The Ascorbate Carrier of Higher Plant Plasma Membranes Preferentially Translocates the Fully Oxidized (Dehydroascorbate) Molecule.

Recently, the uptake of 14C-labeled ascorbate (ASC) into highly purified bean (Phaseolus vulgaris L.) plasma membrane vesicles was demonstrated in our laboratory. However, the question of the redox status of the transported molecule (ASC or dehydroascorbate [DHA]) remained unanswered. In this paper we present evidence that DHA is transported through the plasma membrane. High-performance liquid chromatography analysis of the redox status of ASC demonstrated that freshly purified plasma membranes exhibit a high ASC oxidation activity. Although it is not yet clear whether this activity is enzymatic, it complicates the interpretation of ASC-transport experiments in vitro and in vivo. In an attempt to correlate the ASC redox status to transport of the molecule, the ability of different compounds to reduce DHA was analyzed and their effect on ASC-transport activity tested. Administering of various reductants resulted in different levels of inhibition of ASC uptake (dithiothreitol > dithioerythritol > [beta]-mercaptoethanol > [beta]-mercaptopropanol). Glutathione, cysteine, dithionite, and thiourea did not significantly affect ASC transport. Statistical analysis indicated a strong correlation of the Spearman rank correlation coefficient (Rs) of 0.919 (P = 0.0005, n = 9) between the level of ASC oxidation and the amount of transported molecules into the vesicles. The administering of ASC oxidants such as ferricyanide and ASC oxidase resulted in a stimulated ASC uptake into the plasma membrane vesicles. Together, our results demonstrate that a vitamin C carrier in purified bean plasma membranes translocates DHA from the apoplast to the cytosol.

Solubilization and Separation of a Plant Plasma Membrane NADPH-O2- Synthase from Other NAD(P)H Oxidoreductases.

Solubilization and ion-exchange chromatography of plasma membrane proteins obtained from bean (Phaseolus vulgaris L.) seedlings resulted in a single NAD(P)H-O2--synthase protein peak. This enzyme showed a high preference toward NADPH as a substrate (reaction rate, 27.4 nmol O2- produced min-1 mg-1 protein), whereas NADH reactions ranged from 0 to maximally 15% of the NADPH reactions. The protein functions as an oxidase and it was clearly resolved from NAD(P)H dehydrogenases identified with commonly used strong oxidants (ferricyanide, cytochrome c, DCIP, and oxaloacetate). The involvement of peroxidases in O2- production is excluded on the basis of potassium-cyanide insensitivity and NADPH specificity. The NADPH oxidase is only moderately stimulated by flavins (1.5-fold with 25 [mu]M flavine adenine dinucleotide and 2.5-fold with 25 [mu]M flavin mononucleotide) and inhibited by 100 [mu]M p-chloromercuribenzenesulfonic acid, 200 [mu]M diphenyleneiodonium, 10 mM quinacrine, 40 mM pyridine, and 20 mM imidazole. The presence of flavins was demonstrated in the O2-synthase fraction, but no b-type cytochromes were detected. The effect of these inhibitors and the detection of flavins and cytochromes in the plant O2- synthase make it possible to compare this enzyme with the NADPH O2- synthase of animal neutrophil cells.

The Role of Ascorbate Free Radical as an Electron Acceptor to Cytochrome b-Mediated Trans-Plasma Membrane Electron Transport in Higher Plants.

The action of ascorbate free radical as an electron acceptor to cytochrome b-mediated trans-plasma membrane electron transport is demonstrated. Addition of ascorbate free radical to ascorbate-loaded plasma membrane vesicles caused a rapid oxidation of the cytochrome, followed by a slower re-reduction. The fully reduced dehydroascorbate was ineffective.

Transmembrane electron transport in ascorbate-loaded plasma membrane vesicles from higher plants involves a b-type cytochrome.

The possible involvement of a high-potential b-type cytochrome in plasma membrane electron transport was tested using ascorbate-loaded membrane vesicles. Absorption spectra demonstrated that the cytochrome was about 89% reduced in these preparations. Use of ascorbate oxidase and washing of the vesicles further indicated that reduction was mediated by intra-vesicular ascorbate. Addition of low concentrations of ferricyanide caused a rapid cytochrome oxidation followed by a slower re-reduction. The kinetics of this response indicate that the electron acceptor was fully reduced before re-reduction of the cytochrome occurred. These observations suggest that the b-type cytochrome mediates transmembrane electron transfer.

Carrier mediated uptake of dehydroascorbate into higher plant plasma membrane vesicles shows trans-stimulation.

The activity of the ascorbate (Asc) carrier of purified Phaseolus plasma membranes is demonstrated to be highly stimulated when membrane vesicles are preloaded with Asc. Asc transport is inhibited by DTT but is not affected by glutathione or ferricyanide, indicating that dehydroascorbate (DHA) is the preferred species for uptake. Asc transport in the loaded vesicles showed saturable kinetics with an apparent affinity constant of 24 microM and maximal uptake rate of 94 pmol/mg/min. Addition of DHA stimulated the efflux of Asc molecules from the loaded vesicles. Together these results suggest the presence of an Asc/DHA exchange mechanism in higher plant plasma membranes.

Properties of plasma membranes of Phsp 70-ipt transformed tobacco (Nicotiana tabacum).

Application of 10 successive daily heat shocks reduced the growth of control tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants by about 15%; for Phsp 70-ipt transformed plants this is about 48%. The shoot diameter of these ipt-transformed plants increased by about 75%. In addition, in heat shock treated ipt-plants (IPT-HS) the upper lateral buds grew out due to a reduction of apical dominance. The older leaves of IPT-HS plants had a higher chlorophyll content. In spite of the observed effects due to a higher endogenous cytokinin content in the IPT-HS plants, no significant changes were observed on the plasma membrane fatty acid composition, nor on its fluidity as determined from the steady-state fluorescence anisotropy of DPH. Only a minor change in the plasma membrane free sterol composition was found as evidenced by a 20% decrease in the stigmasterol to sitosterol ratio in IPT-HS, indicative for a possible anti-senescence effect of enhanced endogenous cytokinins, but without significant effects on the plasma membrane function.

Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves.

The plant plasma membrane (PM) contains more than one b-type cytochrome. One of these proteins has a rather high redox potential (can be fully reduced by ascorbate) and is capable of transporting electrons through the PM. Four genes encoding proteins with considerable homology to the sequences of cytochrome b(561) proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b(561) located in the Arabidopsis PM, first PM vesicles were purified by aqueous polymer two-phase partitioning from the leaves of 9-week-old A. thaliana. PM proteins were solubilized by nonionic detergent, and the fully ascorbate-reducible b-type cytochrome was partially purified by anion-exchange chromatography steps. Potentiometric redox titration of the fraction, containing the fully ascorbate-reducible b-type cytochrome after the first anion-exchange chromatography step, revealed the presence of two hemes with redox potentials of 135 mV and 180 mV, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing the fully ascorbate-reducible b-type cytochrome after the second anion-exchange chromatography step revealed the presence of a single polypeptide band at about 120 kDa. However, heat treatment (15 min, 90 degrees C) before electrophoresis was able to split the 120 kDa band into two bands with molecular masses of about 23 and 28 kDa. These values are lower than the apparent molecular mass for the fully ascorbate-reducible b-type cytochrome purified from Phaseolus vulgarishypocotyls (about 52 kDa) but are in good agreement with those characteristic for the cytochrome b(561) proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis genome (24-31 kDa).