Dehydroascorbate uptake activity correlates with cell growth and cell division of tobacco bright yellow-2 cell cultures

Recently, ascorbate (ASC) concentration and the activity of a number of enzymes from the ASC metabolism have been proven to correlate with differences in growth or cell cycle progression. Here, a possible correlation between growth and the activity of a plasma membrane dehydroascorbate (DHA) transporter was investigated. Protoplasts were isolated from a tobacco (Nicotiana tabacum) Bright Yellow-2 cell culture at different intervals after inoculation and the activity of DHA transport was tested with (14)C-labeled ASC. Ferricyanide (1 mM) or dithiothreitol (1 mM) was included in the test to keep the external (14)C-ASC in its oxidized respectively reduced form. Differential uptake activity was observed, correlating with growth phases of the cell culture. Uptake of DHA in cells showed a peak in exponential growth phase, whereas uptake in the presence of dithiothreitol did not. The enhanced DHA uptake was not due to higher endogenous ASC levels that are normally present in exponential phase because preloading of protoplasts of different ages did not affect DHA uptake. Preloading was achieved by incubating cells before protoplastation for 4 h in a medium supplemented with 1 mM DHA. In addition to testing cells at different growth phases, uptake of DHA into the cells was also followed during the cell cycle. An increase in uptake activity was observed during M phase and the M/G1 transition. These experiments are the first to show that DHA transport activity into plant cells differs with cell growth. The relevance of the data to the action of DHA and ASC in cell growth will be discussed.     

Glucose 6-phosphate plays a central role in the regulation of glycogen synthesis in a glycogen-storing liver cell line

Two substrains of the epithelial liver cell line C1I, one storing large amounts of glycogen, the other one being very poor in glycogen were used as a model for studying glycogen synthesis. The glycogen content of glycogen-rich cells doubled during the proliferative phase and remained high in plateau phase although glycogen synthase I activity was not significantly altered during growth cycle and was too low to account for the increase in glycogen. However, the activity of the glucose 6-phosphate (Glc6-P)-dependent synthase rose continuously during growth cycle, and intracellular Glc6-P-concentration increased about 10-fold in log phase cells to 0.72 mumol g-1 wet weight. A0.5 of synthase for Glc6-P was 0.79 mM. It was also found that in contrast to the enzyme from normal liver, glycogen phosphorylase a from C1I cells was inhibited by Glc6-P, the apparent Ki being 0.45 mM. It was concluded that glycogen accumulation in C1I cells was due to stimulation of synthase and inhibition of phosphorylase by Glc6-P. Findings from the glycogen-poor cell line which revealed similar specific activities of synthase and phosphorylase but only low Glc6-P (0.056 mumol g-1 wet weight) supported this conclusion. Addition of glucose to starved cells resulted in a transient activation of synthase in both cell lines. Net glycogen synthesis, was, however, only observed in the cells with a high Glc6-P-content. Thus, modulation of synthase and phosphorylase by Glc6-P and not activation/inactivation of the enzymes seems to play a predominant role in glycogen accumulation in this cell line.     

Ascorbate transport from the apoplast to the symplast in intact leaves

Infiltration of reduced ascorbate (ASC) into the leaves of Betula pendula Roth and subsequent measurement of its loss therein after incubation allowed us to follow ascorbate transport from apoplast to symplast in intact leaves. All of the ascorbate extracted from the native apoplast was in fully oxidized form, dehydroascorbate (DHA). When 5 m M of ASC was infiltrated into the leaves, its intense decay occurred, but only 55% of ASC lost was recovered in apoplast as DHA. When ASC was added to the freshly extracted intercellular washing fluid (IWF), ASC oxidation occurred as well. However, all oxidized ASC was recovered as DHA, indicating that further decomposition of DHA did not occur. Similarly, all of the ASC infiltrated into the leaves was found therein either as ASC or DHA after incubation of leaves for up to 60 min. On this base the ascorbate infiltrated into the leaves and not recovered in the IWF was interpreted as ascorbate taken up into the symplast. The calculated uptake rates of ascorbate at different ASC concentrations followed saturation kinetics with the maximum uptake rate of 300 nmol m⁻² plasma membrane (PM) area min⁻¹ and Michaelis constant of 12.8 m M . The uptake of ascorbate was significantly inhibited by the addition of dithiothreitol or by PM H⁺ ATPase inhibitor erythrosin B. Thus, our results support the previous observations that DHA is preferably transported from the apoplastic to the cytoplasmic side of the membrane and show that this process is dependent upon PM proton gradient.     

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.     

The application of ascorbate or its immediate precursor, galactono-1,4-lactone, does not affect the response of nitrogen-fixing pea nodules to water stress

Nitrogen fixation in legumes is dramatically inhibited by abiotic stresses, and this reduction is often associated with oxidative damage. Although ascorbate (ASC) has been firmly associated with antioxidant defence, recent studies have suggested that the functions of ASC are related primarily to developmental processes. This study examines the hypothesis that ASC is involved in alleviating the oxidative damage to nodules caused by an increase in reactive oxygen species (ROS) under water stress. The hypothesis was tested by supplying 5 mM ASC to pea plants (Pisum sativum L.) experiencing moderate water stress (ca. −1 MPa) and monitoring plant responses in relation to those experiencing the same water stress without ASC. A supply of exogenous ASC increased the nodule ASC+dehydroascorbate (DHA) pool compared to water-stressed nodules without ASC, and significantly modulated the response to water stress of the unspecific guaiacol peroxidase (EC 1.11.1.7) in leaves and nodules. However, ASC supply did not produce recovery from water stress in other nodule antioxidant enzymes, nodule carbon and nitrogen enzymes, or nitrogen fixation. The supply of the immediate ASC precursor, galactono-1,4-lactone (GL), increased the nodule ASC+DHA pool, but also failed to prevent the decline of nitrogen fixation and the reduction of carbon flux in nodules. These results suggest that ASC has a limited role in preventing the negative effects of water stress on nodule metabolism and nitrogen fixation.     

A mechanism accounting for the low cellular level of linoleic acid in cystic fibrosis and its reversal by DHA

Specific fatty acid alterations have been described in the blood and tissues of cystic fibrosis (CF) patients. The principal alterations include decreased levels of linoleic acid (LA) and docosahexaenoic acid (DHA). We investigated the potential mechanisms of these alterations by studying the cellular uptake of LA and DHA, their distribution among lipid classes, and the metabolism of LA in a human bronchial epithelial cell model of CF. CF (antisense) cells demonstrated decreased levels of LA and DHA compared with wild type (WT, sense) cells expressing normal CFTR. Cellular uptake of LA and DHA was higher in CF cells compared with WT cells at 1 h and 4 h. Subsequent incorporation of LA and DHA into most lipid classes and individual phospholipids was also increased in CF cells. The metabolic conversion of LA to n-6 metabolites, including 18:3n-6 and arachidonic acid, was upregulated in CF cells, indicating increased flux through the n-6 pathway. Supplementing CF cells with DHA inhibited the production of LA metabolites and corrected the n-6 fatty acid defect. In conclusion, the evidence suggests that low LA level in cultured CF cells is due to its increased metabolism, and this increased LA metabolism is corrected by DHA supplementation.     

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     

Preferential use of glucose during diauxic growth of Carrot cells growing on glucose and malate

Suspension cells of carrot plants grown on mixed carbon sources of glucose (Glc) and malate preferentially used Glc. The cells started to utilize malate only after Glc was depleted from the medium, thus exhibiting a diauxic growth. The residual concentration of Glc decreased rapidly during the first growth phase, and that of malate decreased only during the second growth phase. Malate uptake was negligible throughout the diauxic growth, suggesting that malate was being utilized via another metabolite. An active metabolic flow from fumarate to pyruvate and oxaloacetate via malate was induced in cells during the second growth phase. These results strongly suggested that malate remained unused in the medium in the first phase, and in the second phase it was converted extracellularly into fumarate, which was subsequently transported into cells and metabolized into malate and further into pyruvate and oxaloacetate. This study presents the second case of diauxic growth in plants and the peculiar mode of malate utilization.     

Localization of ascorbic acid, ascorbic acid oxidase, and glutathione in roots of Cucurbita maxima L

To understand the function of ascorbic acid (ASC) in root development, the distribution of ASC, ASC oxidase, and glutathione (GSH) were investigated in cells and tissues of the root apex of Cucubita maxima. ASC was regularly distributed in the cytosol of almost all root cells, with the exception of quiescent centre (QC) cells. ASC also occurred at the surface of the nuclear membrane and correspondingly in the nucleoli. No ASC could be observed in vacuoles. ASC oxidase was detected by immunolocalization mainly in cell walls and vacuoles. This enzyme was particularly abundant in the QC and in differentiating vascular tissues and was absent in lateral root primordia. Administration of the ASC precursor L-galactono-gamma-lactone markedly increased ASC content in all root cells, including the QC. Root treatment with the ASC oxidized product, dehydroascorbic acid (DHA), also increased ASC content, but caused ASC accumulation only in peripheral tissues, where DHA was apparently reduced at the expense of GSH. The different pattern of distribution of ASC in different tissues and cell compartments reflects its possible role in cell metabolism and root morphogenesis.     

The ascorbate system in recalcitrant and orthodox seeds

Recalcitrant seeds of Ginkgo biloba L., Quercus cerris L., Aesculus hippocastanum L. and Cycas revoluta Thunb. are shed by the plant at a high moisture content, contain a large amount of ascorbic acid (ASA) and maintain high ascorbate (ASC) peroxidase (EC 1.11.1.11) activity. Three proteins showing ASC peroxidase activity are present in G. biloba seeds. Conversely, dry orthodox seeds ( Vicia faba L., Avena sativa L., Pinus pinea L.) are completely devoid of ASA and ASC peroxidase. Experimentally induced rapid variations of the water level in both recalcitrant and orthodox seeds do not affect the ASC peroxidase; slow dehydration affects the ASC peroxidase activity moderately in recalcitrant seeds, but provokes a complete loss of germinability. Another peculiar difference between orthodox and recalcitrant seeds concerns the ascorbate recycling enzymes, ascorbate free radical (AFR) reductase (EC 1.6.5.4) and dehydroascorbate (DHA) reductase (EC 1.8.5.1). The DHA reduction capability is low in recalcitrant seeds, but is high in the orthodox ones. In contrast, AFR reductase activity is high in recalcitrant seeds and low in the orthodox ones. Data reported here concerning the ASC system appear to contribute to better understanding the recalcitrance. The presence of three different proteins showing ASC peroxidase activity in the archaic seed-bearing plant G. biloba and its involvement in the spermatophyte evolution is discussed.     

Zonal changes in ascorbate and hydrogen peroxide contents,peroxidase, and ascorbate-related enzyme activities in onion roots

Onion (Allium cepa) roots growing hydroponically show differential zonal values for intra- (symplastic) and extra- (apoplastic) cellular ascorbate (ASC) and dehydroascorbate (DHA) contents and for related enzyme activities. In whole roots, ASC and DHA concentrations were higher in root apex and meristem and gradually decreased toward the root base. Guaiacol peroxidase, ASC peroxidase, monodehydroascorbate oxidoreductase, DHA reductase, catalase, and glutathione reductase activities showed differential activity patterns depending on the zone of the root and their apoplastic or symplastic origin. An in vivo staining of peroxidase activity also revealed a specific distribution pattern along the root axis. Using electron microscopy, hydrogen peroxide was found at different locations depending on the root zone but was mainly located in cell walls from epidermal and meristematic cells and in cells undergoing lignification. A balanced control of all of these molecules seems to exist along the root axis and may be directly related to the mechanisms in which the ASC system is involved, as cell division and elongation. The role of ASC on growth and development in relation to its presence at the different zones of the root is discussed.     

The redox state of the ascorbate-dehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells

The effects of ascorbate (ASC) and dehydroascorbate (DHA) on cell proliferation were examined in the tobacco Bright Yellow 2 (TBY-2) cell line to test the hypothesis that the ASC-DHA pair is a specific regulator of cell division. The hypothesis was tested by measuring the levels of ASC and DHA or another general redox pair, glutathione (GSH) and glutathione disulfide (GSSG), during the exponential-growth phase of TBY-2 cells. A peak in ASC, but not GSH, levels coincided with a peak in the mitotic index. Moreover, when the cells were enriched with ascorbate, a stimulation of cell division occurred whereas, when the cells were enriched with DHA, the mitotic index was reduced. In contrast, glutathione did not affect the mitotic-index peak during this exponential-growth phase. The data are consistent in showing that the ASC-DHA pair acts as a specific redox sensor which is part of the mechanism that regulates cell cycle progression in this cell line.     

The redox state of the ascorbate-dehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells

Summary The effects of ascorbate (ASC) and dehydroascorbate (DHA) on cell proliferation were examined in the tobacco Bright Yellow 2 (TBY-2) cell line to test the hypothesis that the ASC-DHA pair is a specific regulator of cell division. The hypothesis was tested by measuring the levels of ASC and DHA or another general redox pair, glutathione (GSH) and glutathione disulfide (GSSG), during the exponential-growth phase of TBY-2 cells. A peak in ASC, but not GSH, levels coincided with a peak in the mitotic index. Moreover, when the cells were enriched with ascorbate, a stimulation of cell division occurred whereas, when the cells were enriched with DHA, the mitotic index was reduced. In contrast, glutathione did not affect the mitotic-index peak during this exponential-growth phase. The data are consistent in showing that the ASC-DHA pair acts as a specific redox sensor which is part of the mechanism that regulates cell cycle progression in this cell line.     

Preferential Use of Acetate over Glucose Involves Acetate-Mediated Inhibition of Glucose Uptake during Diauxic Growth of Carrot Cells

It has been reported that suspension-cultured rice cells grownon mixed carbon sources of glucose (Glc) and acetate exhibiteddiauxic growth in which acetate was the preferred carbon source(Lee and Lee 1996). Carrot (Daucus carota L.) suspension cells,showing a diauxic growth very similar to that of rice cells,were used to delineate the mechanisms underlying this preferentialuse of acetate over Glc. Uptakes of both Glc and 3-O-methylglucose(3-OMG), a non-metabolizable Glc analogue, were similarly inhibitedwhen acetate or butylate, weak acids which are capable of transportingprotons into the cytosol, were present in the uptake assay mixturecontaining cells harvested during the Glc-utilizing second growthphase. Inhibition of Glc uptake by these weak acids was similarwhen equivalent experiments were carried out with isolated plasmamembranes. It was further shown that Glc uptake, which requiresa proper proton gradient across the plasma membranes, was inhibitedduring the first growth phase by acetate-mediated alkalizationof growth medium and/or simultaneous acidification of cytosol.This study strongly suggests that Glc utilization in plant cellsis inhibited by co-presenting carbon source(s) which can alterthe proton gradient across the plasma membrane. (Received April 1, 1999; Accepted July 23, 1999)     

Mechanical Force and Cytoplasmic Ca2+ Activate Yeast TRPY1 in Parallel

Diets supplemented with n-3 polyunsaturated fatty acids can promote lipid peroxidation and the propagation of oxygen radicals. These effects can be prevented by taurine, a functional ingredient with antioxidant properties. Here, we examined whether there is a correlation between transepithelial taurine transport, on the one hand, and membrane fatty acid composition and peroxidation in intestinal Caco-2 cells, on the other. Differentiated Caco-2 cells were maintained for 10 days, from the day of confluence, in control conditions or in a medium enriched with docosahexaenoic acid (DHA, 100 μmol/l), taurine (10 mmol/l) or DHA plus taurine. Incubation of the monolayers in a medium enriched with DHA increased the incorporation of this fatty acid into the brush-border membrane, at the expense of total n-6 fatty acids (C20:2n-6, C20:3n-6 and C22:4n-6). This was paralleled by increased membrane lipid peroxidation, which was partially limited by the addition of taurine. Transepithelial taurine transport was estimated from taurine uptake and efflux kinetic parameters at apical and basolateral domains. Cell incubation with DHA increased basolateral taurine uptake through an increase in V _max, whereas incubation with taurine downregulated basolateral uptake as occurred for apical taurine transporter. Moreover, addition of DHA reduced the apical downregulation effect exerted on taurine transport by taurine incubation. Our results suggest that the oxidative status of epithelial cells regulates taurine transport, thus satisfying antioxidant cellular requirements.     

Influence of Drought-Induced Water Stress on Soybean and Spinach Leaf Ascorbate-Dehydroascorbate level and Redox Status

We examined the influence of water stress (water deficit) induced by drought on the steady state levels of ascorbic acid (ASC), dehydroascorbate (DHA), and the ASC&rcolon;DHA redox status in leaflets of Glycine max (soybean) and leaves of Spinacia oleracea (spinach). Two soybean cultivars (cv. Essex and cv. Forrest) and one spinach cultivar (cv. Nordic) were grown in high-light growth chambers ( approximately 1000-1200 μmol m-2 s-1) or in the greenhouse during May, June, and July 1999. The cultivars were supplied with water until approximately 25-29 d postemergence, at which time one-half of the plants were not watered for a period of from 4.5 to 7.5 d; the other half of the plants were provided water daily and served as controls. On designated days, leaf water potential (PsiLeaf) was measured, and leaf disks of constant area were excised in the period between approximately 1230 and 1330 hours. Leaf disk samples were immediately frozen in liquid N2, samples were extracted, and ASC and DHA levels were measured and expressed as μmol per gram dry mass per time point. For the soybean cultivars, low PsiLeaf values ( approximately -3.00 to -3.95 MPa) were accompanied by slight decreases in ASC levels and slight increases in DHA levels per gram dry mass. In some cases, leaflet ASC levels of water-stressed soybeans were similar to controls or were even increased by as much as 1.2 times. In soybeans, the mole fraction of ASC remained at 93-99 mol% of the total ascorbate (ASC+DHA), indicating that most of the total ascorbate remained in the reduced form even at low water potential. In spinach plants subjected to water stress (-1.8 to -2.6 MPa), leaf ASC decreased as much as 38%, but the ASC remained at 96-99 mol% of the total ascorbate. It is concluded that during water stress, enzymes of the ascorbate-glutathione cycle in leaf mesophyll cells, as well as in the system that generates reductant to support DHA to ASC recycling, e.g., photosynthetic electron transport in chloroplasts, is able to remain active enough to maintain reduction of DHA to ASC.     

Dehydroascorbate influences the plant cell cycle through a glutathione-independent reduction mechanism

Glutathione is generally accepted as the principal electron donor for dehydroascorbate (DHA) reduction. Moreover, both glutathione and DHA affect cell cycle progression in plant cells. But other mechanisms for DHA reduction have been proposed. To investigate the connection between DHA and glutathione, we have evaluated cellular ascorbate and glutathione concentrations and their redox status after addition of dehydroascorbate to medium of tobacco (Nicotiana tabacum) L. cv Bright Yellow-2 (BY-2) cells. Addition of 1 mm DHA did not change the endogenous glutathione concentration. Total glutathione depletion of BY-2 cells was achieved after 24-h incubation with 1 mm of the glutathione biosynthesis inhibitor l-buthionine sulfoximine. Even in these cells devoid of glutathione, complete uptake and internal reduction of 1 mm DHA was observed within 6 h, although the initial reduction rate was slower. Addition of DHA to a synchronized BY-2 culture, or depleting its glutathione content, had a synergistic effect on cell cycle progression. Moreover, increased intracellular glutathione concentrations did not prevent exogenous DHA from inducing a cell cycle shift. It is therefore concluded that, together with a glutathione-driven DHA reduction, a glutathione-independent pathway for DHA reduction exists in vivo, and that both compounds act independently in growth control.