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.     

Light-Induced De-Epoxidation of Violaxanthin in Guard Cell Protoplasts of Vicia faba

Photosynthetic pigments of Vicia guard cell protoplasts (GCPs)from abaxial epidermis were analyzed by reverse-phase HPLC.Violaxanthin decreased and zeaxanthin increased in GCPs afterlight illumination. The epoxidation state of GCPs decreasedfrom 0.82 (dark) to 0.37 (light), suggesting operation of thexanthophyll cycle in GCPs of Vicia faba. (Received March 15, 1993; Accepted May 10, 1993)     

Photosynthetic Properties of Guard Cell Protoplasts from Vicia faba L.

Guard cell protoplasts were isolated enzymatically from theepidermis of Vicia faba L. and their photosynthetic activitieswere investigated. Time courses of light-induced changes inthe chlorophyll a fluorescence intensity of these protoplastsshowed essentially the same induction kinetics as found formesophyll protoplasts of Vicia. The transient change in thefluorescence intensity was affected by DCMU, an inhibitor ofphotosystem II; by phenylmercuric acetate, an inhibitor of ferredoxinand ferredoxin NADP reductase; and by methyl viologen, an acceptorof photosystem I. Low temperature (77 K) emission spectra ofthe protoplasts had peaks at 684 and 735 nm and a shoulder near695 nm. A high O₂ uptake (175 µmol mg^–1 Chl hr^–1)was observed in guard cell protoplasts kept in darkness, whichwas inhibited by 2 mM KCN or NaN₃ by about 60%. On illumination,this O₂ uptake was partially or completely suppressed, but itssuppression was removed by DCMU, which indicates that oxygenwas evolved (150 µmol mg^–1 Chl hr^–1) photosynthetically.We concluded that both photosystems I and II function in guardcell chloroplasts and that these protoplasts have high respiratoryactivity. (Received January 30, 1982; Accepted May 15, 1982)     

Abscisic Acid-Induced Phosphoinositide Turnover in Guard Cell Protoplasts of Vicia faba

Guard cell protoplasts of Vicia faba treated with 10 [mu]M (+)abscisic acid (ABA) in the light exhibited a 20% decrease in diameter within 1.5 h, from 24.1 to 19.6 [mu]m. Within 10 s of administration of ABA, a 90% increase in levels of inositol 1,4,5-trisphosphate was observed, provided that cells were treated with Li+, an inhibitor of inositol phosphatase activity, prior to incubation. Concomitantly, levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate decreased 20% compared to levels in control cells; levels of label in the membrane lipids phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol did not change significantly in response to ABA treatment. These results show that phosphoinositide turnover is activated in response to ABA in guard cells. We conclude that phosphoinositide signaling is likely to be a step in the biochemical cascade that couples ABA to guard cell shrinking and stomatal closure.     

Photosynthetic Carbon Fixation in Guard Cell Protoplasts of Vicia faba L. : Evidence from Radiolabel Experiments

Photosynthetic carbon fixation in guard cells was reexamined in experiments with highly purified guard cell protoplasts from Vicia faba L. irradiated with red light. The fate of ¹⁴CO₂ (4.8 microcuries of NaHCO₃; final concentration: 100 micromolar) supplied to these preparations was investigated with two-dimensional paper, and thin layer chromatography. Rates of CO₂ fixation were 5- to 8-fold higher in the light than in darkness. Separation of acid-stable products into water-insoluble, neutral, and anionic fractions showed that more radioactivity was incorporated into the neutral fraction in the light than in the dark. In the dark, malate and aspartate comprised 90% of the radiolabel found in the anionic fraction, whereas in the light, radioactivity was also found in 3-phosphoglyceric acid (PGA), sugar monophosphates, sugar diphosphates, and triose phosphates. Phosphorylated compounds contained up to 60% of the label in the light-treated anionic fraction. Phosphatase treatment and rechromatography of labeled sugar diphosphate showed the presence of ribulose, a specific metabolite of the photosynthetic carbon reduction pathway (PCRP). In time-course experiments, labeled PGA was detected within 5 seconds. With time, the percentage of label in PGA decreased and that in sugar monophosphate increased. We conclude that PGA is a primary carboxylation product of the PCRP in guard cells and that the activity of the PCRP, and phosphoenolpyruvate-carboxylase is metabolically regulated.     

Effect of CO2 on Volume Change of Guard Cell Protoplast from Vicia faba L

Guard cell protoplasts (GCP) were isolated from epidermal stripsof Vicia faba L. by enzymatic digestion. The presence of non-osmoticvolume in the protoplast was suggested by the relationship betweenprotoplast volume and the mannitol concentration of the suspendingmedium. Light illumination caused swelling of GCP only whenKCl was present in the suspending medium. Dark treatment causedshrinking of GCP irrespective of the presence of 10 mM KCl.In the presence of 10 µM abscisic acid (ABA), GCP shrank.Light-induced swelling was suppressed at concentrations of ambientCO₂ higher than that in normal air. Promotion of swelling wasnot always observed at lower CO₂ concentration. These volumechange responses to light, ABA and CO₂ suggest that GCP retainsits physiological activity as a guard cell. The osmotic contributionof K⁺ to volume increase was lower than expected. Ambient CO₂seems to have some effect on the contribution of K⁺ to osmoregulationof GCP. (Received January 30, 1982; Accepted June 25, 1982)     

High Respiratory Activity of Guard Cell Protoplasts from Vicia faba L.

The rate of O₂ uptake was about 29 times higher in guard cellprotoplasts (GCPs) than in mesophyll protoplasts (MGPs) on aChi basis. The O₂ uptake was inhibited by respiratory inhibitors,but stimulated by respiratory uncouplers. On a Chi basis, theactivities of Cyt c oxidase and NADH-Cyt c reductase, mitochondrialenzymes, were about 27 and 35 times higher in GCPs than in MCPs.On a Chi basis, the ATP content was about 9 times higher inGCPs. The amount of ATP in GCPs was decreased by respiratoryinhibitors, an energy transfer inhibitor, and uncouplers ofoxidative phosphorylation. On a volume basis, GCPs had 8- to10-fold higher respiratory activities than MCPs, but had a lowChi content and lacked the activity of NADP-glyceraldehyde-3-phosphatedehydrogenase (NADP-GAPD), the Calvin cycle enzyme. From theseresults, we concluded that oxidative phosphorylation plays amain role in ATP production in guard cells and that guard cellshave a heterotrophic feature. Salicylhydroxamic acid (SHAM)in combination with KCN or NaN₃ strongly inhibited O₂ uptake,indicating the presence of cyanide-resistant respiration inguard cells. Phenylmercuric acetate (PMA), a potent inhibitorof stomatal opening, reduced the ATP content of GCPs by about90%, whereas it had a relatively small effect on the ATP levelof MCPs. The specific effect of PMA on GCPs is discussed. (Received March 24, 1983; Accepted June 8, 1983)     

Photosynthesis-Dependent Volume Regulation in Guard Cell Protoplasts from Vicia faba L.

A rapid and convenient procedure was developed for isolatingguard cell protoplasts (GCPs) from epidermal strips of Viciafaba L. The mean rates of O₂ uptake in the dark and evolutionin light of the isolated GCPs were 200 and 290 µmol O₂mg^–1 Chl h^–1, respectively, showing net O₂ evolutionin light. Photosynthetic O₂ evolution was suppressed completelyby 5 µM DCMU. Addition of 5 µM DCMU to the incubationmedium after 30 min of light exposure also suppressed the light-inducedswelling of GCP, indicating possible participation of PS IIin volume regulation in GCP. ⁴Present address: Division of Environmental Biology, The NationalInstitute for Environmental Studies, Yatabe machi, Tsukuba,Ibaraki 305, Japan. (Received December 17, 1983; Accepted March 21, 1984)     

Isolation of Guard Cell Protoplasts from Mechanically Prepared Epidermis of Vicia faba Leaves

A method for isolating guard cell protoplasts (GCP) from mechanically prepared epidermis of Vicia faba is described. Epidermis was prepared by homogenizing leaves in a Waring blender in a solution of 10% Ficoll, 5 millimolar CaCl₂, and 0.1% polyvinylpyrrolidone 40 (PVP). Attached mesophyll and epidermal cells were removed by shaking epidermis in a solution of Cellulysin, mannitol, CaCl₂, PVP, and pepstatin A. Cleaned epidermis was transferred to a solution of mannitol, CaCl₂, PVP, pepstatin A, cellulase “Onozuka” RS, and pectolyase Y-23 for the isolation of GCP. Preparations made by this method included both adaxial and abaxial GCP and contained ≤0.017% mesophyll protoplasts, ≤0.6% mesophyll fragments, and no epidermal cell contaminants. Yields averaged 9 × 10⁴ protoplasts/leaflet and 98 to 100% of the GCP excluded trypan blue, concentrated neutral red, and hydrolyzed fluorescein diacetate. Isolated GCP increased in diameter by 2.2 micrometers after incubation in darkness in 10 micromolar fusicoccin, 0.4 molar mannitol, 5 millimolar KCl, and 1 millimolar CaCl₂. Illumination of GCP with 800 micromoles per square meter per second of red light resulted in alkalinization of their suspension medium. When 10 micromolar per square meter per second of blue light was superimposed onto the red light background, the medium acidified. Measurements of chlorophyll a fast fluorescence transients from isolated GCP indicated that GCP were capable of electron transport, and slow transients contained the “M” peak usually associated with a functional photosynthetic carbon reduction pathway.     

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.

     

Light-Induced Alkalinization of the Suspending Medium of Guard Cell Protoplasts from Vicia faba L

The light-dependent pH changes in the suspending medium of guard cell protoplasts (GCP) from Vicia faba were studied. Upon illumination, the medium was initially slightly alkalinized and then acidified. The extent of alkalinization was lower in CO₂-free air than in normal air. This initial alkalinization was inhibited by DCMU. Acidification in CO₂-free air became observable in shorter duration of light exposure than that in normal air. The rate of acidification was higher in CO₂-free air than in normal air. The CO₂ level of the medium decreased in the light, and increased in the dark. ¹⁴CO₂ uptake was enhanced 2- to 3-fold by light, but not in the presence of DCMU. These results indicate that photosynthetic CO₂ fixation does take place in GCP and that the initial alkalinization is due to this photosynthetic CO₂ uptake. Diethylstilbestrol, a nonmitochondrial membrane-bound ATPase inhibitor, inhibited the acidification, suggesting that the acidification resulted from H⁺ extrusion by GCP. The acidification in light was also prevented by KCN, and partly by DCMU. Possible mechanisms of alkalinization and acidification are discussed in relation to guard cell metabolism.     

Carbon Dioxide Fixation by Guard Cell Protoplasts of Commelina communis

Biochemical studies of epidermal tissue may not reflect metabolismof the guard cells which represent less than 5% of the tissuevolume. Pure samples of guard cell protoplasts of Commelinacommunis were therefore used to investigate CO₂ fixation ratesand ¹⁴C-labelling patterns of metabolites in the light and thedark. Qualitatively, results were similar in most respects tothose obtained in a previous study (Schnabl, 1980) for guardcell protoplasts of Vicia faba. CO₂ fixation rates by guardcell protoplasts of C. communis were the same in the light andthe dark but about 50 times lower than the values Schnabl obtainedfor V.faba. The ¹⁴C-labelling pattern of metabolites in C. communiswas also similar in the light and the dark: over 60% of thetotal fixed was in malate with only 1% in sugar phosphates.Label was also detected in starch, aspartate, glutamate andcitrate but not in glycollate as previously recorded in V. fabaguard cell protoplasts. The results confirm the view that the reductive pentose phosphatepathway does not occur in guard cells of C. communis. Key words: CO₂ fixation, Guard cell protoplasts, Stomata     

External Protons Enhance the Activity of the Hyperpolarization-activated K Channels in Guard Cell Protoplasts of Vicia faba

Hyperpolarization-activated K channels (K _H channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell mode, we characterized the effect of varying the external pH between 4.4–8.1 on the activity of the K _H channels in isolated guard cell protoplasts from Vicia faba leaves. Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the K _H channels by 30–150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by ∼60% between pH 8.1 and 4.4 with an apparent pK _a of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing of these charges was ∼30 ? and their apparent dissociation constant was 10^−4.6. The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence of all four transition rate constants. Received: 26 April 1996/Revised: 29 June 1996     

Properties of Proton Pumping in Response to Blue Light and Fusicoccin in Guard Cell Protoplasts Isolated from Adaxial Epidermis of Vicia Leaves

Guard cell protoplasts (GCPs) were isolated from the adaxial epidermis of Vicia leaves. The properties of isolated adaxial GCPs (ad GCPs) were compared with those of abaxial GCPs (ab GCPs) with respect to H+-pumping activity. A saturating pulse of blue light (200 [mu]mol m-2 s-1, 30 s) induced H+ pumping in both ad GCPs and ab GCPs under red light. The maximum rate of blue-light-dependent H+ pumping was slightly higher in ad GCPs than in ab GCPs, but the magnitude of H+ pumping in ad GCPs was 68% of that in ab GCPs. H+ pumping was responsive to the second pulse, and the rate and magnitude of the pumping increased with the time between two pulses. The periods required to achieve 50% of the maximum rate were 12 and 22 min for ad GCPs and ab GCPs, respectively. The rates of blue-light-dependent H+ pumping were saturable, with half-saturation at 630 [mu]mol m-2 (21 [mu]mol m-2 s-1, 30 s) for ad GCPs and 105 [mu]mol m-2 (3.5 [mu]mol m-2 s-1, 30s) for ab GCPs. In contrast, fusicoccin, an activator of the plasma membrane H+- ATPase, induced H+ pumping with a slightly higher rate in ad GCPs than in ab GCPs. Both types of protoplast swelled similarly in response to fusicoccin. These results suggest that ad GCPs have almost the same activity for H+ pumping as ab GCPs, whereas ad GCPs require a larger number of photons to activate the H+ pump than ab GCPs.     

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.     

Involvement of Calmodulin and Calmodulin-Dependent Myosin Light Chain Kinase in Blue Light-Dependent H Pumping by Guard Cell Protoplasts from Vicia faba L

Signal transduction processes involved in blue light-dependent proton pumping were investigated using guard cell protoplasts from Vicia faba. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cyclic AMP- and cyclic GMP-dependent protein kinases, had no effect. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, inhibitors of protein kinase C, produced slight inhibition of the blue light-dependent proton pumping. 1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl] -4-phenylpiperazine, a specific inhibitor of Ca²⁺/calmodulin (CaM)-dependent protein kinase II, did not inhibit the proton pumping, but 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine and 1-(5-chloro-naphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), inhibitors of Ca²⁺/CaM-dependent myosin light chain kinase, strongly suppressed the proton pumping. A CaM antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited blue light-dependent proton pumping, whereas its less active structural analog, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had little effect on the response. Other CaM antagonists, trifluoperazine, compound 48/80, prenylamine, and 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4-phenyl-piperidinyl)- propylbenzenesulfonamide inhibited the proton pumping. In accord with these results, light-induced stomatal opening in the epidermis of Commelina benghalensis ssp. was inhibited by ML-9 and W-7, but not by H-7 and W-5. Thus, it is concluded that CaM and Ca²⁺/CaM-dependent myosin light chain kinase are the components of the signal transduction process in blue light-dependent proton pumping in guard cells.     

Ferricyanide Reduction by Guard Cell Protoplasts

Guard cell protoplasts of Commelina communis L. reduced exogenousferricyanide at pH values lower than 5?0; upon addition of NADH,reduction of ferricyanide by guard cell protoplasts was stimulatedover the pH range 4?0 to 9?0 with two peaks of activity at pH5?0 and between pH 8?0 and pH 9?0. Calcium chloride (1?0 molm^–3) and MgCl2 (1?0 mol m^–3) increased the NADH-stimulatedreduction of ferricyanide. Superoxide dismutase and cyanidehad little effect on the NADH-stimulated reduction of ferricyanideby guard cell protoplasts, but, salicylhydroxamic acid completelyinhibited this activity. The NADH-stimulated reduction of ferricyanidealso occurred in the cell-free supernatant. Horseradish peroxidasedid not reduce ferricyanide in the absence of NADH over a broadrange of pH (4?0 to 9?0). However, in the presence of NADH,horseradish peroxidase reduced ferricyanide over the pH range5?0 to 9?0 with maximal activity at pH 8?0. The NADH-stimulatedreduction of ferricyanide by horseradish peroxidase showed similarproperties to those observed with guard cell protoplasts. Mannitol,superoxide dismutase, and cyanide did not inhibit the NADH-stimulatedreduction of ferricyanide by horseradish peroxidase; SHAM, however,completely inhibited the reduction of ferricyanide by horseradishperoxidase. Catalase inhibited the NADH-stimulated reductionof ferricyanide by horseradish peroxidase by 20%, while absenceof oxygen in the assay medium stimulated this activity over60%. We propose that the reduction of ferricyanide in the presenceof NADH by guard cell protoplasts, can be explained in termsof peroxidase activity associated with the plasma membrane andsecreted to the extracellular medium. However, the capacityof guard cell protoplasts to reduce ferricyanide at acid pHvalues where little peroxidase activity occurs may indicatethe presence of a plasma membrane redox system in guard cellsof C. communis. Key words: Commelina, guard cell protoplasts, ferricyanide reduction, peroxidase, redox system     

共聚焦显微技术研究SA对蚕豆气孔保卫细胞的影响

水杨酸(salicylic acid,SA)作为植物体内一种内源性的信号分子,具有多种生理功能.实验表明水杨酸以浓度依赖的方式诱导气孔关闭,抑制气孔张开.20U/mL的CAT与SA共同处理时可逆转SA诱导气孔关闭作用的83%~90%.以H2O2荧光探针H2DCFDA结合激光扫描共聚焦显微术直接检测到SA处理可引起保卫细胞内H2O2的产生;在保卫细胞内显微注射CAT可完全阻止SA导致的DCF荧光增强.表明SA诱导的气孔关闭可能与H2O2的产生有关.