Histochemical Approach to Properties of Vicia faba Guard Cell Phosphoenolpyruvate Carboxylase

Properties of phosphoenolpyruvate carboxylase in guard cells dissected from frozen-dried Vicia faba L. leaflets were studied using quantitative histochemical techniques. Control experiments with palisade cells and whole leaflet extract proved that the single cell approach was valid. Most characteristics of enzyme activity in guard cells were identical to those in the leaflet extract. The activities were highly dependent on temperature, with maximum activity at 25 to 35 C. Half-maximum activity (with 1 millimolar phosphoenolpyruvate [PEP]) was observed at 0.1 millimolar Mg²⁺. Two-hundred millimolar NaCl inhibited the reaction by 50%. With frozen-dried leaflet extract, the apparent K_m(PEP) was 0.15 millimolar at pH 7.7; with guard cells, the values were 1.49, 0.5 to 0.8, and 0.24 millimolar in three successive experiments. Additional experiments showed that apparent K_m(PEP) of guard cell activity from plants within a single growth lot was reproducible and did not change during stomatal opening. Mixed extract experiments proved that soluble compounds were not responsible for the difference observed between leaflet and guard cell activities. The differences in apparent K_m(PEP) of guard cell activity could not be unambiguously interpreted. The physiological implications of the properties of this enzyme in guard cells are discussed.     

Abscisic Acid Accumulation by in Situ and Isolated Guard Cells of Pisum sativum L. and Vicia faba L. in Relation to Water Stress

Isolated guard cells, prepared by sonication of epidermal peels, were used to investigate the endogenous level of abscisic acid (ABA) in the guard cells of turgid and stressed leaves of Vicia faba L. and the argenteum (arg) mutant of Pisum sativum L. The guard cells of V. faba and arg were found to contain 18 and 8 times more ABA, respectively, when isolated from stressed leaves than from turgid leaves. Isolated guard cells of V. faba were also directly stressed with the osmoticum Aquacide III. These guard cells were capable of producing stress-induced ABA to at least 3 times their ABA level when non-stressed.     

Action of growth regulators on the cotyledonary stomata ofCucumis sativus L.: Structure and ontogeny

Anomocytic stomata and stomata with single subsidiary cells are commonly observed Sometimes a stoma appears anisocytic. Double cytoplasmic connections between nearby stomata and division of guard cells with persistent or degenerating nuclei are seen in GA. One or more divisions of guard cells, displaced guard cells and single guard cells with or without pore are noticed in SUC. Formation of single guard cells is a common feature in TIBA. Paracytic stomata, one and a half stomata and persistent stomatal initials are seen in SUL. COUM seems to be not inhibitory inCucumis sativus. In COL stomata with unequal guard cells, unequal stomatal cells with thickening in between but without intervening pore, stoma with double pores, persistent stomatal initials which may be solitary or in groups with varying shapes and with one or two nuclei of different shapes are noticed. The growth regulators affect the frequency of stomata, epidermal cells; stomatal index; size of guard and epidermal cells.     

Chloroplast Function in Guard Cells of Vicia faba L. : Measurement of the Electrochromic Absorbance Change at 518 nm

Stomatal conductance is coupled to leaf photosynthetic rate over a broad range of environmental conditions. We have investigated the extent to which chloroplasts in guard cells may contribute to this coupling through their photosynthetic activity. Guard cells were isolated by sonication of abaxial epidermal peels of Vicia faba. The electrochromic band shift of isolated guard cells was probed in vivo as a means of studying the electric field that is generated across the thylakoid membranes by photosynthetic electron transport and dissipated by photophosphorylation. Both guard cells and mesophyll cells exhibited fast and slow components in the formation of the flash-induced electrochromic change. The spectrum of electrochromic absorbance changes in guard cells was the same as in the leaf mesophyll and was typical of that observed in isolated chloroplasts. This observation indicates that electron transport and photophosphorylation occur in guard cell chloroplasts. Neither the fast nor the slow component of the absorbance change was observed in the presence of the uncoupler carbonylcyanide p-trifluoromethoxy-phenylhydrazone which confirms that the absorbance change was caused by the electric field across the thylakoid membranes. The magnitude of the fast rise was reduced by half in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Therefore, photosystem II is functional and roughly equal in concentration to photosystem I in guard cell chloroplasts. The slow rise was abolished by 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone indicating the involvement of the cytochrome b₆/f complex in electron transport between the two photosystems. Relaxation of the absorbance change was irreversibly retarded in cells treated with the energy transfer inhibitor, N,N′-dicyclohexylcarbodiimide. The slowing of the rapid decay kinetics by N,N′-dicyclohexylcarbodiimide confirms that the electrical potential across the thyalkoid membrane is dissipated by photophosphorylation. These results show that guard cell chloroplasts conduct photosynthetic electron transport in a manner similar to that in mesophyll cells and provide the first evidence that photophosphorylation occurs in guard cells in vivo.     

Effects of O(2) and CO(2) Concentration on the Steady-State Fluorescence Yield of Single Guard Cell Pairs in Intact Leaf Discs of Tradescantia albiflora: Evidence for Rubisco-Mediated CO(2) Fixation and Photorespiration in Guard Cells

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 CO₂ concentrations, as would be expected if either phosphoenolpyruvate carboxylase or ribulose-1, 5 bisphosphate carboxylase/oxygenase (Rubisco)-dependent CO₂ fixation is the sink for photosynthetic ATP and NADPH in guard cells. However, we also found that changing O₂ concentration had a strong effect on fluorescence yield, and that O₂ sensitivity was only evident when the concentration of CO₂ was low. This finding provides evidence that both O₂ and CO₂ 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 C₃ 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.     

Metabolism of Abscisic Acid in Guard Cells of Vicia faba L. and Commelina communis L

Metabolism of abscisic acid (ABA) was investigated in isolated guard cells and in mesophyll tissue of Vicia faba L. and Commelina communis L. After incubation in buffer containing [G-³H]±ABA, the tissue was extracted by grinding and the metabolites separated by thin layer chromatography. Guard cells of Commelina metabolized ABA to phaseic acid (PA), dihydrophaseic acid (DPA), and alkali labile conjugates. Guard cells of Vicia formed only the conjugates. Mesophyll cells of Commelina accumulated DPA while mesophyll cells of Vicia accumulated PA. Controls showed that the observed metabolism was not due to extracellular enzyme contaminants nor to bacterial action.

Metabolism of ABA in guard cells suggests a mechanism for removal of ABA, which causes stomatal closure of both species, from the stomatal complex. Conversion to metabolites which are inactive in stomatal regulation, within the cells controlling stomatal opening, might precede detectable changes in levels of ABA in bulk leaf tissue. The differences observed between Commelina and Vicia in metabolism of ABA in guard cells, and in the accumulation product in the mesophyll, may be related to differences in stomatal sensitivity to PA which have been reported for these species.

     

PHYSIOLOGICAL IMPLICATIONS OF VICIA FABA AND NICOTIANA TABACUM GUARD-CELL ULTRASTRUCTURE

The guard cells of Vicia faba and Nicotiana tabacum contain numerous mitochondria, elements of endoplasmic reticulum, spherosomes, and peroxisome-like microbodies. A full ribosomal complement appears in young but not in fully mature guard cells. Numerous small lipid droplets external to the plasmalemma were noted in mature Vicia guard cells. Chloroplasts were found in both epidermal and guard cells of both species. Full photosynthetic capacity was indicated by the grana fretwork of guard-cell chloroplasts. A specialized peripheral reticulum was observed in the guard-cell chloroplasts of Vicia. Plasmodesmata were observed in both walls between sister guard cells and between guard and epidermal cells. In the latter case plasmodesmata were found primarily in pit fields of transverse walls. It is postulated that the small volume of guard cells allows them an osmotic advantage over larger neighboring cells in generating turgor.     

The mechanism of stomatal movement in Allium cepa L.

In the guard cells of Allium cepa leaves, no starch was found either when the stomata were open or closed. The lack of other soluble polysaccharides that could be hydrolyzed during the opening reaction of the stomata (Schnabl, Planta 1977, in press) leads to the question, how is the osmotic effect, which is the basis of the stomatal movement, achieved in Allium? It is shown in this paper, by histochemical and microprobe analyses, that in Allium — as in other plant species—the K⁺ concentration of the guard cells increases during stomatal opening. The charges of the K⁺ ions in the guard cells seem to be fully compensated by imported Cl^- ions. This could mean that if starch is present in the guard cells, as in the majority of plant species, its major role in the mechanism of stomatal movement is to deliver the cuunteranions for the imported K⁺ ions.     

Effect of Potassium Levels on the Stomatal Behavior of the Hemi-Parasite Striga hermonthica

The hemi-parasite Striga hermonthica, exhibits an anomalous pattern of stomatal response, stomata remaining open in darkness and when subjected to water stress. This suggests irregularity in stomatal response due to malfunction of the stomatal mechanism. To test this suggestion guard cells were isolated from the effects of surrounding cells, by incubating epidermal strips at low pH. These stomata responded rapidly to low CO₂ concentrations, darkness, and ABA. Thus, a paradox exists between stomatal behavior observed in whole leaves and that in isolated guard cells. However, when incubated in the presence of high potassium concentrations (>200 millimolar KCl) stomatal responses in epidermal strips resembled those found in whole leaves, with enhanced opening and reduced closing responses. It is suggested that the anomalous behavior of stomata in Striga and other leafy hemiparasites can be explained by the modulatory effects of high potassium concentrations which accumulate in the leaves as a consequence of high transpiration rates and the lack of a retranslocation system.     

A light and electron microscopy study of the epidermis of Paphiopedilum spp. with emphasis on stomatal ultrastructure

Abstract Light and fluorescence microscopy studies indicated that chlorophyll was absent from the guard cells of the lady slipper orchids, Paphiopedilum insigne (Wall.) Pfitz, P. insigne (hybrid), P. venustum (Wall.) Pfitz and P. harrisseanum Hort. In the guard cells of P. aureum hyeanum Hort., however, very slight red fluorescence suggested that chlorophyll and hence chloroplasts were present. Ultrastructural studies of the lower epidermis of P. insigne (hybrid) confirmed the absence of chloroplasts in guard and epidermal cells although plastids of an unusual structure were found in these cells. In fully developed epidermal cells the plastids contained large amounts of a fibrous, possibly proteinaceous substance, spherical, lightly staining vesicles and an electron-dense material located in reticulate and non-reticulate regions. Additionally, latticed crystalline inclusions and plasto-globuli were occasionally observed in the epidermal cell plastids. In plastids of fully developed guard cells the fibrous material, starch and plastoglobuli were present. From the earliest stages of development of the epidermal tissue starch was present in both epidermal cell and guard cell plastids. At maturity, however, starch had accumulated to greater levels in the guard cell plastids and had entirely disappeared in the epidermal cell plastids. In differentiating epidermal tissue, plasmodesmata were found between neighbouring epidermal cells and between guard and epidermal cells. At maturity, plasmodesmata between guard and epidermal cells were not observed. Mitochondria were particularly abundant in guard cells. Large oil drops developed in guard and epidermal cells, being especially abundant in the former at maturity. Our results confirm the observations of Nelson & Mayo (1975) that certain lady slipper orchids possess functional stomata the guard cells of which do not contain chloroplasts.     

Comparative Studies of Fluorescence from Mesophyll and Guard Cell Chloroplasts in Saxifraga cernua: Analysis of Fluorescence Kinetics as a Function of Excitation Intensity

The chlorophyll fluorescence induction curves from mesophyll and guard cell chloroplasts of Saxifraga cernua, including both the fast (O to P, the transients involved in the rise in variable fluorescence) and slow (P to steady state fluorescence due to quenching) components, were characterized over a range of excitation intensities using microspectrophotometry (with epi-lumination) equipped with apertures designed to eliminate cross contamination of the fluorescence signal between the two chloroplast types. At low excitation intensities, the fast fluorescence kinetics from guard cell plastids showed an extended I to D phase and a more rapid appearance of P while minimal quenching from P to steady state fluorescence was observed compared to the transients from mesophyll chloroplasts suggesting a lower activity of photochemical (electron movement via carriers between donor and acceptor sites) and nonphotochemical (such as membrane conformational changes) events which regulate the fluorescence induction curve kinetics. As the excitation intensity was increased, the quenching rates of guard cells were faster at initiating conditions for photophosphorylation and the fast and slow fluorescence kinetics from guard cells resembled those of the mesophyll cells.

Guard cell chloroplasts of S. cernua from intact epidermal peels showed a low temperature (77 K) fluorescence emission spectrum having three major peaks (at 685, 695, and 730 nanometers when excited at 440 nanometers) which were qualitatively similar to those in the spectrum obtained from mesophyll tissue.

These data suggest that S. cernua guard cell chloroplast photosystems I and II contribute to light-dependent stomatal activity only at high light intensities.

     

Enzymic and substrate basis for the anaplerotic step in guard cells

From the maximum rate of malate accumulation in Vicia faba L. guard cells during stomatal opening the maximum rate of organic anion synthesis is calculated to be 200 millimoles per kilogram dry weight per hour. A minimum estimate for the phosphoenolpyruvate (PEP) carboxylase-catalyzed reaction in guard cells is 650 millimoles per kilogram dry weight per hour which is significantly higher than in any other leaf tissue. The apparent K_mpep of the guard cell enzyme is 60 μm at pH 8.7, but is probably higher at lower pH. The concentration of PEP in guard cells was 270μm (=2.2 × 10⁻¹⁵ moles/guard cell pair) during anion synthesis. These results support the possibility that the carboxylation of PEP is the anaplerotic step in guard cells.     

Tissue-specific localization of an abscisic acid biosynthetic enzyme, AAO3, in Arabidopsis

Arabidopsis aldehyde oxidase 3 (AAO3) is an enzyme involved in abscisic acid (ABA) biosynthesis in response to drought stress. Since the enzyme catalyzes the last step of the pathway, ABA production sites may be determined by the presence of AAO3. Here, AAO3 localization was investigated using AAO3 promoter:AAO3-GFP transgenic plants and by an immunohistochemical technique. AAO3-GFP protein exhibited an activity to produce ABA from abscisic aldehyde, and the transgene restored the wilty phenotype of the aao3 mutant. GFP-fluorescence was detected in the root tips, vascular bundles of roots, hypocotyls and inflorescence stems, and along the leaf veins. Intense immunofluorescence signals were localized in phloem companion cells and xylem parenchyma cells. Faint but significant GFP- and immuno-fluorescence signals were observed in the leaf guard cells. In situ hybridization with antisense AAO3 mRNA showed AAO3 mRNA expression in the guard cells of dehydrated leaves. These results indicate that the ABA synthesized in vascular systems is transported to various target tissues and cells, and also that the guard cells themselves are able to synthesize ABA.     

Calvin-Benson Cycle Enzymes in Guard-Cell Protoplasts from Vicia faba L: Implications for the Greater Utilization of Phosphoglycerate/Dihydroxyacetone Phosphate Shuttle between Chloroplasts and the Cytosol

Activities of Calvin-Benson cycle enzymes were found in protoplasts of guard cells from Vicia faba L. The activities of NADP-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD) and ribulose-1,5-bisphosphate carboxylase (RuBPC) were 2670 and 52 micromoles per milligrams chlorophyll per hour, respectively. Activities of NADP-GAPD and RuBPC in guard cells were increased by red light illumination, and the light activations were inhibited completely by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. Enzymes related to the Calvin-Benson cycle such as 3-phosphoglycerate kinase (PGAK), triose phosphate (TP) isomerase, and fructose-1,6-bisphosphatase (FBPase) were shown to be present in guard-cell chloroplasts. From these results, we conclude that the photosynthetic carbon reduction pathway is present in guard-cell chloroplasts of Vicia faba. We compared these enzyme activities in guard cells with those in mesophyll cells. The activities of NADP-GAPD and PGAK were more than several-fold higher and that of TP isomerase was much higher in guard-cell chloroplasts than in mesophyll chloroplasts. In contrast, activities of RuBPC and FBPase were estimated to be roughly half of those in mesophyll chloroplasts. High activities of PGAK, NAD-GAPD, and TP isomerase were found in fractions enriched in cytosol of guard cells. Illumination of guard-cell protoplasts with red light increased the cellular ATP/ADP ratio from 5 to 14. These results support the interpretation that guard cells utilize a shuttle system (e.g. phosphoglycerate [PGA]/dihydroxyacetone phosphate [DHAP] shuttle) for an indirect transfer of ATP and reducing equivalents from chloroplasts to the cytosol.     

Immunological evidence for the presence of ribulose bisphosphate carboxylase in guard cell chloroplasts

Ribulose bisphosphate carboxylase (Rubisco) has been found in Vicia faba L. guard cell chloroplasts by two immunological methods, using antibodies raised against highly purified subunits of ribulose bisphosphate carboxylase. Indirect cytoimmunofluorescence revealed binding of antibodies against both the small and the large subunits of ribulose bisphosphate carboxylase. Binding was observed only after partial digestion of guard cell walls by 4% Cellulysin to facilitate antibody penetration. After electrophoresis of a homogenate of guard cell protoplasts, the presence of both subunits was also revealed by immunolabeling technique. Positive response required the inhibition of proteolysis which appeared to be active upon homogenization.     

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.     

Rubisco activity in guard cells compared with the solute requirement for stomatal opening

We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5-bisphophate carboxylase/oxygenase (Rubisco) were determined by the [¹⁴C]carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to ¹⁴CO₂ for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19 femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant.     

Absence of Variable Fluorescence from Guard Cell Chloroplasts of Stenotaphrum secundatum

The lack of detectable variable fluorescence from guard cell chloroplasts in both the albino and green portions of variegated leaves of St. Augustine grass (Stenotaphrum secundatum var variegatum A.S. Hitchc.) is reported. Fluorescence was measured either with a highly sensitive, modified fluorescence microscope which was capable of recording fluorescence induction curves from single chloroplasts, or with a spectrofluorometer. Both fast and slow fluorescence transients from S. secundatum guard cells showed a rapid rise and then remained at a steady level. Neither variable fluorescence increase (induction) nor decrease (quenching), properties normally associated with photosystem II, was observed from these chloroplasts. These fluorescence kinetics did not change either with alterations of the specimen preparation procedure or with alterations of the excitation light intensities and wavelengths. These results indicate that guard cell chloroplasts in this variety of S. secundatum do not conduct normal photosystem II electron transport. Light regulation of stomatal conductance in intact leaves of this plant did occur, however, and was similar to light regulation observed in other species. The conductance of the green portion of the leaves was much greater in the light than in the dark, and was much greater than the conductance of the albino portion of the leaves. Stomata in the green portion of the leaves also showed greater opening in blue light than in red light. These results provide evidence that stomatal regulation in this variety of S. secundatum does not rely on photosystem II electron transport in guard cell chloroplasts.     

Negative regulation of abscisic acid-induced stomatal closure by glutathione in Arabidopsis

We found that glutathione (GSH) is involved in abscisic acid (ABA)-induced stomatal closure. Regulation of ABA signaling by GSH in guard cells was investigated using an Arabidopsis mutant, cad2-1, that is deficient in the first GSH biosynthesis enzyme, γ-glutamylcysteine synthetase, and a GSH-decreasing chemical, 1-chloro-2,4-dinitrobenzene (CDNB). Glutathione contents in guard cells decreased along with ABA-induced stomatal closure. Decreasing GSH by both the cad2-1 mutation and CDNB treatment enhanced ABA-induced stomatal closure. Glutathione monoethyl ester (GSHmee) restored the GSH level in cad2-1 guard cells and complemented the stomatal phenotype of the mutant. Depletion of GSH did not significantly increase ABA-induced production of reactive oxygen species in guard cells and GSH did not affect either activation of plasma membrane Ca²⁺-permeable channel currents by ABA or oscillation of the cytosolic free Ca²⁺ concentration induced by ABA. These results indicate that GSH negatively modulates a signal component other than ROS production and Ca²⁺ oscillation in ABA signal pathway of Arabidopsis guard cells.     

Cytochemical and cytofluorometric evidence for guard cell photosystems

Evidence for photosynthetic linear electron transport in guard cells was obtained with two sensitive methods of high spacial resolution. Light-dependent diaminobenzidine oxidation (an indicator of PSI) and DCMU-sensitive, light-dependent thiocarbamyl nitroblue tetrazolium reduction (an indicator of PSII) were observed in guard cell plastids of Hordeum vulgare L. cv Himalaya using electron microscopic cytochemical procedures. DCMU-sensitive Chl a fluorescence induction (an indicator of PSII) was detected in individual guard cell pairs of Vicia faba L. cv Longpod using an ultramicrofluorometer. At least for these species, we conclude these results are proof for the presence of PSII in guard cell chloroplasts, which until now has been somewhat controversial.