Ribulosebisphosphate carboxylase activity and photosynthetic o(2) evolution rate in vicia guard-cell protoplasts

Activities of ribulose-1,5-bisphosphate carboxylase and rates of photosynthetic O₂ evolution were measured in guard-cell and mesophyll protoplasts from Vicia faba. The ribulose-1,5-bisphosphate carboxylase activity of guard-cell protoplasts was 30% of that of mesophyll protoplasts; however, the O₂ evolution rate was 3 times higher in guard-cell protoplasts than in mesophyll protoplasts on a chlorophyll basis. When the dark-adapted, guard-cell protoplasts were illuminated by red light, O₂ was evolved with an induction period, which became shorter when the protoplasts were reilluminated. High activity of irreversible NADP-glyceraldehyde-3-phosphate dehyrogenase was found in guard-cell protoplasts. Several lines of evidence revealed that there was virtually no contamination by mesophyll cells in guard-cell preparations. These results indicate that guard cells fix CO₂ photosynthetically and imply that the cells utilize a considerable proportion of reducing equivalents from water for reactions other than CO₂ fixation.     

A role for fructose 2,6-bisphosphate in regulating carbohydrate metabolism in guard cells

Fructose 2,6-bisphosphate (Fru2,6P₂) appears to function as a regulator metabolite in glycolysis and gluconeogenesis in animal tissues, yeast, and the photosynthetic cells of leaves. We have investigated the role of Fru2,6P₂ in guard-cell protoplasts from Vicia faba L. and Pisum sativum L. (Argenteum mutant), and in epidermal strips purified by sonication from all cells except for the guard cells. Guard-cell protoplasts were separated into fractions enriched in cytosol and in chloroplasts by passing them through a nylon net, followed by silicone oil centrifugation. The cytosol contained a pyrophosphate: fructose 6-phosphate phosphotransferase (involved in glycolysis) which was strongly stimulated by Fru2,6P₂. A cytosolic fructose 1,6-bisphosphatase (a catalyst of gluconeogenesis) was inhibited by Fru2,6P₂. There was virtually no fructose 1,6-bisphosphatase activity in guard-cell chloroplasts of V. faba. It is therefore unlikely that the starch formed in these chloroplasts originates from imported triose phosphates or phosphoglycerate.

The level of Fru2,6P₂ in guard-cell protoplasts and epidermal strips was about 0.1 to 1 attomole per guard cell in the dark (corresponding to 0.05 to 0.5 nanomole per milligram chlorophyll) and increased three- to tenfold within 15 minutes in the light. Within the same time span, hexose phosphate levels in guard-cell protoplasts declined to approximately one-half, indicating that acceleration of glycolysis involved stimulation of reactions using hexose phosphates. The level of Fru2,6P₂ in guard cells appears to determine the direction in which carbohydrate metabolism proceeds.

     

Light Activation of NADP-Malate Dehydrogenase in Guard Cell Protoplasts from Vicia faba L

Light-induced swelling of guard cell protoplasts (GCP) from Vicia faba was accompanied by increases in content of K⁺ and malate. DCMU inhibited the increase of K⁺ and malate, and consequently swelling.

Effect of light on the activity of selected enzymes that take part in malate formation was studied. When isolated GCP were illuminated, NADP-malate dehydrogenase (NADP-MDH) was activated, and the activity reached a maximum within 5 minutes. The enzyme activity underwent 5- to 6-fold increase in the light. Upon turning off the light, the enzyme was inactivated in 5 minutes NAD-MDH and phosphoenolpyruvate carboxylase (PEPC) were not influenced by light. The rapid light activation of NADP-MDH was inhibited by DCMU, suggesting that the enzyme was activated by reductants from the linear electron transport in chloroplasts. An enzyme localization study by differential centrifugation indicates that NADP-MDH is located in the chloroplasts, NAD-MDH in the cytosol and mitochondria, and PEPC in the cytosol. After light activation, the activity of NADP-MDH in guard cells was 10 times that in mesophyll cells on a chlorophyll basis. The physiological significance of light-dependent activation of NADP-MDH in guard cells is discussed in relation to stomatal movement.

     

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.     

Photosystem II in Guard Cells of Vicia faba: Immunological Detection

Antibodies were raised against individual polypeptides of the oxygen-evolving photosystem II (PSII) complex from mesophyll chloroplasts of Vicia faba (Long Pod). These antibodies were used to probe immunologically for the presence of the main structural components of the PSII complex in guard cell chloroplasts, using both immunofluorescence microscopy and Western blotting. Immunofluorescence of epidermal peels with antibodies raised against the extrinsic 33 kilodalton polypeptide, as well as the 47 and the 44 kilodalton subunits and the light-harvesting chlorophyll a/b protein, resulted in intense fluorescence indicating the presence of these polypeptide components in guard cell chloroplasts. Results obtained with Western blot analysis showed that the relative amounts of the 33 kilodalton and light-harvesting complex protein polypeptides are between 60 and 80% of that found in mesophyll cells (on chlorophyll basis). These results provide evidence for the existence of structural components associated with PSII activity in guard cell similar to those of mesophyll chloroplasts.     

Determination of malate levels during the swelling of vacuoles isolated from guard-cell protoplasts

A method for the preparation of vacuoles from guard cells ofVicia faba L. is described. Vacuoles were released from guard-cell protoplasts by osmotic shock and purified on a Ficoll gradient. Contamination of the vacuoles was examined by assaying marker enzymes, such as fumarase, glucose-6-phosphate dehydrogenase, phosphofructokinase, acid phosphatase and mannosidase. Potassium ions in the incubation medium caused increases in the volume of the vacuoles by a factor of about 2.6, while the malate level remained unchanged. In contrast, malate synthesis was stimulated during the swelling phase when complete guard-cell protoplasts were exposed to K⁺. The possible role of K⁺ as an efficient osmotic effector is discussed.Abbreviations DEAE diethylaminoethyl - GCP guard-cell protoplast(s) - GCV guard-cell vacuoles(s) - MCP mesophyll cell protoplast(s) - MCV mesophyll cell vacuole(s)     

Taxonomic survey for the presence of ribulose-1,5-bisphosphate carboxylase activity in guard cells

Guard cell pairs were dissected from freeze-dried leaves of plants representing 15 families, including monocots, dicots, and pteridophytes. All three major photosynthetic carbon pathways (C₂, C₄, and Crassulacean acid metabolism) were represented. These individual guard cell pairs were assayed quantitatively for ribulose-1,5-bisphosphate carboxylase specific activity. Assay sensitivity averaged 0.08 picomoles of ribulose-P₂ dependent P-glycerate formation (i.e. 100-fold more sensitive than required to detect the activity present in a single Vicia faba mesophyll cell). The calculated specific activities for guard cells and mesophyll cells averaged 4 and 472 millimoles per kilogram dry weight per hour, respectively. For all species surveyed, (a) the enzyme activity calculated for guard cells was below the detection limit of the assay, or (b) the specific activity (weight or cell basis) calculated for guard cells was less than 1% of the specific activity calculated for adjacent mesophyll cells. Based on this survey, the generalization is made that the photosynthetic carbon reduction pathway is absent, or virtually so, in guard cell chloroplasts.     

Identification and biochemical characterization of the plasma-membrane H+-ATPase in guard cells of Vicia faba L.

The plasma-membrane H⁺-pump in guard cells generates the driving force for the rapid ion fluxes required for stomatal opening. Since our electrophysio-logical studies revealed a two fold higher pump-current density in guard cells than in mesophyll cells of Vicia faba L. we elucidated the biochemical properties of this proton-translocating ATPase in plasma-membrane vesicles isolated from both cell types. The capability of the H⁺ —ATPase to create an H⁺ gradient is maintained in plasma-membrane vesicles derived from purified guard cells via blender maceration, high-pressure homogenization and polymer separation. The H⁺-pumping activity of these vesicles coincides with the presence of two polypeptides of approx. 100 and 92 kDa which are recognized by a monoclonal antibody raised against the plasma-membrane H⁺-ATPase from Zea mays L. coleoptiles. Comparison of H⁺-pumping activities of isolated membranes revealed an approximately two fold higher activity in guard cells than in mesophyll cells with respect to the total membrane protein content. Furthermore, we demonstrated by western blotting that the difference in pump activities resulted from a higher abundance of the electroenzyme per unit membrane protein in guard-cell plasma membranes. We suggest that the high H⁺-pump capacity is necessary to enable guard cells to respond to sudden changes in the environment by a change in stomatal aperture.     

Electrophoretic Assay for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in Guard Cells and Other Leaf Cells of Vicia faba L

The ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents of guard cells and other cells of Vicia faba L. leaflet were determined. To prevent proteolysis, proteins of frozen protoplast preparations or of cells excised from freeze-dried leaf were extracted directly in a sodium-dodecyl-sulfate-containing solution, which was heated immediately after sample addition. Protein profiles of the different cell types were obtained by electrophoresis of the extracts and subsequent densitometry of the stained protein bands. About one-third of the protein of palisade parenchyma and of spongy parenchyma was Rubisco large subunit. Using chlorophyll (Chl):protein ratios previously obtained, we calculate mesophyll contained ca. 22 millimoles Rubisco per mole Chl. In contrast, guard-cell protoplast preparations were calculated to contain from 0.7 to 2.2 millimoles Rubisco per mole Chl. The upper end of this range is an overestimate resulting from contamination by mesophyll and to the method of peak integration. Extracts of excised guard cells were calculated to contain 0.05 to 0.17 millimole Rubisco per mole Chl. We conclude that Rubisco is absent, or virtually so, in guard cells of V. faba.     

Cyclic and Noncyclic Photophosphorylation in Isolated Guard Cell Chloroplasts from Vicia faba L

High rates of both cyclic and noncyclic photophosphorylation were measured in chloroplast lamellae isolated from purified guard cell protoplasts from Vicia faba L. Typical rates of light-dependent incorporation of ³²P into ATP were 100 and 190 micromoles ATP per milligram chlorophyll per hour for noncyclic (water to ferricyanide) and cyclic (phenazine methosulfate) photophosphorylation, respectively. These rates were 50 to 80% of those observed with mesophyll chloroplasts. Noncyclic photophosphorylation in guard cell chloroplasts was completely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea supporting the notion that photophosphorylation is coupled to linear electron flow from photosystem II to photosystem I. Several lines of evidence indicated that contamination by mesophyll chloroplasts cannot account for the observed photophosphorylation rates.

A comparison of the photon fluence dependence of noncyclic photophosphorylation in mesophyll and guard cell chloroplasts showed significant differences between the two preparations, with half saturation at 0.04 and 0.08 millimole per square meter per second, respectively.

     

Fluorescence Properties of Guard Cell Chloroplasts: EVIDENCE FOR LINEAR ELECTRON TRANSPORT AND LIGHT-HARVESTING PIGMENTS OF PHOTOSYSTEMS I AND II

The presence of chloroplasts in guard cells from leaf epidermis, coleoptile, flowers, and albino portions of variegated leaves was established by incident fluorescence microscopy, thus confirming the notion that guard cell chloroplasts are remarkably conserved. Room temperature emission spectra from a few chloroplasts in a single guard cell of Vicia faba showed one major peak at around 683 nanometers. Low-temperature (77 K) emission spectra from peels of albino portions of Chlorophytum comosum leaves and from mesophyll chloroplasts of green parts of the same leaves showed major peaks at around 687 and 733 nanometers, peaks usually attributed to photosystem II and photosystem I pigment systems, respectively. Spectra of peels of V. faba leaves showed similar peaks. However, fluorescence microscopy revealed that the Vicia peels, as well as those from Allium cepa and Tulipa sp., were contaminated with non-guard cell chloroplasts which were practically undetectable under bright field illumination. These observations pose restrictions on the use of epidermal peels as a source of isolated guard cell chloroplasts. Studies on the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive variable fluorescence kinetics of uncontaminated epidermal peels of C. comosum indicated that guard cell chloroplasts operate a normal, photosystem II-dependent, linear electron transport. The above properties in combination with their reported inability to fix CO₂ photosynthetically may render the guard cell chloroplasts optimally suited to supply the reducing and high-energy phosphate equivalents needed to sustain active ion transport during stomatal opening in daylight.     

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.     

Blue light-modulation of chlorophyll a fluorescence transients in guard cell chloroplasts

Chlorophyll a fluorescence transients from mesophyll and single guard cell pairs of Vicia faba were measured by microspectrofluorometry. In both chloroplast types, fluorescence induction (O to P) was similar under actinic blue and green light. In slow transients from mesophyll cell chloroplasts, blue and green light induced identical, typical rapid quenching from P to S, and the M peak. In contrast, the P to S transient from guard cell (GC) chloroplasts irradiated with blue light showed a much slower quenching rate, and the P to T transition showed no M peak. Actinic green light induced mesophyll-like transients in GC chloroplasts, including rapid quenching from P to S and the M peak. Detection of these transients in single pairs of GC and isolated protoplasts ruled out mesophyll contamination as a signal source. Green light induced a rapid quenching and the M peak in GC chloroplasts from several species. The effect of CO₂ concentration on the fluorescence transients was investigated in the presence of HCO₃⁻ at pH 6.8 and 10.0. In transients induced by green light in both chloroplast types, a pH increase concomitant with a reduction in CO₂ concentration caused an increase in the initial rate of quenching and the elimination of the M peak. Actinic blue light induced mesophyll-like transients from GC chloroplasts in the presence of 10 micromolar KCN, a concentration at which the blue light-induced stomatal opening is inhibited. Addition of 100 to 200 micromolar phosphate also caused large increases in fluorescence quenching rates and a M peak. These results indicate that blue light modulates photosynthetic activity in GC chloroplasts. This blue light effect is not observed in the absence of transduction events connected with the blue light response and in the presence of high phosphate concentrations.     

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 M 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.Abbreviations BL blue light - Chl chlorophyll - DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - GCPs guard-cell protoplasts This research was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada and a University Research Grant from The University of Calgary. Dr. L. Gedamu (University of Calgary) is thanked for providing access to the bioluminometer. Technical assistance by C. Chmielewski, C. Turnnir, S. Ham and K. Meyer is gratefully acknowledged.     

Red Light-Dependent CO(2) Uptake and Oxygen Evolution in Guard Cell Protoplasts of Vicia faba L.: Evidence for Photosynthetic CO(2) Fixation

Suspensions of dark-adapted guard cell protoplasts of Vicia faba L. alkalinized their medium in response to irradiation with red light. The alkalinization peaked within about 50 minutes and reached steady state shortly thereafter. Simultaneous measurements of O₂ concentrations and medium pH showed that oxygen evolved in parallel with the red light-induced alkalinization. When the protoplasts were returned to darkness, they acidified their medium and consumed oxygen. Both oxygen evolution and medium alkalinization were inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In photosynthetically competent preparations, light-dependent medium alkalinization is diagnostic for photosynthetic carbon fixation, indicating that guard cell chloroplasts have that capacity. The striking contrast between the responses of guard cell protoplasts to red light, which induces alkalinization, and that to blue light, which activates proton extrusion, suggests that proton pumping and photosynthesis in guard cells are regulated by light quality.     

Presence of Both Photosystems in Guard Cells of Vicia faba L: IMPLICATIONS FOR ENVIRONMENTAL SIGNAL PROCESSING

A new procedure is reported for high-yield isolation of guard cell protoplasts from Vicia faba L. Delayed light emission and P₇₀₀ content plus absorption and fluorescence emission spectra of these protoplast extracts are reported. It is concluded that both photosystems are present. The presence of photosystem II and the absence of the reductive-step enzyme of the Calvin-Benson Cycle (Outlaw WH Jr, J Manchester, CH DiCamelli, DD Randall, B Rapp, GM Veith 1979 Proc Natl Acad Sci USA 76: 6371-6375) in a cell has no precedent in the literature. It is speculated that noncyclic photosynthetic electron flow is an environmental sensor which causes stomata to remain open in light.     

Guard Cell Starch Biosynthesis Regulated by Effectors of ADP-Glucose Pyrophosphorylase

ADP-glucose pyrophosphorylase catalyzes the regulated step of starch bioynthesis in mesophyll chloroplasts. This enzyme is activated by a high ratio of the concentrations of 3-P-glycerate to inorganic phosphate (Pi) in light. In contrast, starch in guard cell chloroplasts is degraded when stomata open, which usually occurs in light. We have investigated the biochemical causes for this contrasting phenomenon.

Vicia faba L. leaflets were sampled in darkness and after various periods of illumination. The samples were quick-frozen and freeze-dried. Guard cells and other cells were dissected out, weighed, and assayed for ADP-glucose pyrophosphorylase activity, 3-P-glycerate, and Pi. In the pyrophosphorolytic direction, ADP-glucose pyrophosphorylase specific activity in guard cells was 2.7 moles per kilogram protein per hour, which was comparable to the values obtained for palisade and spongy cells. The specific activity in epidermal cells was 4-fold lower. Under our assay conditions, the guard cell enzyme activity was 5-fold higher in the presence of 3-P-glycerate and 5-fold lower with Pi (i.e. similar to the results obtained with extracts of fresh leaflet). During three minutes of illumination, 3-P-glycerate concentration in palisade cells increased 2.5-fold to 10 millimoles per kilogram dry mass. The concentration of 3-P-glycerate in guard cells was 20-fold lower and unaffected by illumination. The concentration of Pi was approximately 17 millimoles per kilogram dry mass in palisade cells, but was 10-fold higher in guard cells. These overall cellular Pi concentrations were unaffected by illumination. We conclude that starch biosynthesis in guard cells is not activated by light because of the low and constant 3-P-glycerate concentration there. We interpret this last to be a consequence of the absence of the photosynthetic carbon reduction pathway in chloroplasts of these cells.

     

Activation of NADP-Malate Dehydrogenase, Pyruvate,Pi Dikinase, and Fructose 1,6-Bisphosphatase in Relation to Photosynthetic Rate in Maize

The activity and extent of light activation of three photosynthetic enzymes, pyruvate,Pi dikinase, NADP-malate dehydrogenase (NADP-MDH), and fructose 1,6-bisphosphatase (FBPase), were examined in maize (Zea mays var Royal Crest) leaves relative to the rate of photosynthesis during induction and under varying light intensities. There was a strong light activation of NADP-MDH and pyruvate,Pi dikinase, and light also activated FBPase 2- to 4-fold. During the induction period for whole leaf photosynthesis at 30°C under high light, the time required to reach half-maximum activation for all three enzymes was only 1 minute or less. After 2.5 minutes of illumination the enzymes were fully activated, while the photosynthetic rate was only at half-maximum activity, indicating that factors other than enzyme activation limit photosynthesis during the induction period in C₄ plants.

Under steady state conditions, the light intensity required to reach half-maximum activation of the three enzymes was similar (300-400 microEinsteins per square meter per second), while the light intensity required for half-maximum rates of photosynthesis was about 550 microEinsteins per square meter per second. The light activated levels of NADP-MDH and FBPase were well in excess of the in vivo activities which would be required during photosynthesis, while maximum activities of pyruvate,Pi dikinase were generally just sufficient to accommodate photosynthesis, suggesting the latter may be a rate limiting enzyme.

There was a large (5-fold) light activation of FBPase in isolated bundle sheath strands of maize, whereas there was little light activation of the enzyme in isolated mesophyll protoplasts. In mesophyll protoplasts the enzyme was largely located in the cytoplasm, although there was a low amount of light-activated enzyme in the mesophyll chloroplasts. The results suggest the chloroplastic FBPase in maize is primarily located in the bundle sheath 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.     

Profile of Basic Carbon Pathways in Guard Cells and Other Leaf Cells of Vicia faba L

Guard cells and three other cell types from Vicia faba L. `Longpod' leaflets were assayed for enzymes that catalyze one step in each of five major carbon pathways in green plants: the photosynthetic carbon reduction pathway (ribulose-bisphosphate carboxylase, EC 4.1.1.39), the photosynthetic carbon oxidation pathway (hydroxypyruvate reductase, EC 1.1.1.81), glycolysis ([NAD] glyceraldehyde-P dehydrogenase, EC 1.2.1.12), the oxidative pentose-P pathway (6-P-gluconate dehydrogenase, EC 1.1.1.44), and the tricarboxylic acid pathway (fumarase, EC 4.2.1.2). Neither ribulose-bisphosphate carboxylase nor hydroxypyruvate reductase could be detected in guard cells or epidermal cells; high levels of these activities were present in mesophyll cells. The specific activity of fumarase (protein basis) was about 4-fold higher in guard cells than in epidermal, palisade parenchyma or spongy parenchyma cells. (NAD) glyceraldehyde-P and 6-P-gluconate dehydrogenases also were present at high protein specific activities in guard cells (2- to 4-fold that in meosphyll cells).

It was concluded that the capacity for metabolite flux through the catabolic pathways is high in guard cells. In addition, other support is provided for the view that photoreduction of CO₂ by these guard cells is absent.