Characterization of in vitro proton pumping by microsomal vesicles isolated from corn coleoptiles

Corn (Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H⁺-transport as measured by [¹⁴C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP·Mg, is temperature-sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Of the divalent cations tested, Mn²⁺ is almost as effective as Mg²⁺, while Ca²⁺ is ineffective. Excess divalent cations, particularly Ca²⁺, reduces the pH gradient. H⁺ transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with ³⁶Cl⁻ indicate that ATP-driven H⁺ transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide-m-chlorophenylhydrazone and the anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, inhibit methylamine accumulation and ³⁶Cl⁻ uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N′-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin.     

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K_m of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg²⁺ present, nor by protonophores and valinomycin (with K⁺ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday     

Pyrophosphate-driven proton transport by microsomal membranes of corn coleoptiles

Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on isopycnic sucrose density gradients. Two peaks of ATP-driven H⁺-transport activity, corresponding to the previously characterized tonoplast (1.07 grams per cubic centimeter) and Golgi (1.13 grams per cubic centimeter) fractions (Chanson and Taiz, Plant Physiol 1985 78: 232-240) were localized. Coincident with these were two peaks of inorganic pyrophosphate (PPi)-driven H⁺-transport. At saturating (3 millimolar) concentrations of Mg²⁺:ATP, the rate of proton transport was further enhanced by the addition of 3 millimolar PPi, and the stimulation was additive, i.e. equal to the sum of the two added separately. The specific PPi analog, imidodiphosphate, antagonized PPi-driven H⁺-transport, but had no effect on ATP-driven transport. Moreover, PPi-dependent proton transport in both tonoplast-enriched and Golgi-enriched fractions was strongly promoted by 50 millimolar KNO₃, unlike the ATP-dependent H⁺-pumps of the same membranes. Taken together, the results indicate that PPi-driven proton transport is mediated by specific membrane-bound H⁺-translocating pyrophosphatases. Both potassium and a permanent anion (NO₃⁻ > Cl⁻), were required for maximum activity. The PPi-driven proton pumps were totally inhibited by N,N′-dicyclohexylcarbodiimide, but were insensitive to 100 millimolar vanadate. The PPi concentration in coleoptile extracts was determined using an NADH oxidation assay system coupled to purified pyrophosphate:fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.90). The total pyrophosphate content of corn coleoptiles was 20 nanomoles/gram fresh weight. Assuming a cytoplasmic location, the calculated PPi concentration is sufficient to drive proton transport at 20% of the maximum rate measured in vitro for the tonoplast-enriched fraction, and 10% of the maximum rate for the Golgi-enriched fraction.     

Auxin movement in corn coleoptiles

Summary Movement of radioactive auxins was analysed in corn coleoptile sections. The results support the idea that processes involved in the transport of indoleacetic acid (IAA) are specific for growth-promoting auxins.Inhibition of IAA transport by triiodobenzoic acid is caused by a reversible block of the exit; the auxin held back remains in the transport pool. The observed increase in immobilization may be a secondary effect caused by the increased concentration of free IAA in the tissue.Auxin molecules are most likely transported by anon-covalent mechanism. IAA and naphthaleneacetic acid (NAA) move through the cell and exit as free molecules. A search for a transient auxin complex, chaseable as required for any transport carrier intermediate, yielded negative results. No¹⁸O was lost from NAA labeled with¹⁸O in the carboxyl group during transport of the auxin through coleoptile tissue.After application of IAA to auxin-depleted tissue, the transport rate undergoes oscillations with a period length of ca. 25 min.The movement of the auxin 2.4-dichlorophenoxyacetic acid which is usually sluggish, increased several times if some IAA was added. Auxin, thus, stimulates its own transport.A model is discussed in which auxin-binding to the plasma membrane and reversible changes of membrane conformation may provide a basis for active secretion and for the observed cooperativity.Leo Brauner zum 70. Geburtstag gewidmet.     

Effect of gravity and centrifugal acceleration on auxin transport in corn coleoptiles

Summary Inversion of corn coleoptile sections resulted in a 10–20% inhibition of basipetal transport of 3-indoleacetic acid (IAA) and a more pronounced inhibition (20–50%) of the transport of 1-naphthaleneacetic acid (NAA).The effect of inversion on basipetal NAA transport was compared in wild-type corn and in the amylomaize mutant which contains smaller and slower sedimenting amyloplasts: the gravity induced inhibition was higher in the wild type coleoptiles (27% versus 9%).In wild type the inhibitory effect on basipetal NAA transport appeared within less than 30 min after inversion; then the effect remained relatively constant over at least 2 hr of transport. When the sections were returned to the upright position the transport rate increased, reaching the level of upright controls within 30 min.An effect of gravity on lateral transport of NAA was also demonstrated and shown to be expressed within 10 min after placing the tissue horizontally.When basipetal transport was tested in the direction of gravity and/or centrifugal acceleration, auxin movement incrased with increasing acceleration. Transport against centrifugal acceleration (10 x g) was less than transport of control sections (inverted at 1 x g).The results agree with the hypothesis that starch statoliths act by a pressure mechanism on the membrane transport system of auxin.     

Patch-clamp studies of ion transport in isolated plant vacuoles

Plant cell vacuoles act as storage depots for substances such as sugars, organic and inorganic ions, many of which are commercially important. We have used the patch-clamp technique to characterize an ATP-dependent proton pump and a non-selective channel in the vacuolar membrane in single isolated barley leaf vacuoles. The large (60 to 80 pS) ion channel shows inward rectification, and carries both K⁺ or malate ions driven by their concentration gradient as well as the electrical gradient established by the ATP-dependent proton pump.     

Initial phases of gravity-induced lateral auxin transport and geotropic curvature in corn coleoptiles

Summary Wild-type corn coleoptiles showed an initial downward bending upon transfer from the vertical to the horizontal position. Strong upward curvature started only 15–30 min after the begin of horizontal exposure.Little, if any at all, initial downward geotropic bending was found with amylomaize coleoptiles at 1 X g. With stronger stimuli (10 or 20 X g) the amylomaize mutant reacted initially strongly in the wrong direction, i.e. opposite to the later response.When wild-type coleoptiles had been symmetrically prestimulated for 60 min with alternating 2-min horizontal exposures from opposite sides, no initial downward bending occurred if the plane of horizontal exposure was maintained from pretreatment to the continuous horizontal stimulation of the test. If, however, the coleoptiles were rotated 90° around their long axis between pretreatment and test, the initial downward bending reaction developed as in the non-prestimulated controls. Thus changes in reactivity remained localized to the site of stimulation.Following the same pretreatments used for the curvature measurements, lateral ³H-IAA transport was measured in coleoptile segments for 10 or 12.5 min. The auxin distribution found was strikingly parallel to the bending for all pretreatments.The dependence of reaction pattern on the duration of prestimulation in the same plane was tested. The function indicates a half life of 10–20 min for the change in sensitivity. The findings are discussed in view of a model of overstimulation and adaptation.     

Direct evidence for a sugar transport mechanism in isolated vacuoles

Sugar transport has been directly observed in isolated higher plant vacuoles for the first time. The latter were released from protoplasts isolated from the mesophyll of Pisum sativum L.     

Sugar transport in isolated corn root protoplasts

Isolated corn (Zea mays L.) root protoplasts were used to study sucrose and hexose uptake. It is found that glucose was preferentially taken up by the protoplasts over sucrose and other hexoses. Glucose uptake showed a biphasic dependence on external glucose concentration with saturable (K_m of 7 millimolar) and linear components. In contrast, sucrose uptake only showed a linear kinetic curve. Sucrose and glucose uptake were linear over a minimum of 1 hour at pH 6.0 and 1 millimolar exogenous sugar concentration. Glucose uptake showed a sharp 42°C temperature optimum, while sucrose uptake showed a lower temperature sensitivity which did not reach a maximum below 50°C. Uptake of both sugars was sensitive to several metabolic inhibitors and external pH. Differences between sucrose and glucose uptake in two different sink tissue (i.e. protoplasts from corn roots and soybean cotyledons) are discussed.     

Partial purification of a tonoplast ATPase from corn coleoptiles

The tonoplast ATPase from corn coleoptile membranes was solubilized using a two-step procedure consisting of a pretreatment with 0.15% (w/v) deoxycholate to remove 60% of the protein, and 40 millimolar octyl-glucoside to solubilize the ATPase. During ultracentrifugation, the solublized ATPase entered a linear sucrose gradient faster than the majority of the protein, resulting in an 11-fold purification over the initial specific activity. The partially purified ATPase was almost completely inhibited by KNO₃ with an estimated K_i of 10 millimolar. The specific activity of the KNO₃-sensitive ATPase was increased 29-fold during purification. N,N′-Dicyclohexylcarbodiimide also completely inhibited the ATPase with half-maximal effects at a concentration of 4 micromolar. Neither vanadate nor azide inhibited enzyme activity. The purified ATPase was stimulated by Cl⁻ and preferred Mg-ATP as substrate. Analysis of frations from the sucrose gradient by sodium dodecyl sulfate-polyacrylamide gel electrophoresis led to the identification of two major polypeptides at 72,000 and 62,000 daltons which were best correlated with ATPase activity. Several minor bands also appeared to copurify with enzyme activity, but were less consistent. Radiation inactivation experiments with intact membranes indicated that the functional molecular size of the tonoplast ATPase was nearly 400,000 daltons. This suggests that the ATPase is composed of several polypeptides, possibly including the 72,000- and 62,000-dalton proteins.     

Linear velocity of cyclic adenosine 3',5'-monophosphate transport in corn coleoptiles

The transport of cyclic adenosine 3′, 5′-monophosphate in corn coleoptile segments is very rapid. The linear velocity of basipetal transport is 183 millimeters per hour, while the velocity of acropetal transport is 79 millimeters per hour. Transport velocity as well as intensity thus appear to be polar in the corn coleoptile. Application of metabolic inhibitors such as cyanide, ouabain, and 2,4-dinitrophenol increase rather than decrease the velocity and intensity of transport. The mechanism of transport in light of these data is discussed.     

Proton and sucrose transport in isolated tonoplast vesicles from sugarcane stalk tissue

Tonoplast vesicles prepared from immature sugarcane ( Saccharum spp., hybrid cv. H65–7052) tissue and purified on a discontinuous dextran gradient take up sucrose. Uptake was stimulated by MgATP. Evidence that the mechanism is linked to proton transport is derived from "pH jump'data and from inhibition of ATP-stimulated sucrose transport by the protonophore carbonyl cyanide m -chlorophenylhydrazone (CCCP) and by the proton-channel blocker of proton-linked ATPases. N. N '-dicyclo-hexylcarbodiimide (DCCD). A saturable phase of sucrose uptake was found at low substrate concentrations, and a linear phase characterized uptake at higher concentrations. Uptake was specific for sucrose, as demonstrated by competition experiments with various sugars. Sucrose uptake by the vesicle fraction was inhibited by KNO₃, protonophores and protein modifying reagents, whereas sodium orthovanadate had no effect. Overall, the evidence suggests an ATP-hydrolysis-dependent tonoplasl antiport for sucrose transport, although a more direct influence of ATP on conformational changes in relevant tonoplast proteins cannot be ruled out.     

The transport of S-adenosyl-L-methionine in isolated yeast vacuoles and spheroplasts.

1. The properties of S-adenosyl-L-methionine accumulating system for both vacuoles and spheroplasts are described. Yeast vacuoles were obtained by a modified metabolic lysis procedure from spheroplasts of Saccharomyces cerevisiae. 2. Isolated vacuoles accumulate S-adenosyl-L-methionine by means of a highly specific transport system as indicated by competition experiments with structural analogs of S-adenosyl-L-methionine. The S-adenosyl-L-methionine transport system shows saturation kinetics with an apparent Km of 68 muM in vacuoles and 11 muM in spheroplasts. 3. S-Adenosyl-L-methionine accumulation into vacuoles does not require glucose, phosphoenolpyruvic acid, ATP, ADP nor any other tri- or di-phosphorylated nucleotides. It is insensitive to azide and 2,4-dinitrophenol which strongly inhibit the glucose-dependent accumulation of S-adenosyl-L-methionine in spheroplasts. 4. The transport of S-adenosyl-L-methionine into vacuoles is optimal at pH 7.4 and is insensitive to nystatin while the uptake of S-adenosyl-L-methionine into spheroplasts is optimal at pH 5.0 and is strongly sensitive to nystatin. On this basis it has thus been possible to measure both the intracytoplasmic and the intravacuolar pool of S-adenosyl-L-methionine. 5. Our results indicate the existence of a highly specific S-adenosyl-L-methionine transport system in the vacuolar membrane which is clearly different from the one present in the plasma membrane of yeast cells.     

Studies on purine transport and on purine content in vacuoles isolated from Saccharomyces cerevisiae.

The transport of purine derivatives into vacuoles isolated from Saccharomyces cerevisiae was studied. Vacuoles which conserved their ability to take up purine compounds were prepared by a modification of the method of polybase-induced lysis of spheroplasts. Guanosine greater than inosine = hypoxanthine greater than adenosine were taken up with decreasing initial velocities, respectively; adenine was not transported. Guanosine and adenosine transporting systems were saturable, with apparent Km values 0.63 mM and 0.15 mM respectively, while uptake rates of inosine and of hypoxanthine were linear functions of their concentrations. Adenosine transport in vacuoles appeared strongly dependent on the growth phase of the cell culture. The system transporting adenosine was further characterized by its pH dependency optimum of 7.1 and its sensitivity to inhibition by S-adenosyl-L-methionine. In the absence of adenosine in the external medium, [14C]adenosine did not flow out from preloaded vacuoles. However, in the presence of external adenosine, a very rapid efflux of radioactivity was observed, indicating an exchange mechanism for the observed adenosine transport in the vacuoles. In isolated vacuoles the only purine derivative accumulated was found to be S-adenosyl-L-homocysteine.     

In-vitro auxin binding to particulate cell fractions from corn coleoptiles

Summary When low concentrations (e.g. 10^-6 M) of labelled 3-indoleacetic acid (¹⁴C-IAA) or -naphthaleneacetic acid (¹⁴C-NAA) are added in vitro to homogenates of corn coleoptiles, radioactivity is reversibly bound to pelletable particles. From the saturation kinetics of the binding it is possible to estimate an apparent K _M between 10^-6 M and 10^-5 M and a concentration of specific sites of 10^-7–10^-6 M per tissue volume.The binding is auxin-specific. Among many compounds tested, only auxins and such auxin analogues that are known to interact directly with auxin in transport and/or growth were found to interfere with this binding. For instance, the growth-active d-dichlorophenoxyisopropionic acid at 10^-4 M inhibits ¹⁴C-NAA binding more than the less active l-isomer.The auxin-binding fractions are practically free of DNA and cytochrome-C oxidase and contain binding sites for 1-naphthylphthalamic acid. The results are discussed in context with the hyothesis—derived mainly from physiological data—that auxin receptors are localized at the plasma membrane.     

In-vitro auxin transport in membrane vesicles from maize coleoptiles

When membrane vesicles from maize (Zea mays L.) coleoptiles are extracted at high buffer strength, a pH-driven, saturable association of [¹⁴C] indole-3-acetic acid is found, similar to the in-vitro auxin-transport system previously described for Cucurbita hypocotyls. The phytotropins naphthylphthalamic acid and pyrenoylbenzoic acid increase net uptake, pressumably by inhibiting the auxin-efflux carrier.Abbreviations IAA indole-3-acetic acid - ION3 ionophore mixture of carbonylcyanide-3-chlorophenylhydrazone, nigericin and valinomycin - 1-NAA, 2-NAA 1-, 2-naphthaleneacetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid     

Studies on purine transport and on purine content in vacuoles isolated from Saccharomyces cerevisiae

The transport of purine derivatives into vacuoles isolated from Saccharomyces cerevisiae was studied. Vacuoles which conserved their ability to take up purine compounds were prepared by a modification of the method of polybase-induced lysis of spheroplasts.Guanosine > inosine = hypoxanthine > adenosine were taken up with decreasing initial velocities, respectively; adenine was not transported.Guanosine and adenosine transporting systems were saturable, with apparent K_m values 0.63 mM and 0.15 mM respectively, while uptake rates of inosine and of hypoxanthine were linear functions of their concentrations.Adenosine transport in vacuoles appeared strongly dependent on the growth phase of the cell culture.The system transporting adenosine was further characterized by its pH dependency optimum of 7.1 and its sensitivity to inhibition by $\text{S-}\text{adenosyl-}\text{l}\text{-methionine}$.In the absence of adenosine in the external medium, [¹⁴C]adenosine did not flow out from preloaded vacuoles. However, in the presence of external adenosine, a very rapid efflux of radioactivity was observed, indicating an exchange mechanism for the observed adenosine transport in the vacuoles.In isolated vacuoles the only purine derivative accumulated was found to be $\text{S-}\text{adenosyl-}\text{l}\text{-homocysteine}$.     

Transport of indoleacetic Acid in intact corn coleoptiles

We have characterized the transport of [³H]indoleacetic acid (IAA) in intact corn (Zea mays L.) coleoptiles. We have used a wide range of concentrations of added IAA (28 femtomoles to 100 picomoles taken up over 60 minutes). The shape of the transport curve varies with the concentration of added IAA, although the rate of movement of the observed front of tracer is invariant with concentration. At the lowest concentration of tracer used, the labeled IAA in the transport stream is not detectably metabolized or immobilized, curvature does not develop as a result of tracer application, and normal phototropic and gravitropic responsiveness are not affected. Therefore we believe we are observing the transport of true tracer quantities of labeled auxin at this lowest concentration.     

The mechanism of oleanolic acid monoglycosides transport into vacuoles isolated from Calendula officinalis leaf protoplasts

The uptake of [3-³H]-oleanolic acid 3-O-monoglucoside and 3-O-monoglucuronide into vacuoles isolated from Calendula officinalis leaf protoplasts was investigated under various conditions. The transport of monoglucoside was stimulated by ATP and pyrophosphate, and sensitive to protein-modifying agent as well as dissipaters of the membrane potential. On the contrary, the transport of monoglucuronide was not dependent on tonoplast energization and was affected only by a protein-modifying factor. Moreover, the kinetics of tonoplast transport of both monoglycosides was characterized as concentration-dependent with saturation phase. The obtained results indicate that oleanolic acid monoglucoside is transported to isolated vacuoles by an active, carrier-mediated and energy-dependent mechanism, whereas the transport of monoglucuronide is a passive, carrier-mediated process.