ABA Uptake in Source and Sink Tissues of Sugar Beet

The mode of abscisic acid (ABA) uptake was studied in excised leaf and root tissue discs of sugar beet (Beta vulgaris L.). Discs were incubated in buffered medium that contained 1 mm CaCl₂ and [¹⁴C]ABA. The sensitivity of ABA uptake to metabolic inhibitors and temperature indicated that the ABA transport system had an energy-dependent component. Energy-dependent uptake was greater in leaf than in root tissue (70% and 50%, respectively). Energy-dependent uptake by both tissues and passive uptake by root tissues were highly pH dependent. Maximal uptake was observed at pH 5.5. Leaf tissue incubated in the dark showed a 50% reduction of uptake as compared with tissue under light. The decrease was due to reduced passive uptake.     

FOLIAR ABSORPTION—AN ACTIVE UPTAKE PROCESS

Mechanisms of foliar absorption of phosphate and rubidium in primary leaves of bean seedlings were resolved by a newly developed leaf-immersion and leaf-washing technique. Uptake was expressed as “specific absorption,” defined as mμmoles/cm² leaf X 24 hr from a known concentration of substance in an external solution maintained in a known environment. Criteria for active uptake included time-course analysis, temperature, oxygen, and energy dependence, sensitivity to metabolic inhibitors, accumulation against a concentration gradient, irreversibility, and pH dependence. The results suggest that the over-all process of foliar absorption of rubidium and phosphate by bean leaves, beginning with zero time and extending over a 24-hr period, is metabolic. It is proposed that carriers play an important role in uptake and that they are proteinaceous.     

Development of pH sensitivity of sucrose uptake during ageing of Vicia faba leaf discs

Uptake of [U-¹⁴C]-sucrose (40 m M ) by fresh and aged peeled leaf discs of broad bean ( Vicia faba L. cv. Aguadulce) has been studied. In fresh discs, uptake was nearly insensitive to external pH, whereas the pH response of absorption in discs aged for 12 h was bell-shaped, with an optimum between pH 5 and 6. At this pH, uptake was nearly twice that in fresh tissue. The passive (insensitive to carbonyl cyanide m -chlorophenylhydrazone and to cold treatment) uptake was the same in fresh or aged discs. The development of pH sensitivity of absorption did not appear when ageing was performed in the presence of 10^−H M cycloheximide or 5.7 × 10⁻⁵ M actinomycin D. Similarly, when the tissues were treated with 10⁻³ M spermidine for 2 h after excision and then aged for 10 h, the development of the pH-sensitive uptake system was inhibited. Ca²⁺ (10⁻² M ) supplied together with spermidine prevented the inhibiting effect of spermidine. The appearance of the pH-sensitive system was also markedly reduced if ageing took place in the presence of 10⁻³ M aminoethoxyvinylglycine. Autoradiographs from fresh discs and from discs aged with or without the inhibitors suggest that pH sensitivity developed more intensively in the parenchyma than in the veins.
The results suggest some caution when using excised leaf discs for studies on sucrose uptake and phloem loading. Development of pH sensitivity of uptake may require the synthesis of both DNA-dependent RNA and protein and could be related to ethylene metabolism.     

Influence of Abscisic acid on K+ absorption by leaf discs ofSolanum tuberosum

It is shown that, contrary to what is generally found, treatment with abscisic acid (ABA) of potato leaf tissues resulted in an increase of K⁺ uptake. Comparison with other hormones was made: BAP induced an inhibition and GA₃ a stimulation of K⁺ uptake. The uptake was sensitive to several metabolic inhibitors, external pH and ATPase inhibitors while p-chloromercuribenzenesulfonic acid (PCMBS) had no effect. Uptake kinetics revealed the presence of both saturable and linear components which were both stimulated by ABA treatment. Our data are consistent with an effect of ABA on the active and passive components of K⁺ uptake. These results are discussed in relation to the action of ABA on foliar senescence and the action on ion partitioning in the whole plant.     

Mechanism for the Uptake of Zinc by Fontinalis antipyretica

The rate of uptake, the effects of carrier ions, temperature, light intensity and dinitrophenol (DNP) upon the uptake of zinc (using zinc-65 as tracer) by Fontinalis antipyretica were investigated. The absorption medium contained 0.5 mW CaSO₄ and the pH was kept constant using boric acid and sodium hydroxide as buffer. About 50 per cent of the zinc absorbed at equilibrium was taken up in the first half hour, Analysis by semi-log plot procedures showed that, at least, three sections may be distinguished in the uptake curve, suggesting that three successive processes (stages) were involved. The first stage was very short, and not influenced by temperature, light intensity and DNP. The second stage, lasting no more than 90 minutes, mi very slightly affected by the same three factors. In this stage freshly killed plants absorb more zinc than living material. The third stage, lasting several days, was very slow and was light, temperature and DNP dependent. Based on these findings, a mechanism for zinc uptake is proposed.     

Zn uptake by Cladophora glomerata

The uptake of zinc by Cladophora glomerata at different pH-values, light conditions and exposure periods is reported. Zinc uptake was pH-dependent, with uptake increasing with rising pH. Uptake was greatest within the first two hours of exposure and was greater in light than in darkness due to increased pH. All results support the dominance of adsorptive uptake of zinc by Cladophora.     

Cobalt transport in a cobalt-resistant strain ofNeurospora crassa

Uptake of Co²⁺ by cobalt-resistant strain is dependent on Co²⁺ concentration in the medium and is linear with time. The uptake is unaffected by metabolic inhibitors and decreased at low pH values. The uptake is independent of temperature in the range 0–40‡ C. The transport system is a passive diffusion process, unlike in the parent wild type strain where it is energy-dependent. It is possible that Mg²⁺ transport system is not involved in Co²⁺ transport in this strain, since the Co²⁺ uptake is not suppressed by Mg²⁺ as in the parent strain.     

Uptake of Amino Acids by Barley Leaf Slices: Kinetics, Specificity, and Energetics

Uptake of ¹⁴C-labelled _L-lysine. _L-arginine, _L-glutamic acid, _L-aspartic acid, and glycine was studied in 0.75 mm wide barley (Hordeum vulgare L. cv. Lise) leaf slices. After an initial period (10 min) of rapid accumulation amino acid uptake proceeded at a steady, lower rate for several hours. Uptake was stimulated by 10^?4M Ca^?2+ ions. Uptake was strongly _pH dependent with the following optima: aspartic acid _pH 3.5. glutamic acid _pH 4.1. glycine _pH 5.8, lysine _pH 6–7, and arginine _pH 5–8 (a broad plateau). The optimal temperature was about 30°C. and the temperature coefficient in the range 0–20gGC was 2.3–2.5. Concentration-dependence data gave uptake isotherms which appeared to be multiphasic for all the amino acids used. The amino acids inhibited each other in a competitive fashion, indicating that they were all transported by a single carrier system. Uptake of lysinc was strongly inhibited by 10^?4M 2.4-dinitrophenol. Lysine uptake was not stimulated by light under aerobic conditions. However, it was much reduced in the dark under anaerobic conditions. This reduction was almost compensated for by light. The light-stimulation of uptake under anaerobic conditions was abolished by 10^?5M 3-(3,4-dichlorophenyl) 1.1-dimethylurea.     

Intestinal zinc uptake in two marine teleosts,squirrelfish (Holocentrus adscensionis) and gulf toadfish (Opsanus beta)

Zinc is a vital micronutrient, yet as an environmental toxicant it can be deleterious to aquatic organisms such as fish. Consequently, the study of zinc uptake mechanisms is essential for understanding nutrition, toxicity, and metabolism of this metal. Intestinal zinc uptake was studied in two marine teleosts, using both in vitro (in vitro perfusion and intestinal sacs) and in vivo techniques (in situ bolus). Female squirrelfish (Holocentrus adscensionis) exhibited significantly increased epithelial zinc uptake associated with enhanced hepatic zinc accumulation. This confirms this zinc-hyperaccumulating teleost as a potential model of zinc absorption. Intestinal zinc uptake in the gulf toadfish (Opsanus beta) was biphasic with respect to zinc concentration (0.3-500 microM), exhibiting both saturable and passive uptake components. In both species, the passage of zinc into the postintestinal compartment was highly dependent on technique. Decreased proportions of postintestinal zinc in vivo, coupled with concentration-dependent distribution of zinc accumulation, suggested mechanisms may act to control the movement of zinc into the circulation. In addition, the results of this study were used to reinterpret previous findings of zinc uptake in freshwater fish and allowed a critique of techniques used to study intestinal metal uptake.     

EFFECT OF DIMETHYL SULFOXIDE ON ZINC65 UPTAKE,RESPIRATION, AND RNA AND PROTEIN METABOLISM IN BEAN (PHASEOLUS VULGARIS) TISSUES

The effect of dimethyl sulfoxide (DMSO) on zinc⁶⁵ uptake, respiration, RNA, and protein metabolism in various tissues of two bean (Phaseolus vulgaris L.) cultivars showing differential growth responses to zinc has been studied. At a concentration of 1%, DMSO stimulated zinc uptake in excised roots, stem-callus tissue, leaf disks, and enzymically isolated leaf cells, but did not significantly alter the uptake and incorporation of C¹⁴-uracil into RNA and C¹⁴-methionine into protein, although a slight inhibition was discernible in some tissues. At a higher concentration (10%) DMSO increased Zn⁶⁵ uptake in excise roots incubated for 2 hr; however, at the same concentration, C¹⁴-uracil and C¹⁴-methionine uptake and incorporation were considerably inhibited in all the tissues. Oxygen uptake as measured with Warburg manometers was impaired, and the inhibition showed a time and concentration dependency. The fact that DMSO inhibited respiration and RNA and protein metabolism, while at the same concentration zinc uptake was increased, suggests that zinc uptake in beans is primarily a non-metabolic process. The possible mechanisms of DMSO action are discussed in the light of its reported effects on membrane permeability and cell metabolism.     

Influence of soil waterlogging on subsequent plant growth and trace metal content

Summary The uptake of trace metals by two plant species (French bean and maize) has been measured on two soils subjected to various waterlogging regimes. Uptake of both manganese and iron was increased due to soil waterlogging, although reoxidation of the soil affected iron more than manganese. Zinc and copper uptake was influenced by a species factor; French bean (Phaseolus vulgaris) showed preferential uptake of zinc, whereas maize (Zea mays) took up copper preferentially. Uptake of cobalt by both species was increased due to waterlogging, following the pattern of manganese.The abilities of these species to take up trace metals from soil followed the pattern predicted by selective extraction of soil for manganese, iron and cobalt, but not for zinc and copper.     

Characterization of sterol uptake in leaf tissues of sugar beet

The uptake of cholesterol has been characterized in leaf discs from mature leaves of sugar beet (Beta vulgaris L.). This transport system exhibited a simple saturable phase with an apparent Michaelis constant ranging from 30 to 190 M depending on the sample. When present at 10 M excess, other sterols were able to inhibit cholesterol uptake. Moreover, binding assays demonstrated the presence of high-affinity binding sites for cholesterol in purified plasma membrane vesicles. In the range 1–60 M, cholesterol uptake showed an active component evidenced by action of the protonophore carbonyl cyanide m-chlorophenylhydrazone. Energy was required as shown by the inhibition of uptake induced by respiration inhibitors (NaN₃), darkness and photosynthesis inhibitors [3-(3,4-dichlorophenyl)-1,1-dimethylurea, methyl viologen]. Moreover, the process was strongly dependent on the experimental temperature. Uptake was optimal at acidic pH (4.0), sensitive to ATPase modulators, inhibited by thiol reagents (N-ethylmaleimide, p-chloromercuribenzenesulfonic acid, Mersalyl) and by the histidyl-group reagent diethyl pyrocarbonate. The addition of cholesterol did not modify H⁺ flux from tissues, indicating that H⁺-co-transport was unlikely to be involved. MgATP did not increase the uptake, arguing against involvement of an ABC cassette-type transporter. By contrast, cryptogein, a sterol carrier protein from the Oomycete Phytophtora cryptogea, greatly increased absorption. Taken together, the results reported in this work suggest that plant cells contain a specific plasma membrane transport system for sterols.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - PCMBS p-chloromercuribenzenesulfonic acid - PMV plasma membrane vesicle - TLC thin-layer chromatography     

Energetics of Amino Acid Uptake by Vicia faba Leaf Tissues

The uptake of [U-¹⁴C]threonine and of (α-¹⁴C]aminoisobutyrate (α-AIB) by Vicia faba leaf discs is strongly pH dependent (optimum: pH 4.0) and exhibits biphasic saturation kinetics. Kinetics of α-AIB uptake at different pH values indicate that acidic pH values decrease the K_m of the carriers while the maximal velocity remains nearly unaffected. Similar results were obtained for both system 1 (from 0.5 to 5 millimolar) and system 2 (from 20 to 100 millimolar).

After addition of amino acids to a medium containing leaf fragments, alkalinizations depending both on the amino acid added and on its concentration have been recorded.

The effects of compounds which increase (fusicoccin) or decrease (uncouplers, ATPase inhibitors, high KCl concentrations) the protonmotive force were studied both on the acidification of the medium and on amino acid uptake by the tissues. There is a close relationship between the time required for the effect of these compounds on the acidification and that needed for inhibition of uptake.

Studies with thiol inhibitors show that 0.1 millimolar N-ethylmaleimide preferentially inhibits uptake by the mesophyll whereas 0.1 millimolar parachloromercuribenzenesulfonate affects rather uptake by the veins.

New evidence was found which added to the electrophysiological data already supporting the occurrence of proton amino acid symport in leaf tissues, particularly in the veins.

     

Characterization of a human plasma membrane heme transporter in intestinal and hepatocyte cell lines

Heme is the most bioavailable form of dietary iron and a component of many cellular proteins. Controversy exists as to whether heme uptake occurs via specific transport mechanisms or passive diffusion. The aims of this study were to quantify cellular heme uptake with a fluorescent heme analog and to determine whether heme uptake is mediated by a heme transporter in intestinal and hepatic cell lines. A zinc-substituted porphyrin, zinc mesoporphyrin (ZnMP), was validated as a heme homolog in uptake studies of intestinal (Caco-2, I-407) and hepatic (HepG2) cell lines. Uptake experiments to determine time dependence, heme inhibition, concentration dependence, temperature dependence, and response to the heme synthesis inhibitor succinylacetone were performed. Fluorescence microscope images were used to quantify uptake and determine the cellular localization of ZnMP; ZnMP uptake was seen in intestinal and hepatic cell lines, with cytoplasmic uptake and nuclear sparing. Uptake was dose- and temperature dependent, inhibited by heme competition, and saturated over time. Preincubation with succinylacetone augmented uptake, with an increased initial uptake rate. These findings establish a new method for quantifying heme uptake in individual cells and provide strong evidence that this uptake is a regulated, carrier-mediated process.     

Uptake and Release of Abscisic Acid by Isolated Photoautotrophic Mesophyll Cells, Depending on pH Gradients

Uptake and release of abscisic acid (AbA) by isolated mesophyll cells of Papaver somniferum is characterized by the following observations: (a) Uptake rate is a linear function of the external AbA concentration in the range from 10⁻⁶ to 5 × 10⁻⁵ molar, and decreases with increasing pH. At any pH, uptake rate is linearly related to the concentration of undissociated abscisic acid, calculated from the pK = 4.7 according to the Henderson-Hasselbalch equation. At low external pH (5.0), AbA accumulation in the cells is about 10-fold. (b) Uptake of AbA is completely inhibited by salts such as KNO₂ or sodium acetate, which decrease the pH gradient between medium and cells. KCN or m-chlorocarbonylcyanide phenylhydrazone inhibits AbA uptake only after longer incubation periods (20-40 minutes). (c) Uptake rate as well as equilibrium concentration is significantly higher in light than in darkness. (d) At low external pH, release of AbA from preloaded cells is strongly stimulated by KNO₂. It is concluded that AbA is distributed between leaf cells and free space according to pH gradients, with the undissociated abscisic acid being the main penetrating species. Uptake and release occur via diffusion, without participation of a carrier.     

Establishment of low extracellular pH is essential for uptake of the fluorescent anionic dye hydroxypyrenetrisulfonate by suspension-cultured carrot cells

Uptake of the fluorescent dye hydroxypyrenetrisulfonate by carrot (Daucus carota L.) suspension cells only occurs when the external (medium) pH falls to below 4.0. Uptake of the dye was shown to be inhibited by a range of compounds, including the ‘Good’ buffers MES, HEPES, HEPPSO and HEPPS, the growth medium component coconut water (CW) and probenecid, an organic anion translocator inhibitor. Tris(hydroxymethyl)aminomethane stimulated transport. Buffer effects were not correlated with pK_a values. Inhibitors of dye uptake (MES, probenecid, CW) also inhibited medium acidification by the cells and Tris stimulated acidification and uptake. Uptake of HPTS correlated strongly with external pH and was restored when external pH was experimentally reduced to below 4.0 even in the presence of inhibitors. This suggests that inhibitors of HPTS uptake at the plasma membrane act primarily by preventing the establishment of a low external pH required for transport. The implications of this on the mechanism of HPTS transport are discussed.     

Uptake of abscisic acid by isolated mesophyll protoplasts from Vicia Faba

Mesophyll protoplasts were isolated from Vicia faba (cv. exhibition longpod) leaf tissue and [³H] abscisic acid ([³H]ABA) uptake was measured as a function of time, concentration, pH and temperature. [³H] ABA uptake with time was linear for 30 s and then reached equilibrium. The uptake rate was a linear function of the external ABA concentration and had a pH optimum of 4–5. Various metabolic inhibitors did not effect the rate of uptake. The Q₁₀ value was less than 1.5. The results suggested that initial uptake was not a metabolically dependent or carrier mediated process but diffusive.     

Transport of Glycerate across the Envelope Membrane of Isolated Spinach Chloroplasts

Uptake of d, l-glycerate into the chloroplast stroma has been studied using the technique of silicone oil filtering centrifugation. Glycerate uptake was 3 to 5 times higher in the light than in darkness, the stimulation by light being abolished by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The pH optimum for uptake was 7.0 at 2°C and 8.5 at 20°C, but at all pH values the rate of uptake was higher at 20°C than at 2°C. Uptake was concentration dependent, saturating above 8 millimolar glycerate. At 2°C, the K_m was 0.3 millimolar and the V_max was 13 micromoles per milligram of chlorophyll per hour. At 20°C initial rates of glycerate uptake were higher than 40 micromoles per milligram of chlorophyll per hour.     

Characterization of Glutathione Uptake in Broad Bean Leaf Protoplasts

Transport of reduced glutathione (GSH) and oxidized glutathione (GSSG) was studied with broad bean (Vicia faba L.) leaf tissues and protoplasts. Protoplasts and leaf discs took up GSSG at a rate about twice the uptake rate of GSH. Detailed studies with protoplasts indicated that GSH and GSSG uptake exhibited the same sensitivity to the external pH and to various chemical reagents. GSH uptake was inhibited by GSSG and glutathione conjugates. GSSG uptake was inhibited by GSH and GS conjugates, and the uptake of metolachlor-GS was inhibited by GSSG. Various amino acids (L-glutamic acid, L-glutamine, L-cysteine, L-glycine, L-methionine) and peptides (glycine-glycine, glycine-glycine-glycine) affected neither the transport of GSH nor GSSG. Uptake kinetics indicate that GSH is taken up by a single saturable transporter, with an apparent Km of 0.4 mM, whereas GSSG uptake exhibits two saturable phases, with an apparent Km of 7 [mu]M and 3.7 mM. It is concluded that the plasma membrane of leaf cells contains a specific transport system for glutathione, which takes up GSSG and GS conjugates preferentially over GSH. Proton flux measurements and electrophysiological measurements indicate that GSH and GSSG are taken up with proton symport. However, a detailed analysis of these measurements suggests that the ion movements induced by GSSG differ from those induced by GSH.     

Light-mediated Oxygen Uptake Measured in Wheat Etioplasts

An in vitro O₂ assay was used to measure early response of wheat (Triticum aestivum L.) etioplasts to light. A transient photoinducible O₂ uptake occurred when dark-grown etioplasts were initially exposed to light. The rate of inducible O₂ consumption was dependent on both the intensity of light and the quantity of organelle protein present. Higher light intensities resulted in greater O₂ utilization per minute, and a greater quantity of organelle protein in the sample resulted in an increased rate of O₂ uptake under the same light intensity conditions. Experiments with various plant tissues as well as with mitochondrial respiratory inhibitors indicated that etioplasts are the organelles responsible for the photoinduced O₂ uptake phenomenon. A preliminary action spectrum study revealed that wavelengths 640 to 680 nm resulted in maximum O₂ uptake. This indicated the presence of an etioplast red light receptor pigment which induces O₂ uptake in etioplasts.