The apoplastic antioxidant enzymatic system in the wood-forming tissues of trees

The complete apoplastic enzymatic antioxidant system, composed by class I ascorbate peroxidases (class I APXs), class III ascorbate peroxidases (class III APXs), ascorbate oxidases (AAOs), and other class III peroxidases (PRX), of wood-forming tissues has been studied in Populus alba, Citrus aurantium, and Eucalyptus camaldulensis. The aim was to ascertain whether these enzymatic systems may regulate directly (in the case of APXs), or indirectly (in the case of AAOs), apoplastic H₂O₂ levels in lignifying tissues, whose capacity to produce and to accumulate H₂O₂ is demonstrated here. Although class I APXs are particularly found in the apoplastic fraction of P. alba (poplar), and class III APXs are particularly found in the apoplastic fraction of C. aurantium (bitter orange tree), the results showed that the universal presence of AAO in the extracellular cell wall matrix of these woody species provokes the partial or total dysfunction of apoplastic class I and class III APXs, and of the whole plethora of non-enzymatic redox shuttles in which ascorbic acid (ASC) is involved, by the competitive and effective removal of ASC. In fact, the redox state (ASC/ASC+DHA) in intercellular wash fluids (IWFs) of these woody species was zero, and thus strongly shifted towards DHA (dehydroascorbate), the oxidized product of ASC. This imbalance of the apoplastic antioxidant enzymatic system apparently results in the accumulation of H₂O₂ in the apoplast of secondary wood-forming tissues, as can be experimentally observed. Furthermore, it is hypothesized that since AAO uses O₂ to remove ASC, it could regulate O₂ availability in the lignifying xylem and, thorough this mechanism, AAO could also control the activity of NADPH oxidase (the enzyme responsible for H₂O₂ production in lignifying tissues) at substrate level, by controlling the tension of O₂. That is, the presence of AAO in the extracellular cell wall matrix appears to be essential for finely tuning the oxidative performance of secondary wood-forming tissues.     

Methane emissions from six crop species exposed to three components of global climate change: temperature, ultraviolet-B radiation and water stress

The l ‐ascorbate (AsA) content and the expression of six l ‐galactose pathway‐related genes were analyzed in peach flesh during fruit development. Fluctuation of AsA during peach fruit development was divided into four phases based on the overall total AsA (T‐AsA) content per fruit: AsA I, 0–36 days after full bloom (DAFB); AsA II, 37–65 DAFB; AsA III, 66–92 DAFB and AsA IV, 93–112 DAFB. Phase AsA III was a lag phase for AsA accumulation, but did not coincide with the lag phase for fruit development. The T‐AsA concentration was highest at the early stage until 21 DAFB [2–3μmol per gram of fresh weight (g^?1 FW)], and decreased to 1/4 and 1/15 of this value at 50 and 92 DAFB, respectively. T‐AsA then remained at 0.15–0.20μmol g^?1 FW until harvest at 112 DAFB. More than 90% of the T‐AsA was in the reduced form until 21 DAFB. The proportion of reduced form of AsA then decreased concomitantly with the decrease in AsA concentration. To determine the main pathway of AsA biosynthesis and the AsA biosynthetic capacity of peach flesh, several precursors were incubated with immature whole fruit (59 DAFB). The AsA concentration increased markedly with l ‐galactono‐1,4‐lactone or l ‐galactose (Gal), but d ‐galacturonate and l ‐gulono‐1,4‐lactone failed to increase AsA, indicating dominance of the Gal pathway and potent AsA biosynthetic capabilities in immature peach flesh. The expression of genes involved in the last six steps of the Gal pathway was measured during fruit development. The genes studied included GDP‐d ‐mannose pyrophosphorylase (GMPH), GDP‐ d ‐mannose‐3′,5′‐epimerase (GME), GDP‐ l ‐galactose guanylyltransferase (GGGT), l ‐galactose‐1‐phosphate phosphatase (GPP), l ‐galactose‐1‐dehydrogenase (GDH) and l ‐galactono‐1,4‐lactone dehydrogenase (GLDH). GMPH, GME and GGGT had similar expression patterns that peaked at 43 DAFB. GPP, GDH and GLDH also had similar expression patterns that peaked twice at 21 and 91 DAFB, although the expression of GDH was quite low. High level of T‐AsA concentration was roughly correlated with the level of gene expression in the early period of fruit development (AsA I), whereas no such relationships were apparent in the other periods (e.g. AsA III and IV). On the basis of these findings, we discuss the regulation of AsA biosynthesis in peach fruit.     

Antioxidant defences of the apoplast

Summary The apoplast of barley and oat leaves contained superoxide dismutase (SOD), catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase activities. The activities of these enzymes in the apoplastic extracts were greatly modified 24 h after inoculation with the biotrophic fungal pathogenBlumeria graminis. The quantum efficiency of photosystem II, which is related to photosynthetic electron transport flux, was comparable in inoculated and healthy leaves during this period. Apoplastic soluble acid invertase activity was also modified in inoculated leaves. Inoculation-dependent increases in apoplastic SOD activity were observed in all lines. Major bands of SOD activity, observed in apoplastic protein extracts by activity staining of gels following isoelectric focusing, were similar to those observed in whole leaves but two additional minor bands were found in the apoplastic fraction. The apoplastic extracts contained substantial amounts of dehydroascorbate (DHA) but little or no glutathione (GSH). Biotic stress decreased apoplastic ascorbate and DHA but increased apoplastic GSH in resistant lines. The antioxidant cycle enzymes may function to remove apoplastic H₂O₂ with ascorbate and GSH derived from the cytoplasm. DHA and oxidized glutathione may be reduced in the apoplast or returned to the cytosol for rereduction.Abbreviations AA reduced ascorbate - APX ascorbate peroxidase - DHA dehydroascorbate (oxidised ascorbate) - DHAR dehydroascorbate reductase - G6PDH glucose-6-phosphate dehydrogenase - GSH reduced glutathione - GSSG glutathione disulphide - GR glutathione reductase - MDHA monodehydroascorbate - MDHAR monodehydroascorbate reductase - SOD superoxide dismutase     

Oxidative stress responses and shock proteins in the unicellular cyanobacterium Synechococcus R2 (PCC-7942)

Oxidative stress responses were tested in the unicellular cyanobacterium Synechococcus PCC 7942 (R2). Cells were exposed to hydrogen peroxide, cumene hydroperoxide and high light intensities. Activities of ascorbate peroxidase and catalase were correlated with the extent and time-course of oxidative stresses. Ascorbate peroxidase was found to be the major enzyme involved in the removal of hydrogen peroxide under the tested oxidative stresses. Catalase activity was inhibited in cells treated with high H₂O₂ concentrations, and was not induced under photo-oxidative stress. Regeneration of ascorbate in peroxide-treated cells was found to involve mainly monodehydroascorbate reductase and to a lesser extent dehydroascorbate reductase. The induction of the antioxidative enzymes was dependent on light and was inhibited by chloramphenicol. Peroxide treatment was found to induce the synthesis of eight proteins, four of which were also induced by heat shock.Abbreviations ASC ascorbate - DHA dehydroascorbate - MDA monodehydroascorbate - GSH reduced glutathione - GSSG oxidized glutathione - ASC Per ascorbate peroxidase - DHA red. dehydroascorbate reductase - MDA red. monodehydroascorbate reductase - GSSG red. glutathione reductase - HSP heat shock proteins - PSP peroxide shock proteins - C_m chloramphenicol     

Ascorbate-glutathione metabolism during PEG-induced water deficit in <Emphasis Type="Italic">Trifolium repens</Emphasis>

In order to elucidate the response of the ascorbate-glutathione (ASC-GSH) cycle to drought stress, the activities of antioxidant enzymes and the levels of molecules involved in the ASC-GSH metabolism were studied in Trifolium repens L. seedlings subjected to PEG-induced water deficit. Compared to the control, the contents of H₂O₂, thiobarbituric acid reactive substances (TBARS), ascorbate (ASC), dehydroascorbate (DHA), and glutathione disulfide (GSSG) increased in PEG-treated seedlings, whereas the glutathione (GSH) content kept constant during the drought period. Further more, the ASC/DHA and GSH/GSSG ratios decreased in the presence of PEG. Except for that of monodehydroascorbate reductase (MDHAR), the activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were up-regulated during water deficit, and the increases in APX and DHAR activities were much higher than those in GR activity. These data indicate that fluctuations in the ASC-GSH metabolism resulted from PEG treatment may have a positive effect on drought stress mitigation in T. repens.     

Rooting hastened in onions by ascorbate and ascorbate free radical

Treatment of onion bulbs with ascorbate or its free radical hastened root emergence on the basal plate in relation to treatments with water or dehydroascorbate. This stimulation was accompanied by a significant increase of DNA synthesis per primordium. After a 24-h imbibition, ascorbate and ascorbate free radical also increased cell length. Ascorbate and ascorbate free radical apparently activated the onset of cell proliferation in root primordia, resulting in a shortening in G₁-S transition. The possible action of the ascorbate system at the plasma membrane level is discussed.Abbreviations ASC ascorbic acid - AFR ascorbate free radical - DHA dehydroascorbate     

Detection and Isolation of Preformed Antifungal Compounds from the Peel of Pyrus bretschneideri Rehd. cv. Pingguoli at Different Stages of Maturity

Ethanol‐dichloromethane crude extract from peel of pear (Pyrus bretschneideri Rehd. cv. Pingguoli) was separated by thin layer chromatographic plates and bioassayed with conidia of Alternaria alternata. The inhibition zones differed significantly in retention factor (R_f) at expanding stage, harvest time and after 100 days of cold storage. The compounds in the inhibition zones were isolated and identified with gas chromatography and mass spectroscopy. Palmitate methyl, oleic acid methyl, linolenic acid methyl and squalene were present at all stages. The concentration of these chemicals was the highest in expanding stage fruit peel and decreased rapidly with fruit development. It is suggested that these compounds may be the main antifungal compounds in the growing fruit. The phthalate alkyl esters occurred at relatively higher concentrations in pear peel at harvest and after 100 days of cold storage. Six phthalate alkyl esters were identified from peel of pear fruit after 100 days of cold storage. It is also supposed that these esters may be the antifungal compounds in postharvest pear.     

Energized Uptake of Ascorbate and Dehydroascorbate From the Apoplast of Intact Leaves in Relation to Apoplastic Steady State Concentrations of Ascorbate

Abstract: Transport of ascorbate (AA) and dehydroascorbate (DHA) through the petiole into detached leaves of Lepidium sativum and other plant species via the transpiration stream, and energized uptake into leaf tissue, were measured indirectly by recording changes in membrane potential and apoplastic pH simultaneously with substrate‐stimulated respiration and transpiratory water loss. When 25 mM AA or DHA was fed to the leaves, steady state respiration at 25 °C was transiently increased by more than 50 % with AA and 70 % with DHA. Stimulation of respiration was accompanied by a transient breakdown of membrane potential followed by alkalinization of the leaf apoplast suggesting energized uptake at the expense of the transmembrane proton motive force. The average CO₂/AA ratio calculated from stimulated respiration during ascorbate uptake was 0.76 ± 0.26 (n = 17). The corresponding ratio for DHA was 1.38 ± 0.28 (n = 11). Far lower CO₂/substrate ratios were observed when NaCl or KCl were fed to leaves. The differences indicate either partial metabolism of AA and DHA in addition to energized transport, or less likely, higher energy requirement for transport of AA and DHA than for the inorganic salts. Maximum rates of energized AA transport into leaf tissue (deduced from maxima of extra respiration and calculated on the basis of CO₂/AA = 0.76) were close to 650 nmol m^‐2 leaf area s^‐1, i.e. far higher than most previously reported rates of transport. When the apoplastic concentration of AA was decreased below steady state levels during infiltration/centrifugation experiments, AA was released from leaf cells into the apoplast. This suggests that AA oxidation to DHA in the apoplast (as occurs during extracellular ozone detoxification) triggers energized transport of the DHA into the symplast and simultaneously AA release from the symplast into the apoplast, perhaps together with protons in a reversal of the energized uptake process.     

Metamorphic changes in localization of sugars in skin of the leopard frog,Rana pipiens

A lectin histochemical study was carried out to determine the distribution of specific sugars in glycoconjugates within an important osmoregulatory organ, amphibian skin. Paraffin sections were made of Rana pipiens skin from dorsal and ventral regions of aquatic larvae in representative developmental stages as well as from several body regions of semiaquatic adult frogs. Sections were incubated with horseradish peroxidase (HRP)‐conjugated lectins, which bind to specific terminal sugar residues of glycoconjugates. Such sites were visualized by DAB‐H₂O₂. The following HRP‐lectins were used: UEA‐1 for α‐L ‐fucose, SBA for N‐acetyl‐D ‐galactosamine, WGA for N‐acetyl‐β‐D ‐glucosamine, PNA for β‐galactose, and Con A for α‐mannose. We found that lectin binding patterns in larvae change during metamorphic climax as the skin undergoes extensive histological remodeling; this results in adult skin with staining patterns that are specific for each lectin and are similar in all body regions. Such findings in R. pipiens provide additional insight into the localization of molecules involved in osmoregulation in amphibian skin. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Stable Form of Ascorbate Peroxidase from the Red Alga Galdieria partita Similar to Both Chloroplastic and Cytosolic Isoforms of Higher Plants

Depletion of the electron donor ascorbate causes rapid inactivation of chloroplastic ascorbate peroxidase (APX) of higher plants, while cytosolic APX is stable under such conditions. Here we report the cloning of cDNA from Galdieria partita, a unicellular red alga, encoding a novel type of APX (APX-B). The electrophoretic mobility, K _m values, k _cat and absorption spectra of recombinant APX-B produced in Escherichia coli were measured. Recombinant APX-B remained active for at least 180 min after depletion of ascorbate. The amino-terminal half of APX-B, which forms the distal pocket of the active site, was richer in amino acid residues conserved in chloroplastic APXs of higher plants rather than cytosolic APXs. In contrast, the sequence of the carboxyl-terminal half, which forms the proximal pocket, was similar to that of the cytosolic isoform. The stability of APX-B might be due to its cytosolic isoform-like structure of the carboxyl-terminal half.     

Transport of ascorbate into protoplasts ofNicotiana tabacum Bright Yellow-2 cell line

Summary The uptake of ascorbate into protoplasts isolated from aNicotiana tabacum Bright Yellow-2 (BY-2) cell suspension culture was investigated. Addition of¹⁴C-labelled ascorbate to freshly isolated protoplasts resulted in a time- and substrate-dependent association of radioactive molecules with the protoplasts. The kinetic characterisation of this presumptive uptake revealed kinetics of Michaelis-Menten type with an apparent maximal uptake activity of 24 pmol/min·10⁶ protoplasts and an apparent affinity constant of 139 M. The amount of ascorbate molecules transported into_{N. tabacum} protoplasts decreased when nonlabelled dehydroascorbate or iso-ascorbate were added but was not affected by addition of 5,6-o-cyclohexylidene ascorbate or ascorbate-2-sulfate. These data indicate a carrier-mediated uptake of ascorbate into the protoplasts that shows a high structural specificity. To investigate which redox status of ascorbate is preferentially taken up by theN. tabacum protoplasts, transport was tested in the presence of various compounds that can affect the redox status of ascorbate. Testing uptake in the presence of a reductant, dithiothreitol, resulted in a significant and concentration-dependent inhibition of the amount of ascorbate molecules transported into the protoplasts. On the other hand, ascorbate uptake was significantly stimulated in the presence of the enzyme ascorbate oxidase. Ferricyanide did not affect ascorbate transport. Inhibition studies revealed that ascorbate uptake in the protoplasts is sensitive to addition of sulfhydryl reagents N-ethyl maleimide andp-chloro-mercuribenzenesulfonic acid and to a disruption of the proton gradient by the protonophore carbonylcyanide-3-chlorophenylhydrazone. The uptake of ascorbate was also inhibited by addition of cytochalasin B but not sensitive to addition of phloretin or sulfinpyrazone. Taken together these data indicate the presence of an ascorbate transport system in the plasma membrane ofN. tabacum protoplasts and suggest dehydroascorbate as the preferentially transported redox species. The putative presence of different carriers for reduced and oxidised ascorbate in the plasma membrane is discussed.Abbreviations Asc ascorbate - BY-2 Bright Yellow 2 - CCCP carbonylcyanide-3-chlorophenylhydrazone - DHA dehydroascorbate - DTT dithiothreitol - MS medium Murashige and Skoog medium - NEM N-ethylmaleimide - pCMBS p-chloromercuribenzenesulfonic acid     

Engineering the acceptor specificity of trehalose phosphorylase for the production of trehalose analogs

Trehalose (α‐D ‐glucopyranosyl‐(1,1)‐α‐D ‐glucopyranoside) is widely used in the food industry, thanks to its protective effect against freezing and dehydration. Analogs of trehalose have the additional benefit that they are not digested and thus do not contribute to our caloric intake. Such trehalose analogs can be produced with the enzyme trehalose phosphorylase, when it is applied in the reverse, synthetic mode. Despite the enzyme's broad acceptor specificity, its catalytic efficiency for alternative monosaccharides is much lower than for glucose. For galactose, this difference is shown here to be caused by a lower K_m whereas the k_cat for both substrates is equal. Consequently, increasing the affinity was attempted by enzyme engineering of the trehalose phosphorylase from Thermoanaerobacter brockii, using both semirational and random mutagenesis. While a semirational approach proved unsuccessful, high‐throughput screening of an error‐prone PCR library resulted in the discovery of three beneficial mutations that lowered K_m two‐ to three‐fold. In addition, it was found that mutation of these positions also leads to an improved catalytic efficiency for mannose and fructose, suggesting their involvement in acceptor promiscuity. Combining the beneficial mutations did not further improve the affinity, and even resulted in a decreased catalytic activity and thermostability. Therefore, enzyme variant R448S is proposed as new biocatalyst for the industrial production of lactotrehalose (α‐D ‐glucopyranosyl‐(1,1)‐α‐D ‐galactopyranoside). © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Substrate specificity of a recombinant d‐lyxose isomerase from Serratia proteamaculans that produces d‐lyxose and d‐mannose

Aims: Characterization of substrate specificity of a d ‐lyxose isomerase from Serratia proteamaculans and application of the enzyme in the production of d ‐lyxose and d ‐mannose. Methods and Results: The concentrations of monosaccharides were determined using a Bio‐LC system. The activity of the recombinant protein from Ser. proteamaculans was the highest for d ‐lyxose among aldoses, indicating that it is a d‐ lyxose isomerase. The native recombinant enzyme existed as a 54‐kDa dimer, and the maximal activity for d‐ lyxose isomerization was observed at pH 7·5 and 40°C in the presence of 1 mmol l^?1 Mn²⁺. The K_m values for d ‐lyxose, d ‐mannose, d ‐xylulose, and d ‐fructose were 13·3, 32·2, 3·83, and 19·4 mmol l^?1, respectively. In 2 ml of reaction volume at pH 7·5 and 35°C, d ‐lyxose was produced at 35% (w/v) from 50% (w/v) d ‐xylulose by the d‐ lyxose isomerase in 3 h, while d ‐mannose were produced at 10% (w/v) from 50% (w/v) d ‐fructose in 5 h. Conclusions: We identified the putative sugar isomerase from Ser. proteamaculans as a d ‐lyxose isomerase. The enzyme exhibited isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left‐hand configuration. High production rates of d‐ lyxose and d ‐mannose by the enzyme were obtained. Significance and Impact of the Study: A new d‐ lyxose isomerase was found, and this enzyme had higher activity for d ‐lyxose and d ‐mannose than previously reported enzymes. Thus, the enzyme can be applied in industrial production of d ‐lyxose and d ‐mannose.     

Protoplast isolation and culture from carob (Ceratonia siliqua) hypocotyls: ability of regenerated protoplasts to produce mannose-containing polysaccharides

The aim of this study was to isolate protoplasts from carob (Ceratonia siliqua L.) embryonic tissues with the ability to regenerate cell walls, divide and synthesize galactomannan, a valuable polysaccharide for industry. Protoplasts isolated from carob hypocotyl hooks regenerated cell walls within 24 h. The first divisions of the regenerated cells were observed after 2 days of culture. The highest percentage that successfully divided was achieved when the seedlings were grown under diffuse light, the hypocotyl hooks were plasmolysed for 1 h before incubation in the protoplast isolation solution and the protoplasts were cultured under diffuse light. After 9 days of culture, cell clusters, consisting of eight cells, had been produced, which underwent further mitotic divisions and which were expected to lead to callus formation. Polysaccharide and oligosaccharide synthesis during protoplast regeneration was studied by radiolabelling with exogenous d ‐[U‐¹⁴C]glucose, d ‐[U‐¹⁴C]mannose or d ‐[2‐³H]mannose, which gave rise to uniform, moderately specific and highly specific labelling, respectively. As revealed by the radioactivity distribution in cell wall monosaccharides, the regenerants deposited new wall polymers that differed markedly from those being synthesized by the hypocotyls from which the protoplasts had been isolated. The regenerants deposited large amounts of callose and smaller amounts of galactose‐, arabinose‐ and mannose‐containing polymers. The latter included glucuronomannan, as demonstrated by a new method involving partial acid hydrolysis followed by β‐glucuronidase (EC 3.2.1.31) digestion. The regenerating protoplasts also released soluble extracellular carbohydrates: polysaccharides which appeared to be mainly acidic arabinogalactans, and oligosaccharides which were mainly neutral and contained glucose, galactose and mannose. We conclude that regenerating carob protoplasts are a useful system for studying carbohydrate secretion, including mannose‐rich poly‐ and oligosaccharides.     

Differential Implication of Glutathione, Glutathione-Metabolizing Enzymes and Ascorbate in Tomato Resistance to Pseudomonas syringae

We studied the response of glutathione‐ and ascorbate‐related antioxidant systems of the two tomato cultivars to Pseudomonas syringae pv. tomato infection. In the inoculated susceptible A 100 cultivar a substantial decrease in reduced glutathione (GSH) content, oxidised glutathione accumulation and GSH redox ratio decline as well as glutathione peroxidase activity increase were found. The enhanced glutathione reductase activity was insufficient to keep the glutathione pool reduced. A transiently increased dehydroascorbic acid (DHA) content and ascorbic acid (AA) redox ratio decrease together with ascorbate peroxidase activity suppression were observed. Adversely to the progressive reduction in GSH pool size, AA content tended to increase but the changes were more modest than those of GSH. By contrast, in interaction with the resistant Ontario cultivar the glutathione pool homeostasis was maintained throughout P. syringae attack and no significant effect on the ascorbate pool was observed. Moreover, in the resistant interaction there was a significantly higher constitutive and pathogen‐induced glutathione‐S‐transferase (GST) activity. The relationship between GST activity and DHA content found in this study indicates that this enzyme could also act as dehydroascorbate reductase. These results reflect the differential involvement of GSH and AA in tomato‐P. syringae interaction and, in favour of the former, they clearly indicate the role of GSH and GSH‐utilizing enzymes in resistance to P. syringae. The maintenance of glutathione pool homeostasis and GST induction appear to contribute to tissue inaccessibility to bacterial attack.     

Effect of Selenium on Ascorbate�CGlutathione Metabolism During PEG-induced Water Deficit in Trifolium repens L.

To elucidate the effect of selenium (Se) on the ascorbate?Cglutathione (ASC?CGSH) cycle under drought stress, the activities of antioxidant enzymes and the levels of molecules involved in ASC?CGSH metabolism were studied in Trifolium repens seedlings subjected to polyethylene glycol (PEG)-induced water deficit alone or combined with 5???M Na₂SeO₄. Compared to the control, H₂O₂, thiobarbituric acid reactive substances (TBARS), ascorbate (ASC), dehydroascorbate (DHA), and glutathione disulfide (GSSG) contents increased, whereas a constant content of glutathione (GSH) and decreases in ASC/DHA and GSH/GSSG ratios were observed in the presence of PEG. The activities of ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) were upregulated, except for monodehydroascorbate reductase (MDHAR) activity during PEG-induced water deficit. Se application decreased the contents of H₂O₂, TBARS, DHA, and GSSG, increased the levels of GSH and ASC, and inhibited the decreases of ASC/DHA and GSH/GSSG ratios. Although it did not affect APX activity significantly, Se addition improved the activities of MDHAR, DHAR, and GR. Furthermore, GR activity showed the highest increase followed by that of DHAR and MDHAR in decreasing order. These data indicated that fluctuations in ASC?CGSH metabolism resulting from Se may have a positive effect on drought stress mitigation, and the regulation in the ASC?CGSH cycle can be attributed mainly to GR and DHAR in PEG?+?Se-treated T. repens seedlings.     

Transport of ascorbate into plasma membrane vesicles ofPhaseolus vulgaris L.

Summary Incubation of bean hook plasma membrane vesicles in the presence of L-[¹⁴C]ascorbate (ASC) resulted in a specific recovery of significant levels of the ligand with the vesicles. The strong decrease in radioactive ASC detected after hypotonic disruption of the vesicles or after an assay at 4 °C indicated that ASC was probably transported from the medium into the lumen of the membrane vesicles. The concentration kinetics of this presumptive transport process revealed a saturation curve which best fitted a biphasic model. Each phase in this model showed Michaelis-Menten type kinetics. The kinetic parameters for the different phases were calculated to be 14 and 79 M (K _m1 andK _m2) and 26 and 53 pmol/min · mg protein (V _max1 andV _max2). High concentrations of iso-ascorbate, dehydroascorbate (DHA) or non-labelled ASC significantly reduced the uptake of the radioactive vitamin. It was demonstrated that sugar or amino acid carriers are not involved in the ASC transport reaction. Generation of transmembrane cation gradients (H⁺, K⁺, Ca²⁺, Na⁺) or addition of sulfhydryl reagents (pCMBS or NEM) did not affect the ASC uptake in any way. It is suggested that ASC is taken up by a facilitated diffusion mechanism.Abbreviations ASC ascorbate - DHA dehydroascorbate - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - NEM N-ethylmaleimide - pCMBS p-chloromercuribenzenesulfonic acid