Effect Of Sodium Hexanitrocobaltate (III) Decomposition On Its Staining Of Intracellular Potassium Ions

The effect was examined of the chemical decomposition of the potassium stain, sodium hexanitrocobaltate (III) (SHC), on its ability to produce stain granules of consistent size that could be used to estimate the K⁺ contents of stomafal guard cells. Stomata in detached epidermis from leaves of Vicia faba (fava bean) were stimulated to accumulate K⁺ by treating them with fusicoccin. Stomatal apertures and the fraction of guard cell area covered by K⁺ precipitate granules (K⁺ score) were measured by digitizing photographic enlargements, and K* scores were correlated with the age of stain that had been stored either in open or closed containers. The ability of stain aged in open containers to produce consistent fractional cell coverage was compared to 1) the ability of identically treated stain to precipitate K⁺ from solutions of KCl, and to 2) the kinetics of decomposition of SHC. It was found that the fractional coverage of guard cells of stomata opened to the same apertures decreased with a first order rate constant of 2.3 × 10^-8/sec. The mass of precipitate formed by treatment of KCl solutions was unchanged for 2 hr after initial preparation of the SHC, and decreased thereafter with a first order rate constant of 1.0 × 10^-5/sec. When stored in tightly sealed containers, nearly 100 hr were required for an occasionally opened bottle of SHC to decay to the same efficacy as a solution left open to the air for 8 hr.     

Expression of a Cs(+)-resistant guard cell K+ channel confers Cs(+)-resistant, light-induced stomatal opening in transgenic arabidopsis.

Inward-rectifying K+ (K+in) channels in the guard cell plasma membrane have been suggested to function as a major pathway for K+ influx into guard cells during stomatal opening. When K+in channels were blocked with external Cs+ in wild-type Arabidopsis guard cells, light-induced stomatal opening was reduced. Transgenic Arabidopsis plants were generated that expressed a mutant of the guard cell K+in channel, KAT1, which shows enhanced resistance to the Cs+ block. Stomata in these transgenic lines opened in the presence of external Cs+. Patch-clamp experiments with transgenic guard cells showed that inward K+(in) currents were blocked less by Cs+ than were K+ currents in controls. These data provide direct evidence that KAT1 functions as a plasma membrane K+ channel in vivo and that K+in channels constitute an important mechanism for light-induced stomatal opening. In addition, biophysical properties of K+in channels in guard cells indicate that components in addition to KAT1 may contribute to the formation of K+in channels in vivo.     

ACTION OF FUSICOCCIN ON THE POTASSIUM BALANCE OF GUARD CELLS OF PHASEOLUS VULGARIS

Fusicoccin induces stomatal opening in both the light and dark. The stomatal aperture and K content of guard cells was measured to determine whether the action of fusicoccin in inducing stomatal opening is directly related to the uptake of K by the guard cells. Both detached and attached epidermis was treated with fusicoccin and the K content was determined by staining with cobalt sodium nitrite or by electron probe microanalysis. The K content of guard cells in detached epidermal strips floated on 10 μm fusicoccin in 10 mm KCl and aqueous CH₃OH (0.02%, v/v) increased in the light and dark as the stomata opened. After exposure to fusicoccin for 6 hr in the light, however, the stomata were closed and no K could be detected in the guard cells. The K content of guard cells of attached epidermis painted with fusicoccin also increased as the stomata opened, but the concentration of K in the subsidiary cells was not significantly altered by fusicoccin-stimulated opening. Moreover, painting with fusicoccin did not significantly change the Ca and P content of the guard or subsidiary cells. Stomata of epidermal strips, opened to their maximum width by fusicoccin, showed only a small and temporary closure when transferred to a solution of 10 μm abscisic acid. The use of metabolic inhibitors suggested that energy for the uptake of the K may be provided by both photophosphorylation and oxidative phosphorylation.     

Light quality and osmoregulation in vicia guard cells : evidence for involvement of three metabolic pathways

Osmoregulation in opening stomata of epidermal peels from Vicia faba L. leaves was investigated under a variety of experimental conditions. The K⁺ content of stomatal guard cells and the starch content of guard cell chloroplasts were examined with cobaltinitrite and iodine-potassium iodide stains, respectively; stomatal apertures were measured microscopically. Red light (50 micromoles per square meter per second) irradiation caused a net increase of 3.1 micrometers in aperture and a decrease of −0.4 megapascals in guard cell osmotic potential over a 5 hour incubation, but histochemical observations showed no increase in guard cell K⁺ content or starch degradation in guard cell chloroplasts. At 10 micromoles per square meter per second, blue light caused a net 6.8 micrometer increase in aperture over 5 hours and there was a substantial decrease in starch content of chloroplasts but no increase in guard cell K⁺ content. At 25 micromoles per square meter per second of blue light, apertures increased faster (net gain of 5.7 micrometers after 1 hour) and starch content decreased. About 80% of guard cells had a higher K⁺ content after 1 hour of incubation but that fraction decreased to 10% after 5 hours. In the absence of KCl in the incubation medium, stomata opened slowly in response to 25 micomoles per square meter per second of blue light, without any K⁺ gain or starch loss. In dual beam experiments, stomata irradiated with 50 micomoles per square meter per second of red light for 3 hours opened without detectable starch loss or K⁺ gain; addition of 25 micomoles per square meter per second of blue light caused a further net gain of 4.4 micometers in aperture accompanied by substantial K⁺ uptake and starch loss. Comparison of K⁺ content in guard cells of opened stomata in epidermal peels with those induced to open in leaf discs showed a substantially higher K⁺ content in the intact tissue than in isolated peels. These results are not consistent with K⁺ (and its counterions) as the universal osmoticum in guard cells of open stomata under all conditions; rather, the data point to sugars arising from photosynthesis and from starch degradation as additional osmotica. Biochemical confirmation of these findings would indicate that osmoregulation during stomatal opening is the result of three key metabolic processes: ion transport, photosynthesis, and sugar metabolism.     

The poplar K+ channel KPT1 is associated with K+ uptake during stomatal opening and bud development

To gain insights into the performance of poplar guard cells, we have measured stomatal conductance and aperture, guard cell K+ content and K+-channel activity of the guard cell plasma membrane in intact poplar leaves. In contrast to Arabidopsis, broad bean and tobacco grown under same conditions, poplar stomata operated just in the dynamic range - any change in conductance altered the rate of photosynthesis. In response to light, CO2 and abscisic acid (ABA), the stomatal opening velocity was two to five times faster than that measured for Arabidopsis thaliana, Nicotiana tabacum and Vicia faba. When stomata opened, the K+ content of guard cells increased almost twofold, indicating that the very fast stomatal opening in this species is mediated via potassium uptake. Following impalement of single guard cells embedded in their natural environment of intact leaves with triple-barrelled microelectrodes, time-dependent inward and outward-rectifying K+-channel-mediated currents of large amplitude were recorded. To analyse the molecular nature of genes encoding guard cell K+-uptake channels, we cloned K+-transporter Populustremula (KPT)1 and functionally expressed this potassium channel in a K+-uptake-deficient Escherichia coli mutant. In addition to guard cells, this K+-transporter gene was expressed in buds, where the KPT1 gene activity strongly correlated with bud break. Thus, KPT1 represents one of only few poplar genes associated with bud flush.     

Potassium Chloride as Stomatal Osmoticum in Allium cepa L., a Species Devoid of Starch in Guard Cells

K⁺ and Cl⁻ contents of guard cells and of ordinary epidermal cells were determined in epidermal samples of Allium cepa L. by electron probe microanalysis; malate contents of the same samples were determined by enzymic oxidation. KCl was, in general, the major osmoticum in guard cells, irrespective of whether stomata had opened on leaves or in epidermal strips floating on solutions. The solute requirement varied between 50 and 110 femtomoles KCl per micrometer increase in aperture per pair of guard cells. Stomata did not open on solutions of K iminodiacetate, presumably because its anion could not be taken up. Stomata opened if KCl or KBr was provided. Taken together, the results indicate that the absence of starch from guard cells deprived them of the ability to produce malate in amounts of osmotic consequence and that the presence of absorbable Cl⁻ (or Br⁻) was necessary for stomatal opening.     

Orthochromatic, Species-Limited Staining of Mast Cells With Night Blue

Night blue will stain the mast cells of rat, mouse and hamster selectively if alcohol differentiation is controlled. The technical steps are: Dewax paraffin sections with xylene, 2 changes; air dry; 2% Na₂SO₄, 3-5 sec; 0.5% night blue in 10% ethanol, 1 hr at 60°C; rinse in water; 9% HNO₃, 15 sec; water 1-5 min; 70% ethanol, 2 changes, 30 sec each; wash; 0.01% safranin, 3-5 sec; rinse, blot, air dry, mount in synthetic resin. A clear orthochromatic stain of the mast-cell granules occurs. Acid fixation prevents the staining reaction.     

Central Roles for Potassium and Sucrose in Guard-Cell Osmoregulation

Osmoregulation in guard cells of intact, attached Vicia faba leaves grown under growth chamber and greenhouse conditions was studied over a daily light cycle of stomatal movements. Under both growth conditions guard cells had two distinct osmoregulatory phases. In the first (morning) phase, opening was correlated with K+ uptake and, to a lesser extent, sucrose accumulation. In the second (afternoon) phase, in which apertures were maximal, K+ content declined and sucrose became the dominant osmoticum. Reopening of the stomata after a CO2-induced closure was accompanied by accumulation of either K+ or sucrose, depending on the time of day, indicating that a single environmental signal may use multiple osmoregulatory pathways. Malate accumulation, correlated with K+ uptake, was detected under growth chamber but not greenhouse conditions, whereas Cl- was the main K+ counterion in the greenhouse. These results indicate that guard-cell osmoregulation in the intact leaf depends on at least two different osmoregulatory pathways, K+ transport and sucrose metabolism. Furthermore, the relative importance of the K+ counterions malate and Cl- appears to be environment-dependent.     

Thick Sections for Embryology

Night blue will stain the mast cells of rat, mouse and hamster selectively if alcohol differentiation is controlled. The technical steps are: Dewax paraffin sections with xylene, 2 changes; air dry; 2% Na₂SO₄, 3-5 sec; 0.5% night blue in 10% ethanol, 1 hr at 60°C; rinse in water; 9% HNO₃, 15 sec; water 1-5 min; 70% ethanol, 2 changes, 30 sec each; wash; 0.01% safranin, 3-5 sec; rinse, blot, air dry, mount in synthetic resin. A clear orthochromatic stain of the mast-cell granules occurs. Acid fixation prevents the staining reaction.     

The Demonstration of Beta Cells in Pancreatic Islets and Their Tumors

The stain is applied routinely to tissues fixed in 10% buffered formalin (pH near 7.0) or in Bouin's fluid. Bring paraffin section to water as usual and mordant 72 hr in 5% CrCl₃ dissolved in 5% acetic acid. Wash in water and in 70% alcohol and stain 6 hr. Formula of staining solution: new fuchsin, 1% in 70% alcohol, 100 ml; HCl, conc., 2 ml and paraldehyde, 2 ml, mixed together and added to the dye solution; let stand 24 hr before use. After staining, wash in running tap water 5-10 min, rinse in distilled water and counterstain if desired. Dehydration in alcohol, clearing and covering completes the process. When the paraldehyde is obtained from a freshly opened bottle, standardized staining times can be used and thus eliminate the necessity of differentiating individual slides. The granules of beta cells stained deep blue to purple and were demonstrated in the pancreatic islet of man, dog, mouse, frog, guinea pig and rabbit.     

The Arabidopsis small G protein ROP2 is activated by light in guard cells and inhibits light-induced stomatal opening

ROP small G proteins function as molecular switches in diverse signaling processes. Here, we investigated signals that activate ROP2 in guard cells. In guard cells of Vicia faba expressing Arabidopsis thaliana constitutively active (CA) ROP2 fused to red fluorescent protein (RFP-CA-ROP2), fluorescence localized exclusively at the plasma membrane, whereas a dominant negative version of RFP-ROP2 (DN-ROP2) localized in the cytoplasm. In guard cells expressing green fluorescent protein-ROP2, the relative fluorescence intensity at the plasma membrane increased upon illumination, suggesting that light activates ROP2. Unlike previously reported light-activated factors, light-activated ROP2 inhibits rather than accelerates light-induced stomatal opening; stomata bordered by guard cells transformed with CA-rop2 opened less than controls upon light irradiation. When introduced into guard cells together with CA-ROP2, At RhoGDI1, which encodes a guanine nucleotide dissociation inhibitor, inhibited plasma membrane localization of CA-ROP2 and abolished the inhibitory effect of CA-ROP2 on light-induced stomatal opening, supporting the negative effect of active ROP2 on stomatal opening. Mutant rop2 Arabidopsis guard cells showed phenotypes similar to those of transformed V. faba guard cells; CA-rop2 stomata opened more slowly and to a lesser extent, and DN-rop2 stomata opened faster than wild-type stomata in response to light. Moreover, in rop2 knockout plants, stomata opened faster and to a greater extent than wild-type stomata in response to light. Thus, ROP2 is a light-activated negative factor that attenuates the extent of light-induced changes in stomatal aperture. The inhibition of light-induced stomatal opening by light-activated ROP2 suggests the existence of feedback regulatory mechanisms through which stomatal apertures may be finely controlled.     

Visualizing Changes in Cytosolic-Free Ca2+ during the Response of Stomatal Guard Cells to Abscisic Acid

In this paper, we report the results of a detailed investigation into abscisic acid (ABA)[mdash]stimulated elevations of guard cell cytosolic-free Ca2+ ([Ca2+]cyt). Fluorescence ratio photometry and ratio imaging techniques were used to investigate this phenomenon. Guard cells of open and closed (opened to 10 to 12 [mu]m before treatment with ABA) stomata were microinjected with the fluorescent Ca2+ indicator Indo-1. Resting [Ca2+]cyt ranged from 50 to 350 nM. ABA (100 nM) stimulated an increase in [Ca2+]cyt in 68 and 81% of guard cells microinjected in the open and closed configuration, respectively. All stomata were observed to close in response to ABA. Increases ranged from 100 to 750 nM above the resting concentration and were arbitrarily grouped into five "classes." ABA-stimulated increases in [Ca2+]cyt were not uniformly distributed across the cytosol of guard cells. Rapid transient increases in [Ca2+]cyt were also observed in the guard cells of stomata microinjected in the closed configuration. We concluded that the ABA-induced turgor loss in guard cells is a Ca2+-dependent process.     

Critical Staining of Pancreatic Alpha Granules with Phosphotungstic Acid Hematoxylin

Mammalian pancreatic alpha granules were differentially stained with phosphotungstic acid haematoxylin. Paraffin sections were dewaxed and hydrated, oxidised 5-40 sec in freshly prepared 0.3% KMnO₄ acidified with 0.3% (w/v) H₂SO₄, decolourised in 4% potassium metabisulphite, mordanted 20 min to 2 hr in 4% iron alum, stained in phosphotungstic acid haematoxylin 16-48 hr, rinsed in 95% ethanol until no stain runs from the tissue, dehydrated in absolute ethanol, cleared in xylene, and covered in synthetic resin. Advantages of this procedure are: (1) consistent, reproducible staining; (2) applicability to all the common laboratory mammals and man; (3) wide latitude at each stage, permitting its use as a routine method; and (4) superior visualization of alpha granules, due to suppression of background staining and absence of glare. For fixation, formalin-acetic or Bouin's solution is recommended.     

Accumulation of malate in guard cells of Vicia faba during stomatal opening

Summary The level of malate in the epidermis from illuminated leaves of Vicia faba was greater than in that from dark-treated leaves. A difference in the malate level was still detected after the epidermis had been treated by rolling so that only the guard cells remained alive. The results suggest that malate may accumulate in guard cells on illumination. In subsequent experiments, stomatal apertures were measured, and potassium as well as malate was analysed in extracts of epidermis. In illuminated leaves, the potassium content of rolled epidermis increased from about 90 to about 335 picoequivalents mm^-2 of epidermis whele malate increased from about zero to about 71 pmoles mm^-2 and the stomata opened; in dark-treated leaves, the potassium content of rolled epidermis decreased slightly, the malate level remained about zero, and the stomata showed very slight further closure. The measured increase in potassium is likely to represent an increase in potassium concentration in the guard cells of about 0.4 Eq l^-1 with stomatal opening; the increase in malate could correspond to 0.23 Eq l^-1 (with respect to potassium) in the guard cells. Thus, malate accumulating in guard cells could balance about half of the potassium taken up by guard cells when stomata open in the light.     

The stomata of the fern Adiantum capillus-veneris do not respond to CO2 in the dark and open by photosynthesis in guard cells

The stomata of the fern Adiantum capillus-veneris lack a blue light-specific opening response but open in response to red light. We investigated this light response of Adiantum stomata and found that the light wavelength dependence of stomatal opening matched that of photosynthesis. The simultaneous application of red (2 micromol m(-2) s(-1)) and far-red (50 micromol m(-2) s(-1)) light synergistically induced stomatal opening, but application of only one of these wavelengths was ineffective. Adiantum stomata did not respond to CO2 in the dark; the stomata neither opened under a low intercellular CO2 concentration nor closed under high intercellular CO2 concentration. Stomata in Arabidopsis (Arabidopsis thaliana), which were used as a control, showed clear sensitivity to CO2. In Adiantum, stomatal conductance showed much higher light sensitivity when the light was applied to the lower leaf surface, where stomata exist, than when it was applied to the upper surface. This suggests that guard cells likely sensed the light required for stomatal opening. In the epidermal fragments, red light induced both stomatal opening and K+ accumulation in guard cells, and both of these responses were inhibited by a photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The stomatal opening was completely inhibited by CsCl, a K+ channel blocker. In intact fern leaves, red light-induced stomatal opening was also suppressed by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. These results indicate that Adiantum stomata lack sensitivity to CO2 in the dark and that stomatal opening is driven by photosynthetic electron transport in guard cell chloroplasts, probably via K+ uptake.     

pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions: cellular responses to stress and their implication for plant water relations

A paradigm for the response of plants to stress is presented which suggests that plants move towards a state of minimal metabolic activity as a stress intensifies and remain in that state until that stress is relieved. The paradigm is based on the proposition that cells that interface with the transpiration stream employ variations on the following theme to move towards that state. Tension on the apoplastic water opens a mechanosensitive Ca2+ channel, a response that is augmented by apoplastic ABA. The resulting elevated cytoplasmic Ca2+ deactivates a plasmalemma H+/ATPase and also activates a K(+)-H+ symport. The inflow of K+ and H+ depolarizes the membrane and renders the apoplast less acidic, the protons being removed to the vacuole and the K+ ions being re-exported via the K+ outward rectifying channel. The onset of darkness in guard and mesophyll cells deactivates the plasmalemma H+/ATPase and then the events outlined above ensue except that these cells do not appear to utilize either Ca2+ or ABA during these changes. In stressed cells it is proposed that elevated cytoplasmic Ca2+ activates the release of an ABA precursor from a stored form. ABA is then released in the apoplast after export of the precursor if the activity of the K(+)-H+ symport has brought the apoplastic pH close to 7.0. It is proposed that aquaporins in the xylem parenchyma and mesophyll cells are opened by elevated cytoplasmic Ca2+ when the water potential of the transpiration stream is high so that water can be stored in the 'xylem parenchyma reservoir'. The water in this reservoir is then used to increase the water potential in the transpiration stream when the water column is under tension and to help repair embolisms by a mechanism that resembles stomatal closure.     

Staining Mitochondria in Fixed Blood Smears

Wet blood smears are placed immediately in Helly's fluid for 24 hr, transferred directly to a saturated solution of potassium dichromate for 48 hr and washed in running water for 2-4 hr. The slides are then treated with iodine and sodium thiosulf ate and washed several hours or overnight. Excess water is removed by blotting the slide around the smear, Altmann's aniline fuchsin is placed on the smear and the slide is heated over a spirit lamp until white fumes appear. After the slide cools the stain is poured off and the excess removed by washing with distilled water. Methyl green (1% aqueous) is dropped on the smear and left for approximately 30 sec. It is then passed rapidly through 2 changes of absolute ethanol and into xylene, from which it is mounted in Permount. This stains mitochondria, red blood corpuscles and specific granules of eosinophilic granulocytes red on a green background.     

Quantitative changes in microtubule distribution correlate with guard cell function in Arabidopsis

Radially arranged cortical microtubules are a prominent feature of guard cells. We observed guard cells expressing GFP-tubulin (GFP-TUA6) with confocal microscopy and found recognizable changes in the appearance of microtubules when stomata open or close (Eisinger et al., 2012). In the present study, analysis of fluorescence distribution showed a dramatic increase in peak intensities of microtubule bundles within guard cells as stomata open. This increase was correlated with an increase in the total fluorescence that could be attributed to polymerized tubulin. Adjacent pavement cells did not show similar changes in peak intensities or integrated fluorescence when stomatal apertures changed. Imaging of RFP-tagged end binding protein 1 (EB1) and YFP-tagged α-tubulin expressed in the same cell revealed that the number of microtubules with growing ends remained constant, although the total amount of polymerized tubulin was higher in open than in closed guard cells. Taken together, these results indicate that the changes in microtubule array organization that are correlated with and required for normal guard cell function are characterized by changes in microtubule clustering or bundling.     

Role of Ca and EGTA on Stomatal Movements in Commelina communis L

Ca²⁺ (0.1-1.0 millimolar) accelerated dark-induced stomatal closure and reduced stomatal apertures in the light in epidermal peels of Commelina communis L. In contrast, ethyleneglycol-bis-(β-aminoethyl ether) N,N′tetraacetic acid (EGTA) (2 millimolar), a Ca²⁺ chelator, prevented closure in the dark and accelerated opening in the light. EGTA did not promote significant opening in the dark. It is therefore concluded that EGTA does not increase ion uptake into guard cells, but rather prevents ion efflux. Addition of EGTA to incubating solutions with 10 millimolar KCl resulted in steady state apertures of 15.6 micrometers, whereas in the absence of EGTA similar apertures required 55 millimolar KCl and 150 millimolar KCl was needed in the presence of 1 millimolar CaCl₂. The results demonstrate the importance of Ca²⁺ in the regulation of stomatal closure and point to a role of Ca²⁺ in the regulation of K⁺ efflux from stomatal guard cells.     

High-temperature Disruption of Guard Cells of Vicia faba: EFFECT ON STOMATAL APERTURE

Increased variability in stomatal aperture at high temperatures can be attributed, in part, to the differential sensitivity of guard cells to thermal damage. Individual stomata become increasingly open at higher temperatures until guard cells are lethally damaged; at that temperature, apertures decrease. The extent of irreversible damage causing closure was estimated by K⁺ uptake, neutral red accumulation, and visual scoring of chloroplasts.