameter and chloroplast number increased with ploidy, the use of the sporophytic parameter appears to provide a more satisfactory means of estimating ploidy status in Musa.

Role of zeaxanthin in blue light photoreception and the modulation of light-CO2 interactions in guard cells

The stomatal response to blue light is an intrinsic component of the sensory transducing processes mediating light-stimulated stomatal movements. Guard cell chloroplasts have a specific blue light response with an action spectrum that resembles the action spectrum for blue light-stimulated stomatal opening, suggesting a role of guard cell chloroplasts in the sensory transduction of blue light. The xanthophyll, zeaxanthin has recently been identified as a blue light photoreceptor in guard cells. The inhibitor of zeaxanthin formation, dithiothreitol, inhibits zeaxanthin formation and the stomatal response to blue light in a concentration-dependent fashion. In greenhouse-grown leaves, guard cell zeaxanthin content closely tracks incident radiation and it is positively correlated with stomatal apertures. The sensitivity of guard cells to blue light co-varies with guard cell zeaxanthin content. A zeaxanthin-less mutant of Arabidopsis is devoid of a typical stomatal response to blue light. At constant light and temperature, changes in ambient [CO 2 ] in a growth chamber caused large changes in stomata aperture and in guard cell zeaxanthin. The aperture-zeaxanthin changes were linearly related over a wide range of [CO 2 ]. Experiments with detached epidermis showed a similar relation among [CO 2 ], stomatal apertures and guard cell zeaxanthin, and DTT inhibited the CO 2 response in the light without altering the CO 2 response in the dark. These results indicate that blue light sensing by guard cell zeaxanthin has a regulatory role in the light response of stomata. Zeaxanthin also appears to mediate light-CO 2 interactions in guard cells.

Chromosome doubling via tuber disc culture in dihaploid potato as determined by confocal microscopy.

The potential of tuber disc culture for chromosome doubling was investigated in somaclonal populations of four dihaploid genotypes and one tetraploid cultivar of potato (Solanum tuberosum). Laser scanning confocal microscopy (LSCM) was used for rapid determination of the ploidy level based on the number of chloroplasts in stomatal guard cells of leaves. Factorial analysis of chloroplast number in 58 clones and two leaf types showed that somaclones were clearly divided in two groups. Clones with 5-7 chloroplasts per cell as observed in tuber derived diploid controls were classified as 2X (not doubled), while those with 9-14 chloroplasts resembled the tuber derived tetraploid controls and were considered 4X (doubled). A high frequency of spontaneous chromosome doubling, 42% - 50%, was detected in 3 dihaploid genotypes, whereas no doubling was observed in one of the dihaploids as well as the tetraploid cultivar Yukon Gold. Effects of leaf type on chloroplast number was also significant. The middle leaf showed significantly higher chloroplast number than the younger leaves.

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 develop CAM under water or salt stress. When ice plants shift from C3 metabolism to CAM, their stomata remain closed during the day and open at night. Recent studies have shown that the stomatal response of ice plants in the C3 mode depends solely on the guard cell response to blue light. Recent evidence for a possible role of the xanthophyll, zeaxanthin in blue light photoreception of guard cells led to the question of whether changes in the regulation of the xanthophyll cycle in guard cells parallel the shift from diurnal to nocturnal stomatal opening associated with CAM induction. In the present study, light-dependent stomatal opening and the operation of the xanthophyll cycle were characterized in guard cells isolated from ice plants shifting from C3 metabolism to CAM. Stomata in epidermis detached from leaves with C3 metabolism opened in response to white light and blue light, but they did not open in response to red light. Guard cells from these leaves showed light-dependent conversion of violaxanthin to zeaxanthin. Induction of CAM by NaCI abolished both white light- and blue light-stimulated stomatal opening and light-dependent zeaxanthin formation. When guard cells isolated from leaves with CAM were treated with 100 mM ascorbate, pH 5.0 for 1 h in darkness, guard cell zeaxanthin content increased at rates equal to or higher than those stimulated by light in guard cells from leaves in the C3 mode. The ascorbate effect indicates that chloroplasts in guard cells from leaves with CAM retain their competence to operate the xanthophyll cycle, but that zeaxanthin formation does not take place in the light. The data suggest that inhibition of light-dependent zeaxanthin formation in guard cells might be one of the regulatory steps mediating the shift from diurnal to nocturnal stomatal opening typical of plants with CAM.

Development and accumulation of ABA in fluridone‐treated and drought‐stressed Vicia faba plants under different light conditions

The effects of fluridone on guard cell morphology, chloroplast ultrastructure and accumulation of drought stress‐induced abscisic acid (ABA) were studied in Vicia faba L. plants grown under different light conditions. Drought stress was induced by allowing the leaves to lose 12% of their fresh weight. The appearance of defective and undeveloped stomata, and chloroplasts with a destroyed thylakoid membrane system was found in fluridone‐treated plants grown at a photosynthetic photon flux (PPF) of 600 μmol m‐2 s‐1. Plants grown at a PPF of 40 μmol m‐2 s‐1 had diminished levels of ABA after imposition of dehydration. Fluridone treatment reduced the level of ABA in both unstressed and dehydrated leaves. Accumulation of ABA in the control plants was considerably reduced when they were exposed to dark periods of 24, 48 and 72 h just before imposition of the stress. Twenty‐four hours after the dark treatment dehydration of the leaves resulted in a 3‐fold decrease in the level of stress‐induced ABA, and 72 h after dark treatment the amount of stress‐induced ABA approximated the prestressed values. Fluridone‐treated plants failed to accumulate ABA under water stress. In addition to functionally active chloroplasts, well‐developed and functional stomata are required for drought stress to elicit a rise in ABA.

Close correspondence between the action spectra for the blue light responses of the guard cell and coleoptile chloroplasts, and the spectra for blue light-dependent stomatal opening and coleoptile phototropism.

Fluorescence spectroscopy was used to characterize blue light responses from chloroplasts of adaxial guard cells from Pima cotton (Gossypium barbadense) and coleoptile tips from corn (Zea mays). The chloroplast response to blue light was quantified by measurements of the blue light-induced enhancement of a red light-stimulated quenching of chlorophyll a fluorescence. In adaxial (upper) guard cells, low fluence rates of blue light applied under saturating fluence rates of red light enhanced the red light-stimulated fluorescence quenching by up to 50%. In contrast, added blue light did not alter the red light-stimulated quenching from abaxial (lower) guard cells. This response pattern paralleled the blue light sensitivity of stomatal opening in the two leaf surfaces. An action spectrum for the blue light-induced enhancement of the red light-stimulated quenching showed a major peak at 450 nm and two minor peaks at 420 and 470 nm. This spectrum matched closely an action spectrum for blue light-stimulated stomatal opening. Coleoptile chloroplasts also showed an enhancement by blue light of red light-stimulated quenching. The action spectrum of this response, showing a major peak at 450 nm, a minor peak at 470 nm, and a shoulder at 430 nm, closely matched an action spectrum for blue light-stimulated coleoptile phototropism. Both action spectra match the absorption spectrum of zeaxanthin, a chloroplastic carotenoid recently implicated in blue light photoreception of both guard cells and coleoptiles. The remarkable similarity between the action spectra for the blue light responses of guard cells and coleoptile chloroplasts and the spectra for blue light-stimulated stomatal opening and phototropism, coupled to the recently reported evidence on a role of zeaxanthin in blue light photoreception, indicates that the guard cell and coleoptile chloroplasts specialize in sensory transduction.

A Simple Method for Estimating Ploidy of in Vitro Watermelon Plantlets

Ploidy of in vitro watermelon plantlets was estimated by painting the lower epidermis of leaves with fluorescein diacetate (FDA) and observing fluorescence of guard cell chloroplasts with a microscope and UV light. Leaves from shoot-tip cultures of known diploid and tetraploid cultivars were used to establish the mean number of chloroplasts per guard cell pair for in vitro plantlets. Leaves from diploid and tetraploid plantlets had 9.7 and 17.8 chloroplasts per guard cell pair, respectively. This method was used to estimate ploidy of shoots regenerated from cotyledon explants of the diploid cultivar Minilee. Approximately 10.6% of regenerated shoots were classified as tetraploid while still in vitro. Putative tetraploids were transplanted to the field and self-pollinated. A majority of polyploids identified in vitro were true breeding, nonchimeric tetraploids. This study demonstrate that FDA can be used to estimate ploidy of in vitro shoots of watermelon prior to acclimatization and transfer of plants to the greenhouse or field.

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 μmol m(-2) s(-1). Zeaxanthin formation in corn mesophyll chloroplasts was insensitive to low fluence rates and saturated at around 1800 μmol 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.

Characterization of somatic hybrids of potato by use of RAPD markers and isozyme analysis

Electrofusion of mesophyll protoplasts from two male sterile dihaploid Solanum tuberosum genotypes. DHAK‐11 and DHAK‐33, was performed. Selection of putative fusion products was based on vigorous callus growth. Regeneration of rooted putative hybrid plants was scored 14 weeks after fusion. Characterization of hybrids was performed by use of morphological assessment, random amplified polymorphic DNA (RAPD), and cytological and isozyme analysis. The rate of regenerated hybrids from callus was ca 6%. Of the putative hybrids, 45% were confirmed as true hybrids. Morphological assessment of the putative hybrids revealed that tetraploid and neartetraploid hybrids were vigorous plants with intermediate characteristics between the two parental phenotypes in respect to internode length, leaf size and shape, and purple pigmentation on the abaxial side of the leaves. Near‐hexaploid hybrids were slender plants with small leaves and short petioles. Selected RAPD primers showed unique marker bands for the two parental genotypes. Hybrid plants revealed the unique marker bands from both parents. A total of 53 randomly chosen decamer primers were tested and 26 primers (49%) detected polymorphism between the two dihaploid parentals. Two primers revealed that one parental marker band was missing in two aneuploid hybrids. However, of 51 putative hybrids, a double test with two independently chosen primers showed unequivocally the hybrid character of 23 plants. The ploidy level of the hybrids was analysed by chromosome numbers in root tip cells and by number of guard cell chloroplasts. A strong correlation between the chromosome number and the number of chloroplasts was obtained. The hybrid nature of all RAPD‐verified hybrids was confirmed by isozyme analysis with malate dehydrogenase.

A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana.

A nuclear recessive mutant of Arabidopsis thaliana, arc5, has been isolated in which there is no significant increase in chloroplast number during leaf mesophyll cell expansion and in which there are only 13 chloroplasts per mesophyll cell compared with 121 in wild-type cells. Mature arc5 chloroplasts in fully expanded mesophyll cells are 6-fold larger than in wild-type cells. A large proportion of arc5 chloroplasts also show some degree of central constriction, suggesting that the mutation has prevented the completion of the chloroplast division process. To examine the interaction of arc loci, a double mutant was constructed between arc1, a mutant possessing many small chloroplasts, and arc5. A second double mutant was also constructed between arc3, a previously discovered mutant also possessing few large chloroplasts per cell, and arc1. Analysis of these double mutants shows that chloroplast number per mesophyll cell is greater when arc5 and arc3 mutations are expressed in the arc1 background than when expressed alone. The cell-specific nature of arc mutants was also analyzed. The phenotypic traits characteristic of arc3 and arc5 are a reduction in chloroplast number and an increase in chloroplast size in mesophyll cells: these changes are also observed in reduced form in the epidermal and guard cell chloroplasts of arc3 and arc5 plants. Analysis of parenchyma sheath cell chloroplasts suggests that in leaves of arc1 plants the normal developmental distinction between mesophyll and parenchyma sheath chloroplasts is perturbed. The relevance of these findings to the analysis of the control of chloroplast division in mesophyll cells is discussed.

Photosynthesis by Guard Cell Chloroplasts of <italic>Vicia faba</italic> L.: Effects of Factors Associated with Stomatal Movement

Photosynthesis by Guard Cell Chloroplasts of <italic>Vicia faba</italic> L.: Effects of Factors Associated with Stomatal Movement

Ploidy variation of pronamide-treated maize calli during long term culture

Anther-derived calli of corn were treated with 10 μM pronamide for 2, 3 and 4 days. The ploidy level of the calli was then evaluated using flow cytometry, at different times after the treatment. Untreated haploid calli did not change in ploidy level for 97 days but by 466 days, there were up to 50% diploid or higher ploidy cells thus showing that spontaneous doubling may occur during corn calli subculture with this genotype. Pronamide treatment did increase the percentage of diploid and tetraploid cells and by 466 days, all of the lines showed an additional change toward higher ploidy levels. This change may be due to spontaneous chromosome doubling or to differential cell cycle times of cells with different ploidy levels. The ploidy level of plants regenerated from the cultures was determined by counting the guard cell chloroplast numbers and the correlation with the ploidy level of the cultures was r(2)=0.84. These studies show that pronamide treatments can increase haploid maize callus chromosome numbers and that spontaneous chromosome doubling can occur with time in maize callus.

Regulation of blue-light-induced proton pumping by Vicia faba L. guard-cell protoplasts: Energetic contributions by chloroplastic and mitochondrial activities

An initial response during signal transduction in guard cells, following absorption of blue light, is the extrusion of protons. Translocation of protons across the guard-cell plasmalemma is an energy-requiring activity. The present study has investigated the energetic contribution from guard-cell chloroplasts and mitochondria to blue-light-induced proton pumping by Vicia faba guard-cell protoplasts. The addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea to the protoplast suspension had a minimal effect on rates of acidification when oxygen concentrations of the medium were maintained close to near-saturating levels. Under the same conditions, oligomycin reduced both the rates of blue-light-induced acidification and total proton efflux. Lowering the oxygen concentration of the suspending medium to approximately 20 μM resulted in complete inhibition of blue-light-induced acidification activity. Swelling of protoplasts induced by blue light was also inhibited by low oxygen levels. Levels of ATP from whole-protoplast extracts were reduced by about 64% when exposed to low levels of oxygen. Increasing oxygen levels to near-saturating levels restored both blue-light-induced acidification rates and swelling of the protoplasts within a 60-min recovery period. Levels of ATP also increased during the recovery period. Addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea or oligomycin to the suspending medium prior to increasing the oxygen concentration caused a reduction in acidification rates after the recovery period by 40 and 80%, respectively. Levels of ATP in guard-cell protoplasts were also reduced by both inhibitors after a 60-min recovery period. The results demonstrate that both guard-cell chloroplasts and mitochondria contribute energetically to blue-light-induced proton pumping by guard-cell protoplasts. Furthermore, both energy sources are inhibited by low oxygen concentrations, suggesting coordinated metabolic regulation between photo- and oxidative phosphorylation in guard cells.

Phosphate Translocator of Isolated Guard-Cell Chloroplasts from Pisum sativum L. Transports Glucose-6-Phosphate.

Chloroplasts were isolated from ruptured guard-cell protoplasts of the Argenteum mutant of Pisum sativum L. and purified by centrifugation through a Percoll layer. The combined volume of the intact plastids and the uptake of phosphate were determined by silicone oil-filtering centrifugation, using tritiated water and [14C]sorbitol as membrane-permeating and nonpermeating markers and [32P]phosphate as tracer for phosphate. The affinities of the phosphate translocator for organic phosphates were assessed by competition with inorganic phosphate. The affinities for dihydroxyacetone phosphate, 3-phosphoglycerate (PGA), and phosphoenolpyruvate were in the same order as those reported for mesophyll chloroplasts of several species. However, the guard-cell phosphate translocator had an affinity for glucose-6-phosphate that was as high as that for PGA. Guard-cell chloroplasts share this property with amyloplasts from the root of pea (H.W. Heldt, U.I. Flugge, S. Borchert [1991] Plant Physiol 95: 341-343). An ability to import glucose-6-phosphate enables guard-cell chloroplasts to synthesize starch despite the reported absence of a fructose-1,6-bisphosphatase activity in the plastids, which would be required if only C3 phosphates could enter through the translocator.

Fast Fluorescence Quenching from Isolated Guard Cell Chloroplasts of Vicia faba Is Induced by Blue Light and Not by Red Light

Chlorophyll a fluorescence transients from isolated Vicia faba guard cell chloroplasts were used to probe the response of these organelles to light quality. Guard cell chloroplasts were isolated from protoplasts by passing them through a 10-mum nylon net. Intact chloroplasts were purified on a Percoll gradient. Chlorophyll a fluorescence transients induced by actinic red or blue light were measured with a fluorometer equipped with a measuring beam. Actinic red light induced a monophasic quenching, and transients induced by blue light showed biphasic kinetics having a slow and a fast component. The difference between the red and blue light-induced transients could be observed over a range of fluence rates tested (200-800 mumol m(-2) s(-1)). The threshold fluence rate of blue light for the induction of the fast component of quenching was 200 mumol m(-2) s(-1), but in the presence of saturating red light, fluence rates as low as 25 mumol m(-2) s(-1) induced the fast quenching. These results indicate that guard cell chloroplasts have a specific response to blue light.

Effects of O2 and CO2 Concentration on the Steady-State Fluorescence Yield of Single Guard Cell Pairs in Intact Leaf Discs of Tradescantia albiflora

A procedure for following changes in the steady-state yield of chlorophyll a fluorescence (F(s)) from single guard cell pairs in variegated leaves of Tradescantia albiflora is described. As an indicator of photosynthetic electron transport, F(s) is a very sensitive indirect measure of the balance of adenosine 5'-triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), producing reactions with the sink reactions that utilize those light-generated products. We found that F(s) under constant light is sensitive to manipulation of ambient CO(2) concentrations, as would be expected if either phosphoenolpyruvate carboxylase or ribulose-1, 5 bisphosphate carboxylase/oxygenase (Rubisco)-dependent CO(2) fixation is the sink for photosynthetic ATP and NADPH in guard cells. However, we also found that changing O(2) concentration had a strong effect on fluorescence yield, and that O(2) sensitivity was only evident when the concentration of CO(2) was low. This finding provides evidence that both O(2) and CO(2) can serve as sinks for ATP and NADPH produced by photosynthetic electron transport in guard cell chloroplasts. Identical responses were observed with mesophyll cell chloroplasts in intact leaves. This finding is difficult to reconcile with the view that guard cell chloroplasts have fundamentally different pathways of photosynthetic metabolism from other chloroplasts in C(3) plants. Indeed, Rubisco has been detected at low levels in guard cell chloroplasts, and our studies indicate that it is active in the pathways for photosynthetic carbon reduction and photorespiration in guard cells.