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1. Glutamine synthetase [l-glutamate: ammonia ligase (ADP), E.C. 6.3.1.2]was purified to apparent homogeneity from the shoots of light-grown pea seedlings. It was found to be quite unstable, but. could be partially stabilized by the addition of divalent cation (Mg2+ or Mn2+), and still further by the addition of sucrose (0.5–1.5 m), fructose (1–2.5 m), or ethyleneglycol (20–30%). Stability varied considerably depending on pH and whether Mg2+ or Mn2+ was used. Under certain incubation conditions the inactivated enzyme could be partially reactivated. 2. The pH optimum for glutamine synthesis varied widely (5.0–8.2) depending on the type and concentration of divalent cation (Mn2+, Co2+, Mg2+) and the ATP concentration. 相似文献
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Light-induced changes in the fluorescence of the pH-indicating dyes pyranine or 5-(and 6-)carboxy-2, 7-dichlorofluorescein (CDCF) which had been fed to leaves were examined to monitor cellular pH changes. After short-term feeding of pyranine (pK 7.3) to leaves of Amaranthus caudatus L., a NAD-malic-enzyme-type C4 plant, vascular bundles and surrounding cells became fluorescent. Fluorescence emission from mesophyll cells required longer feeding times. In CO2-free air, pyranine fluorescence increased much more on illumination after mesophyll cells had become fluorescent than when only the vascular bundles and the bundle sheath of Amaranthus leaves had been stained. After short feeding times and in the absence of actinic illumination, CO2 decreased pyranine fluorescence very slowly in Amaranthus and rapidly in C3 leaves. After prolonged feeding times, the extent of the light-dependent increase in pyranine fluorescence was several times greater in different C4 plants than in C3 species. The kinetics of the fluorescence changes were also remarkably different in C3 and C4 plants. Carbon dioxide (500 l · l–1) suppressed the light-induced increase in pyranine fluorescence more in C4 than in C3 leaves. Light-dependent changes in light scattering, which are indicative of chloroplast energization, and in 410-nm transmission, which indicate chloroplast movement, differed kinetically from those of the changes in pyranine fluorescence. Available evidence indicated that light-dependent changes in pyranine fluorescence did not originate from the apoplast of leaf cells. Microscopic observation led to the conclusion that, after prolonged feeding times or prolonged incubation, changes in pyranine fluorescence emitted from C4 leaves reflect pH changes mainly in the cytosol of mesophyll cells. A transient acidification reaction indicated by quenching of pyranine fluorescence in the dark-light transient and not observed in C3 species is attributed to the carboxylation of phosphoenolpyruvate. After short feeding times and in the absence of actinic illumination, CO2 (250 l l–1) decreased pyranine fluorescence very slowly in Amaranthus and more rapidly in C3 leaves. After prolonged feeding times, both the rate and the extent of CO2-dependent quenching of pyranine fluorescence increased, but the increase was insufficient to indicate the presence of highly active carbonic anhydrase in the compartment from which pyranine fluorescence was emitted. In contrast to pyranine, CDCF (pK 4.8) did not increase but rather decreased its fluorescence on illumination of an Amaranthus leaf, indicating acidification of an acidic compartment, most probably the vacuole of green leaf cells. The pattern of the acidification reaction was similar in C4 and C3 leaves. The remarkably large extent of the light-dependent increase in pyranine fluorescence from leaves of C4 species and its slow kinetics are proposed to be caused by an alkalization of the cytosol which in the absence of CO2 is larger in the mesophyll than in the bundle sheath. It gives rise to deprotonation of dye originally located in the mesophyll and, in addition, of dye which diffuses from the bundle sheath into the mesophyll following a pH gradient. Implications of slow diffusional transport of pyranine and CO2 between mesophyll and bundle-sheath cells and the fast metabolite transport required in C4 photosynthesis are discussed.Abbreviations CDCF
5-(and 6-)carboxy-2,7-dichlorofluorescein
- DHAP
dihydroxyacetone phosphate
- PGA
3-phosphoglycerate
This work was supported by the Sonderforschungsbereiche 176 and 251 of the University of Würzburg and by the Gottfried-Wilhelm-Leibniz Program of the Deutsche Forschungsgemeinschaft. A.S.R. was the recipient of a fellowship of the Alexander-von-Humboldt Foundation. We are grateful to Mrs. S. Neimanis for cooperation. 相似文献
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Illumination of leaves of C3 plants caused cytosolic alkalization and vacuolar acidification in the mesophyll cells. Both phenomena were particularly pronounced when CO2 was absent, were suppressed by CO2, and were related to the activation state of the photosynthetic apparatus. The cytosolic alkalization reaction has at least two major components. Trivalent cytosolic phosphoglycerate must be protonated before it can be transferred into the chloroplasts for reduction. Pumping of protons from the cytosol into the vacuole also contributes to cytosolic alkalization. The dependence of light scattering by chloroplast thylakoids on the energy fluence rate was closely related to that of vacuolar acidification under different conditions for chloroplast energization. This indicates (i) transport of energy from the chloroplasts to the cytosol in the light and (ii) use of this energy for the transport of protons into the vacuoles. The light-dependent vacuolar acidification is interpreted to be caused by the increase in the activity of a proton-translocating enzyme of the tonoplast. The decrease of vacuolar acidification during photosynthetic carbon reduction or photorespiration is indicative of decreased cytosolic energization. In low light, the light-dependent vacuolar acidification was stimulated in the absence of CO2 when photorespiration was inhibited. The data do not support the view that photorespiration is capable of increasing the cytosolic energy state in the light.This work was supported by the Sonderforschungsbereiche 176 and 251 of the University of Würzburg. Z.-H. Y. acknowledges support by the Leibniz program of the Deutsche Forschungsgemeinschaft and by the Committee for Education of the People's Republic of China. 相似文献
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Action spectra in the red region of the spectrum for light-dependent cytosolic alkalization in leaves of C3 plants which also received a low background of blue light differed from the action spectra for light-dependent vacuolar acidification. Light above 680 nm was less effective in supporting the cytosolic alkalization reaction than light below 680 nm. In contrast, in leaves illuminated in CO2-free air the light-dependent vacuolar acidification exhibited a maximum at or even above 700 nm. When photorespiratory carbohydrate oxidation was suppressed in low oxygen, a substantially changed action spectrum of the acidification reaction resembled in shape that of the cytosolic alkalization with the exception that it was extended towards the far-red. From the presented data and from previously published data (Yin et al., 1990b, Planta 182, 253–261; Yin et al., 1990c, Planta 182, 262–269) it is concluded that in the presence of a weak background of blue light, and in the absence of CO2 which drains electrons from the electron transport chain, cyclic photophosphorylation induced by far-red light permits increased export of dihydroxyacetone phosphate from the chloroplasts into the cytosol where its oxidation increases the cytosolic energy state giving rise to increased proton transport across the tonoplast. The data do not lend support to the view that export of malate from the chloroplasts and its oxidation in the mitochondria contribute significantly to cytosolic energization in the light.Abbreviations CDCF
5-(and-6)-carboxy-2,7-dichlorofluorescein
- DHAP
dihydroxyacetone phosphate
- OAA
oxaloacetate
- PGA
phosphoglycerate
This work was performed within the Sonderforschungsbereiche 176 and 251 of the University of Würzburg. Z.-H. Y. acknowledges support by the Leibniz program of the Deutsche Forschungs-gemeinschaft. 相似文献
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The regulation of pH in the apoplast, cytosol and chloroplasts of intact leaves was studied by means of fluorescent pH indicators
and as a response of photosynthesis to acid stress. The apoplastic pH increased under anaerobiosis. Aeration reversed this
effect. Apoplastic responses to CO2, HCl or NH3 differed considerably. Whereas HCl and ammonia caused rapid acidification or alkalinization, the return to initial pH values
was slow after cessation of fumigation. Addition of CO2 either did not produce the acidification expected on the basis of known apoplastic buffering or even caused some alkalinization.
Removal of CO2 shifted the apoplastic pH into the alkaline range before the pH returned to initial steady-state levels. In the presence
of vanadate, the alkaline shift was absent and the apoplastic pH returned slowly to the initial level when CO2 was removed from the atmosphere. In contrast to the response of the apoplast, anaerobiosis acidified the cytosol or, in some
species, had little effect on its pH. Acidification was rapidly reversed upon re-admission of oxygen. The CO2-dependent pH changes were very fast in the cytosol. Considerable alkalinization was observed after removal of CO2 under aerobic, but not under anaerobic conditions. Rates of the re-entry of protons into the cytosol during recovery from
CO2 stress increased in the presence of oxygen with the length of previous exposure to high CO2. Effective pH regulation in the chloroplasts was indicated by the recovery of photosynthesis after the transient inhibition
of photosynthetic electron flow when CO2 was increased from 0.038% to 16% in air. As photosynthesis became inhibited under high CO2, reduction of the electron transport chain increased transiently. The time required for recovery of photosynthesis from inhibition
during persistent CO2 stress was similar to the time required for establishing steady-state pH values in the cytosol under acid stress. The high
capacity of leaf cells for the rapid re-attainment of pH homeostasis in the apoplast and the cytoplasm under acid or alkaline
stress suggested the rapid activation or deactivation of membrane-localised proton-transporting enzymes and corresponding
ion channel regulation for co-transport of anions or counter-transport of cations together with proton fluxes. Acidification
of the cytoplasm appeared to activate energy-dependent proton export primarily into the vacuoles whereas apoplastic alkalinization
resulted in the pumping of protons into the apoplast. Proton export rates from the cytosol into the apoplast after anaerobiosis
were about 100 nmol (m2 leaf area)−1 s−1 or less. Proton export under acid stress into the vacuole was about 1200 nmol m−2 s−1. The kinetics of pH responses to the addition or withdrawal of CO2 indicated the presence of carbonic anhydrase in the cytosol, but not in the apoplast.
Received: 19 July 1999 / Accepted: 29 December 1999 相似文献
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FITC-dextran for measuring apoplast pH and apoplastic pH gradients between various cell types in sunflower leaves 总被引:8,自引:0,他引:8
The liquid in the free space of leaf cell walls, the apoplast, is in direct contact with the plasma membrane and its nutrient uptake systems. Therefore, the pH of the apoplast is of utmost interest. We have elaborated a non-destructive method by which excised sunflower leaves ( Helianthus annuus cv. Erika) were perfused with fluorescein isothiocyanate-dextran (FITC-dextran) (4 000 Da) via the transpiration stream. We showed that leaf apoplast pH can be measured by using the fluorescence ratio technique together in conjunction with this dye. Evidence is provided that FITC-dextran does not penetrate the plasma membrane over a period of ca 17 h from the beginning of dye perfusion. Dye enrichment in the leaf apoplast did not cause an 'inner filter effect' and thus the fluorescence ratio was only dependent on pH. In vivo calibration yielded a pKa of 5.92, which was virtually identical to the pKa of 5.93 calculated for dye solutions. Hence, FITC-dextran can be detected in complex environments and covers a pH range prevailing in the leaf apoplast.
Based on this method we developed a microscope image technique visualizing pH gradients between various cell types. The pH in the lumen of the xylem vessel was ca 0.3–0.5 units lower than that of the apoplast of surrounding cells. Nitrate present in the leaf apoplast caused an increase in pH, especially in the dark. Under these conditions, in the intercostal area, the apoplast pH around the stomata was ca 0.5–1.0 units higher than that of the surrounding epidermal cells. 相似文献
Based on this method we developed a microscope image technique visualizing pH gradients between various cell types. The pH in the lumen of the xylem vessel was ca 0.3–0.5 units lower than that of the apoplast of surrounding cells. Nitrate present in the leaf apoplast caused an increase in pH, especially in the dark. Under these conditions, in the intercostal area, the apoplast pH around the stomata was ca 0.5–1.0 units higher than that of the surrounding epidermal cells. 相似文献
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Mhling K.H.; Plieth C.; Hansen U-P.; Sattelmacher B. 《Journal of experimental botany》1995,46(4):377-382
The fluorochrome FITC-dextran was used to measure the effectof light on the apoplastic pH of intact Vicia faba leaves withthe ratio imaging technique. In darkadapted leaves the apoplasticpH varied depending on the leaf between 5.2 and 5.9. Red light(660 nm, 412 W m2) leads to multiphasic responses:in the first seconds an alkalinization ({small tilde}0.3 pHunits), and thereafter an acidification of the leaf apoplast({small tilde}0.4 pH units) were observed. Both effects couldbe inhibited by DCMU. While variation of CO2 concentration revealedno effect on light-induced apoplastic pH changes, a decreasein O2 concentration decreased the effect. On the basis of ourdata it is suggested that the influence of photosynthesis onplasmalemma H+ ATPase is responsible for the observed effects,rather than altered CO2 uptake. Key words: Leaf apoplast, apoplastic pH, light, ratio imaging, pH-sensitive fluorescent dye, Vicia fab 相似文献
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In plants, the extracellular space (apoplast) is one of the main places where exchange of molecules occurs between cells. Not only is this compartment involved in the storage of multiple metabolites and ions, including calcium and protons, but it also plays a role in the transmission of signaling molecules for cell-to-cell communication. It has recently been shown multiple times that these two aspects are linked and can influence each other. In particular, apoplast pH was shown as a primary regulator of auxin (IAA) transport in Arabidopsis thaliana. To prove the role of apoplastic pH, we have developed a protocol for apoplastic fluid extraction from Arabidopsis leaves, followed by pH determination using the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) fluorescent dye. This technique successfully allows one to monitor apoplastic pH variations among different plant lines and to link changes in apoplastic pH to cellular responses in the plant. 相似文献
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Salinity affects water availability in the soil and subsequently the plant uptake capacity. Upon exposure to salt stress, leaf growth in monocot plants has been shown to be reduced instantaneously, followed by a gradual acclimation. The growth reactions are caused by an initial water deficit and an accompanied osmotic effect, followed by an IAA-induced sequestration of protons into the apoplast that increases leaf growth again as explained by the acid growth theory. In this study, we investigated the dynamics of growth reactions and apoplastic pH in leaves of the dicot Vicia faba in the presence of NaCl during the initiation of salt stress. Concurrent changes in apoplastic pH were detected by ratiometric fluorescence microscopy using the fluorescent dye fluorescein tetramethylrhodamine dextran. To elucidate the possible relation between the dynamics of leaf growth and apoplastic pH, results of the ratio imaging technique were combined with an in vivo growth analysis imaging approach. Leaf growth rate of V. faba was highest in the dusk and the early night phase; at this time a concomitant decrease of the apoplastic pH was observed. Under salinity, the apoplastic pH in leaves of V. faba increased with a simultaneous decrease of leaf growth towards increasing developmental stages, but with complex aberrations in the 24-h-leaf-growth pattern compared to control leaves. In conclusion, these results show that salt stress leads to an increase in apoplastic pH and to a declined leaf growth activity with complex 24-h-interactions of growth and pH in V. faba. 相似文献
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The K+-sensitive fluorescent dye benzofuran isophthalate (PBFI) and the pH-sensitive fluorescein isothiocyanate dextran (FITC-Dextran)
were used to investigate the influence of light/dark transitions on apoplastic pH and K+ concentration in intact leaves of Vicia faba L. with fluorescence ratio imaging microscopy. Illumination by red light led to an acidification in the leaf apoplast due
to light-induced H+ extrusion. Similar apoplastic pH responses were found on adaxial and abaxial sides of leaves after light/dark transition.
Stomatal opening resulted only in a slight pH decrease (0.2 units) in the leaf apoplast. Gradients of apoplastic pH exist
in the leaf apoplast, being about 0.5–1.0 units lower in the center of the xylem veins as compared with surrounding cells.
The apoplastic K+ concentration in intact leaves declined during the light period. A steeper light-induced decrease in apoplastic K+, possibly caused by higher apoplastic K+, was found on the abaxial side of leaves concentration. Simultaneous measurements of apoplastic pH and K+ demonstrated that a light-induced decline in apoplastic K+ concentration indicative of net K+ uptake into leaf cells occurs independent of apoplastic pH changes. It is suggested that the driving force that is generated
by H+ extrusion into the leaf apoplast due to H+-ATPase activity is sufficient for passive K+ influx into the leaf cells.
Received: 7 March 2000 / Accepted: 12 May 2000 相似文献
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After opening the stomata in CO2-free air, darkened leaves of several plant species were titrated with CO2 at concentrations between 1 and 16%, in air in order to reversibly decrease cellular pH values and to calculate buffer capacities
from pH changes and bicarbonate accumulation using both gas-exchange and fluorescence methods for analysis. After equilibration
with CO2 for times ranging between 4.4 and 300 s, fast CO2 release from bicarbonate indicated catalysis by highly active carbonic anhydrase. Its time constant was below 2.5 s. Additional
CO2 was released with time constants of about 5, 15 and approximately 300 s. With CO2 as the acidifying agent, calculated buffer capacities depend on assumptions regarding initial pH in the absence of an acid
load. At an initial stroma pH of 7.7, the stromal buffer capacity was about 20 mM pH-unit−1 in darkened spinach leaves. At an initial pH of 7.5 it would be only 12 mM pH-unit−1, i.e. not higher than expected solely on the basis of known stromal concentrations of phosphate and phosphate esters, disregarding
the contribution of other solutes. At a concentration of 16%, CO2 reduced the stromal pH by about 1 pH unit. Buffering of the cytosol was measured by the CO2-dependent quenching of the fluorescence of pyranine which was fed to spinach leaves via the petiole. Brief exposures to high
CO2 minimized interference by effective cytosolic pH regulation. Cytosolic buffering appeared to be similar to or only somewhat
higher than chloroplast buffering if the initial cytosolic pH was assumed to be 7.25, which is in accord with published cytosolic
pH values. The difference from chloroplast pH values indicates the existence of a pH gradient across the chloroplast envelope
even in darkened leaves. Apoplastic buffering was weak as measured by the CO2-dependent quenching of dextran-conjugated fluorescein isothiocyanate which was infiltrated together with sodium vanadate
into potato leaves. In the absence of vanadate, the kinetics of apoplastic fluorescence quenching were more complex than in
its presence, indicating fast apoplastic pH regulation which strongly interfered with the determination of apoplastic buffering
capacities. At an apoplastic pH of 6.1 in potato leaves, apoplastic buffering as determined by CO2 titration with and without added buffer was somewhat below 4 mM pH-unit−1. Thus the apoplastic and cytosolic pH responses to additions of CO2 indicated that the observed cytoplasmic pH regulation under acid stress involves pumping of protons from the cytosol into
the vacuole of leaf cells, but not into the apoplast.
Received: 27 November 1998 / Accepted: 22 March 1999 相似文献
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Felle HH Herrmann A Hanstein S Hückelhoven R Kogel KH 《Molecular plant-microbe interactions : MPMI》2004,17(1):118-123
To investigate apoplastic responses of barley (Hordeum vulgare L.) to the barley powdery mildew fungus Blumeria graminis f. sp. hordei, noninvasive microprobe techniques were employed. H(+)- and Ca(2+)-selective microprobes were inserted into open stomata of barley leaves inoculated with Blumeria graminis f. sp. hordei race A6 conidia. Resistance gene-mediated responses of barley genotype Ingrid (susceptible parent line) and the near-isogenic resistant Ingrid backcross lines (I-mlo5, I-Mla12, and I-Mlg) were continuously monitored from 20 min to 4 days after inoculation. The main events were categorized as short-term responses around 2 h after inoculation (hai), intermediate responses around 8 and 12 hai, and long-term responses starting between 21 and 24 hai. Short-term responses were rapid transient decreases of apoplastic H(+)- and Ca2+ activities that lasted minutes only. Kinetics were similar for all genotypes tested, and thus, these short-term responses were attributed as nonspecific first encounters of fungal surface material with the host plasma membrane. This is supported by the observation that a microinjected chitin oligomer (GlcNAc)8 yielded similar apoplastic alkalinization. Intermediate responses are trains of H+ (increase) spikes that, being different in susceptible Ingrid and penetration-resistant I-mlo5 (or I-Mlg), were interpreted as accompanying specific events of papillae formation. Long-term events were massive slow and long-lasting alkalinizations up to two pH units above control. Since these latter changes were only observed with near-isogenic hypersensitive reaction (HR)-mounting genotypes I-Mla12 and I-Mlg but not with I-mlo5 or, to a smaller extent, with susceptible Ingrid, both lacking significant rates of HR, they were rated as cell death specific. It is concluded that apoplastic pH changes are important indicators of host-pathogen interactions that correlate with both the different stages of fungal development and the different types of host defense response. 相似文献
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Several peptide hydrolases have been isolated in a partiallypurified form from green wheat leaves by a combination of gelchromatography and chromatography on DEAE-cellulose. These hydrolasesall degrade haemoglobin and other substrates tested at an acidpH. Three of the enzymes have been identified as proteinasesby measurement of the total N: -amino N ratio of the degradationproducts. These three enzymes have a similar optimum assay pHof about 4.5 with haemoglobin as substrate, but differ markedlyin their stability at high temperatures.
1 Nomenclature in this paper follows the recommendation of theCommission on Biochemical Nomenclature (1972). (Received November 30, 1977; ) 相似文献
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A method was elaborated by which the pH in leaf apoplast can be measured. The technique is based on the pH dependent fluorescence of 5-carboxyfluorescein (5-CF) or fluorescein isothiocyanate (FITC). The fluorescein isothiocyanate is coupled with a macromolecular dextran molecule (FITC-dextran). For eliminating the effect of the absolute dye concentration the dual excitation technique was applied. It was shown that the ratio of fluorescence excited by light of 491 nm and 463 nm was virtually independent of the concentration of 5-CF and that this fluorescence ratio was related to the pH. The plasmalemma is practically impermeable to FITC-dextran and in the test we carried out over a period of 6 h not the slightest indication was found that it may penetrate the plasma membrane. For 5-CF this cannot be ruled out completely. It is possible that at pH values below 4.5 it may penetrate biological membranes at low rates.
Experiments with leaves of sunflower ( Helianthus animus cv. Erika) perfused with 5-carboxyfluorescein and supplied with different nitrogen forms showed that NH+ 4 application resulted in a decrease and NO+ 3 application in an increase of the leaf apoplast pH. Leaf spraying with fasicoccin was followed by a pH decrease, while leaf spraying with the protonophores p -trifluoromethoxy carbonytcyanide phenylhydra-zon (FCCP) or nigericin resulted in neutral apoplastic pH. These results provide evidence that the method is well suited for measuring the response of the leaf apoplast pH to changing physiological conditions. 相似文献
Experiments with leaves of sunflower ( Helianthus animus cv. Erika) perfused with 5-carboxyfluorescein and supplied with different nitrogen forms showed that NH
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We examined microbial colonization, exoenzyme activity, and processing of leaves of yellow poplar (Liriodendron tulipifera), red maple (Acer rubrum), and white oak (Quercus alba) in three streams on the Allegheny Plateau of West Virginia, United States. Leaf packs were placed in streams that varied in their underlying bedrock geology, and therefore in their sensitivity to the high level of acidic precipitation that occurs in this region. The mean pH of the streams was 4.3 in the South Fork of Red Run (SFR), 6.2 in Wilson Hollow Run (WHR), and 7.7 in the North Fork of Hickman Slide Run (HSR). Through time, the patterns of microbial biomass and exoenzyme activity were generally similar among leaf species, but the magnitude of microbial biomass and exoenzyme activity differed among leaf species. Pectinase activity was greatest in HSR, the most alkaline stream, whereas the activity of exocellulase and xylanase was greatest in WHR and SFR, the intermediate and acidic streams. This variation in the activity of different exoenzymes was consistent with published pH optima for these exoenzymes. Variation in processing rates, both among leaf species and among streams, seems to be related to the level of microbial exoenzyme activity on the leaf detritus. 相似文献