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1.
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. 相似文献
2.
Changes in neutral sugar and organic acid content of guard cells were quantitated by high-performance liquid chromatography during stomatal opening in different light qualities. Sonicated Vicia faba epidermal peels were irradiated with 10 [mu]mol m-2 s-1 of blue light, a fluence rate insufficient for the activation of guard cell photosynthesis, or 125 [mu]mol m-2 s-1 of red light, in the presence of 1 mM KCl, 0.1 mM CaCl2. The low-fluence-rate blue light stimulated an average net stomatal opening of 4.7 [mu]m in 2 h, whereas the saturating fluence rate of red light stimulated an average net opening of 3.8 [mu]m in 2 h. Under blue light, the malate content of guard cells increased to 173% of the initial level during the first 30 min of opening and declined as opening continued. Sucrose levels continuously rose throughout the blue light-stimulated opening, reaching 215% of the initial level after 2 h. The starch hydrolysis products maltose and maltotriose remained elevated at all times. Under red light, guard cells showed very little increase in organic acid or maltose levels, whereas sucrose levels increased to 208% of the initial level after 2 h. Total measured organic metabolite concentrations were correlated with stomatal apertures in all cases except where substantial malate increases occurred. These results support the hypothesis that light quality modulates alternative mechanisms of osmotic accumulation in guard cells, including potassium uptake, photosynthetic sugar production, and starch breakdown. 相似文献
3.
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. 相似文献
4.
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 Ca 2+/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 Ca 2+/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 Ca 2+/CaM-dependent myosin light chain kinase are the components of the signal transduction process in blue light-dependent proton pumping in guard cells. 相似文献
5.
Concentrations of soluble sugars in guard cells in detached, sonicated epidermis from Vicia faba leaves were analyzed quantitatively by high performance liquid chromatography to determine the extent to which sugars could contribute to changes in the osmotic potentials of guard cells during stomatal opening. Stomata were illuminated over a period of 4 hours with saturating levels of red or blue light, or a combination of red and blue light. When stomata were irradiated for 3 hours with red light (50 micromoles per square meter per second) in a solution of 5 millimolar KCl and 0.1 millimolar CaCl 2, stomatal apertures increased a net maximum of 6.7 micrometers and the concentration of total soluble sugar was 289 femtomoles per guard cell (70% sucrose, 30% fructose). In an identical solution, 2.5 hours of irradiation with 25 micromoles per square meter per second of blue light caused a maximum net increase of 7.1 micrometers in stomatal aperture and the total soluble sugar concentration was 550 femtomoles per guard cell (91% sucrose, 9% fructose). Illumination with blue light at 25 micromoles per square meter per second in a solution lacking KCl caused a maximum net increase in stomatal aperture of 3.5 micrometers and the sugar concentration was 382 femtomoles per guard cell (82% sucrose, 18% fructose). In dual beam experiments, stomata irradiated with 50 micromoles per square meter per second of red light opened steadily with a concomitant increase in sugar production. Addition of 25 micromoles per square meter per second of blue light caused a further net gain of 3.7 micrometers in stomatal aperture and, after 2 hours, sugar concentrations had increased by an additional 138 femtomoles per guard cell. Experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were performed with epidermis illuminated with 50 micromoles per square meter per second of red light or with 25 micromoles per square meter per second of blue light in solutions containing or lacking KCl. DCMU completely inhibited sugar production under red light, had no effect on guard cell sugar production under blue light when KCl was present, and inhibited sugar production by about 50% when guard cells were illuminated with blue light in solutions lacking KCl. We conclude that soluble sugars can contribute significantly to the osmoregulation of guard cells in detached leaf epidermis of V. faba. These results are consistent with the operation of two different sugar-producing pathways in guard cells: a photosynthetic carbon reduction pathway and a pathway of blue light-induced starch degradation. 相似文献
6.
The properties of photosynthetic O 2 evolution by mesophyll cellchloroplasts (MCC) and guard cell chloroplasts (GCC) isolatedfrom protoplasts of Vicia faba L. have been studied and effectson O 2 evolution of factors known to regulate stomatal movementshave been compared. The O 2 evolution of GCC was CO 2-dependent.The saturating light intensity for O 2 evolution was between150 and 200 µmol m 2s 1 for MCC and was between400 and 1,000µmol m 2s 1 for GCC. Light quality(red vs. blue) had no significant effect on O 2 evolution byeither MCC or GCC. The O 2 evolution rate of MCC was stronglydependent on external K + concentration, but GCC did not respondsignificantly to variations in external K + concentration between0 and 250 mM. The optimal external pH for O 2 evolution by MCCwas approximately 7.5, and either higher or lower external pHsignificantly inhibited O 2 evolution. However, O 2 evolutionby GCC was only slightly enhanced when external pH was increasedfrom 6.0 to 8.0. Our observation of differential sensitivityof MCC and GCC to light intensity and to variations of cytoplasmicK + and pH may indicate differential regulation of photosynthesisin MCC and GCC.
1Current address: Biology Department, Pennsylvania State University,208 Mueller Laboratory, University Park, PA 16802, U.S.A. 相似文献
8.
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 O 2 uptake (175 µmol mg 1 Chl hr 1)was observed in guard cell protoplasts kept in darkness, whichwas inhibited by 2 mM KCN or NaN 3 by about 60%. On illumination,this O 2 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) 相似文献
9.
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. 相似文献
10.
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. 相似文献
11.
The rate of O 2 uptake was about 29 times higher in guard cellprotoplasts (GCPs) than in mesophyll protoplasts (MGPs) on aChi basis. The O 2 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 NaN 3 strongly inhibited O 2 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) 相似文献
12.
A rapid and convenient procedure was developed for isolatingguard cell protoplasts (GCPs) from epidermal strips of Viciafaba L. The mean rates of O 2 uptake in the dark and evolutionin light of the isolated GCPs were 200 and 290 µmol O 2mg 1 Chl h 1, respectively, showing net O 2 evolutionin light. Photosynthetic O 2 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) 相似文献
13.
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 CaCl 2, and 0.1% polyvinylpyrrolidone 40 (PVP). Attached mesophyll and epidermal cells were removed by shaking epidermis in a solution of Cellulysin, mannitol, CaCl 2, PVP, and pepstatin A. Cleaned epidermis was transferred to a solution of mannitol, CaCl 2, 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 × 10 4 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 CaCl 2. 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. 相似文献
14.
研究了NADPH氧化酶在ABA(abscisic acid)诱导蚕豆气孔关闭信号转导网络中的作用,荧光光谱实验表明,在嗜中性白血球NADPH氧化酶抑制剂DPI(diphenyleneio-donium)存在的条件下,与对照相比,大大逆转了由ABA引起HPTS(8-hydroxypyrene-1,3,6-trisulfonic acid,trisodium salt)的荧光强度下降。表皮生物分析法显示,10^-6mol/L的DPI和10^3unit/mL的过氧化氢酶(catalase,CAT)在一定程度上也逆转了ABA诱导张开气孔的关闭。因此推测:在ABA诱导蚕豆气孔保卫细胞过程中,质膜上的NADPH气体酶可能催化形成超氧自由基O^-2,再经歧化反应形成H2O2,而形成的H2O2参与了气孔运动调节。 相似文献
15.
The stomata in the abaxial epidermis of Vicia faba were examined for the location of redox systems using tetrazolium salts. Three distinct redox systems could be demonstrated: chloroplast, mitochondrial, and plasmalemma. The chloroplast activity required light and NADP. Mitochondrial activity required added NADH and was suppressed by preincubation with KCN. The plasmalemma redox system in guard cells also required NADH, but was insensitive to KCN and was stimulated by blue light. The involvement of an NADH dehydrogenase in the blue light stimulated redox system in guard cells was suggested by the sensitivity to plantanetin, an inhibitor of NADH dehydrogenase. The redox system of mitochondria was the most active followed by that of plasmalemma. The activity of chloroplasts was the least among the three redox systems. The plasmalemma mediated tetrazolium reduction was stimulated by exogenous flavins and suppressed by Kl or phenylacetate, inhibitors of flavin excitation. We therefore conclude that an NADH-dependent, flavin mediated electron transport system, sensitive to blue light, operates in the plasmalemma of guard cells. 相似文献
16.
Intact chloroplasts isolated from leaves of Vicia faba L. var. the Sutton show a decline in the endogenous level of α-ketoglutarate upon illumination. α-Ketoglutarate supplied to the chloroplasts is similarly utilized in this light-dependent reaction, and its consumption is paralleled by a concomitant increase in the level of glutamate. There is no photostimulation of glutamate synthesis in chloroplasts broken by osmotic shock, but it can be somewhat restored by addition of ferredoxin and NADP. These results suggest that in the isolated chloroplast the synthesis of glutamate from α-ketoglutarate is regulated by the availability of reduced pyridine nucleotide generated by photosynthetic electron transport. This conclusion is supported by the finding of an apparent competition between the photoreduction of phosphoglycerate to triose phosphate and the photoutilization of α-ketoglutarate. 相似文献
17.
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 Mg 2+. Two-hundred millimolar NaCl inhibited the reaction by 50%. With frozen-dried leaflet extract, the apparent Km(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 Km(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 Km(PEP) of guard cell activity could not be unambiguously interpreted. The physiological implications of the properties of this enzyme in guard cells are discussed. 相似文献
18.
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) 相似文献
19.
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. 相似文献
20.
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 CO 2-free air than in normal air. This initial alkalinization was inhibited by DCMU. Acidification in CO 2-free air became observable in shorter duration of light exposure than that in normal air. The rate of acidification was higher in CO 2-free air than in normal air. The CO 2 level of the medium decreased in the light, and increased in the dark. 14CO 2 uptake was enhanced 2- to 3-fold by light, but not in the presence of DCMU. These results indicate that photosynthetic CO 2 fixation does take place in GCP and that the initial alkalinization is due to this photosynthetic CO 2 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. 相似文献
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