Upregulation of gamma-glutamate-cysteine ligase as part of the long-term adaptation process to iron accumulation in neuronal SH-SY5Y cells

Reactive iron is an important prooxidant factor, whereas GSH is a crucial component of a long-term adaptive system that allows cells to function during extended periods of high oxidative stress. In this work, the adaptive response of the GSH system to prolonged iron loads was characterized in human dopaminergic SH-SY5Y neuroblastoma cells. After the initial death of a substantial portion of the cell population, the surviving cells increased their GSH content by up to fivefold. This increase was traced to increased expression of the catalytic and modulatory subunits of -glutamate-cysteine ligase. Under conditions of high iron load, cells maintained a low GSSG content through two mechanisms: 1) GSSG reductase-mediated recycling of GSSG to GSH and 2) multidrug resistant protein 1-mediated extrusion of GSSG. Increased GSH synthesis and low GSSG levels contributed to recover the cell reduction potential from –290 mV at the time of cell death to about –320 mV. These results highlight the fundamental role of GSH homeostasis in the antioxidant response to cellular iron accumulation and provide novel insights into the adaptive mechanisms of neurons subjected to increased iron loads, such as those observed in Parkinson's disease. oxidative stress; glutathione; multidrug resistance protein 1; oxidixed glutathione reductase; neurodegenerative diseases     

Modelling Temperature Effects in Breaking Rest in Red-osier Dogwood (Cornus sericea L.)

Temperature requirements for bud development after a rest period(breaking rest) from maximum rest to end of rest were determinedto develop an empirical model for predicting rest developmentin terminal vegetative buds of red-osier dogwood (Cornus sericeaL.). One-year-old plants at maximum rest were exposed to temperaturesfrom 5 to 20 °C a 12 h photoperiod (SD) in growth chambers.Depth of rest was measured by days to bud break in either 16h photoperiod (LD) or natural daylength at 20/15 °C/nighttemperature. Developmental stages during rest development wereexpressed by degree growth stage (°GS). Chilling was effective breaking rest after plants attained maximumrest (270 °GS). Development during rest (breaking rest)increased with decreasing temperature. No significant developmentoccurred at 20 °C. Rate of rest development (°GS h^–1)at all temperatures varied during the breaking rest period anddepended on developmental stage (°GS). A °GS model describedand quantified rest development (°GS). Using temperatureand developmental stage, the model predicted end of rest (315°GS)within 3 days and daily rest development (°GS) in both years. Cornus sericea L, Cornus stolonifera Michx, dogwood, bud development, dormancy, temperature effects, chilling, degree growth stage     

Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.     

Temporal changes of multiple redox couples from proliferation to growth arrest in IEC-6 intestinal epithelial cells

Changes in intracellular redox couples and redox reactive molecules have been implicated in the regulation of a variety of cellular processes, including cell proliferation and growth arrest by contact inhibition. However, the magnitude, direction, and temporal relationship of redox changes to cellular responses are incompletely defined. The present work sought to characterize redox and metabolic changes associated with proliferative stages to contact inhibition of growth in rat IEC-6 intestinal epithelial cells. From the first day of culture until 1 day before confluence, an increase in GSH concentrations and a significant reduction in the redox potential of the GSSG/2GSH couple were observed. These changes were accompanied by a decrease in relative reactive oxygen species (ROS) and nitric oxide (NO) concentrations and oxidation of the redox potential of the NADP⁺/reduced NADP and NAD⁺/NADH couples. Postconfluent cells exhibited a significant decrease in GSH concentrations and a significant oxidation of the GSSG/2GSH couple. When cell proliferation decreased, relative ROS concentrations increased (P < 0.01), whereas NO concentrations remained unchanged, and the NAD⁺/NADH couple became more reduced. Together, these data indicate that the redox potential of distinct couples varies differentially in both magnitude and direction during successive stages of IEC-6 growth. This finding points out the difficulty of defining intracellular redox status at particular stages of cell growth by examining only one redox species. In addition, the data provide a numerical framework for future research of regulatory mechanisms governed by distinct intracellular redox couples. cell proliferation; contact inhibition; glutathione     

Gradual Drought Under Field Conditions Influences the Glutathione Metabolism,Redox Balance and Energy Supply in Spring Wheat

Glutathione (GSH) metabolism, redox balance and energy supply in spring wheat (Triticum aestivum L.) during gradual drought stress under field conditions were investigated. Although levels of total and reduced GSH were decreased, the ratio of GSH/GSSG (glutathione disulfide) was markedly increased by drought. Levels of GSH biosynthetic precursors, cysteine (Cys) and -glutamylcysteine (-GC), and the activities of their biosynthetic enzymes, -glutamylcysteine synthetase (-GCS) and glutathione synthetase (GSHS) were also significantly increased in stressed plants. Glutathione reductase (GR) activity, which is responsible for the conversion of GSSG to GSH, was also increased under this field stress. However, two other important enzymes in GSH metabolism, glutathione peroxidase (GP) and glutathione S-transferase (GST), showed decreased activity in the droughted plants. These results suggest that the higher ratio of GSH/GSSG, the rate of GSH biosynthesis and the capacity of its redox cycling rather than GSH accumulation might be essential for drought resistance of plants. Activities of the two key Calvin-cycle enzymes possessing exposed sulfhydryl groups, NADP⁺-dependent glyceraldehydes-3-phosphate dehydrogenase (G3PD) and fructose-1,6-bisphosphatase (FBPase) were not affected by drought stress, whereas, activity of the key enzyme in the pentose-phosphate pathway (PPP), 6-phosphogluconate dehydrogenase (6-PGD), increased in the droughted plants. The ratios of NADPH/NADP⁺, NADH/NAD⁺ and ATP/ADP increased in the droughted plants, indicating that an up-regulation of the reduced redox state and the energy supply in the plant cells might be an important physiological strategy for plants responding to drought stress. A simple correlation between the high ratio of GSH/GSSG, the rate of GSH biosynthesis and the redox cycle and the high reduction states of redox status in the plant cells was also observed under field drought.     

Release of glutathione from erythrocytes and other markers of oxidative stress in carbon monoxide poisoning

Thom, Stephen R., Melissa Kang, Donald Fisher, and HarryIschiropoulos. Release of glutathione from erythrocytes and othermarkers of oxidative stress in carbon monoxide poisoning. J. Appl. Physiol. 82(5):1424-1432, 1997.Rats exposed to CO in a manner known to causeoxidative stress in brain exhibited a twofold increase in plasma levelsof oxidized proteins, thiobarbituric acid-reactive substances (TBARS),oxidized glutathione (GSSG), and reduced glutathione(GSH). Changes were neither directly related to hypoxicstress from carboxyhemoglobin nor significantly influenced bycirculating platelets or neutrophils. Treatment with the nitric oxidesynthase inhibitorN-nitro-L-arginine methylester inhibited elevations in GSH and GSSG but not changesin oxidized proteins or TBARS, suggesting that two oxidative mechanismsmay be operating in this model and that GSH and GSSG elevationsinvolved nitric oxide-derived oxidants. Elevations of blood GSH andGSSG occurred at different anatomic sites, indicating that no singleorgan was the source of the increased peptides. Animals that underwentexchange transfusion with a hemoglobin-containing saline solution didnot exhibit elevations in GSH and GSSG, suggesting that blood-bornecells released these peptides in response to oxidative stress. In invitro studies, erythrocytes, but not platelets and leukocytes,responded to oxidative stress from peroxynitrite by releasing GSH,whereas no release was observed in response to nitric oxide orsuperoxide. Glucose, maltose, and cytochalasin B, agents that protectextracellular components of the hexose transport protein complex fromoxidative stress, prevented GSH release. The data indicate that nitricoxide-derived oxidants are involved in CO-mediated oxidative stresswithin the vascular compartment and that elevations of severalcompounds may be useful for identifying exposures to CO likely toprecipitate brain injury.

     

The Effects of Partially Drying Part of the Root System of Helianthus annuus on the Abscisic Acid Content of the Roots, Xylem Sap and Leaves

Plants of Helianthus annuus were grown in soil in pots suchthat approximately 30% of the root system protruded throughthe base of the pot. After 7 d further growth in aerated nutrientsolution, the attached, protruding roots were air-dried for10–15 min and thereafter surrounded with moist still air,in the dark, for 49 h, whilst the soil was kept at field capacity.The roots of the control plants remained in the nutrient solutionthroughout the experiment. This treatment rapidly reduced the water content of protrudingroots from 20.5 to 17.8 g g^–1 dry mass (DM), which remainedless than that of the control roots for the rest of the experiment.This treatment also reduced root turgor and water potential.The abscisic acid (ABA) concentrations in the protruding roots,xylem sap and leaves of the treated plants increased significantly,compared to values recorded for control plants. In treated roots, the ABA concentration was significantly increased4 h after treatment, with a maximum of 4.4+0.1 nmol g^–1(DM) after 25 h. The ABA concentration in the xylem sap of thetreated plants was significantly greater than in the controls25 h, 30 h, and 49 h after the partial drying of the roots,with a maximum concentration of approximately 970 pmol ABA cm-3at 49 h. Initially, the ABA concentration in the leaves was0.45 nmol g^–1 (DM) which increased significantly to 1.1±0.1 nmol g^–1 at 25 h, to 1.7±0.3 nmol g^–1at 49 h. Leaf conductance was significantly less in plants with air-driedroots than in the controls 8 h after the start of the treatmentand thereafter. The water relations of the leaves of the treatedplants did not differ from those of the control plants. These results confirm previous reports that ABA is rapidly generatedin partially-dried and attached root systems and demonstratesa concomitant large increase in the ABA content of the xylemsap. It is suggested that partial dehydration of some of theroots of Helianthus annuus, increases ABA concentration in thetranspiration stream and decreases leaf conductance in the absenceof changes in leaf water status. As these responses were initiatedin free-growing roots the stimulus is independent of any increasesin soil shear strength that are associated with soil drying. Key words: Soil drying, roots, ABA, leaf conductance, water relations     

Contrasting effects of thiol-modulating agents on endothelial NO bioactivity

The bioactivity of endothelium-derived nitric oxide(NO) is an important component of vascular homeostasis that issensitive to intracellular redox status. Because glutathione (GSH) is a major determinant of intracellular redox state, we sought to define itsrole in modulating endothelial NO bioactivity. In porcine aorticendothelial cells (PAECs), we depleted intracellular GSH (>70%) using1) buthionine-(S,R)-sulfoximine (BSO), whichinhibits GSH synthesis; 2) diamide, which oxidizes thiols;or 3) 1-chloro-2,4-dinitrobenzene (CDNB), which putativelydepletes GSH through glutathione S-transferase activity.Cellular GSH depletion with BSO had no effect on endothelial NObioactivity measured as A-23187-induced cGMP accumulation. In contrast,oxidation of intracellular thiols with diamide inhibited bothA-23187-induced cGMP accumulation and the cGMP response to exogenousNO. Diamide treatment of either PAECs, PAEC membrane fractions, orpurified endothelial nitric oxide synthase (eNOS) resulted insignificant inhibition (~75%) of eNOS catalytic activity measured asL-[³H]arginine-to-L-[³H]citrullineconversion. This effect appeared related to oxidation of eNOS thiols asit was completely reversed by dithiothreitol. Glutathione depletionwith CDNB inhibited A-23187-stimulated cGMP accumulation but not thecGMP response to exogenous NO. Rather, CDNB treatment impaired eNOScatalytic activity in intact PAECs, and this effect was reversed byexcess NADPH in isolated purified eNOS assays. Consistent with theseresults, we found spectral evidence that CDNB reacts with NADPH andrenders it inactive as a cofactor for either eNOS or glutathionereductase. Thus thiol-modulating agents exert pleiotropic effects onendothelial NO bioactivity, and these data may help to resolve a numberof conflicting previous studies linking GSH status with endothelialcell NO bioactivity.

     

Nitrate Nutrition of Grasses from Steady-State Supplies in Flowing Solution Culture following Nitrate Deprivation and/or Defoliation: II. ASSIMILATION OF AND SHORT-TERM EFFECTS ON UPTAKE

Plants of two genotypes of Lolium perenne L. cv. S23 and a L.perenne ? L. multiflorum Lam. hybrid cv. Augusta were grownin flowing solution culture. N was suppled in one treatmentat 10 mmol m^–3 NO^–3 throughout (HN), and in another(LN) the N supply was terminated after 10 d for 11 d. When was re-supplied both LN and HN plants were leftentire or defoliated. The two genotypes showed similar responsesto all treatments. The concentration of N in shoot dry matterdeclined from 4.4% to 2.0% and in the root from 2.8% to 1.0%over the 11 d of N deprivation, with 95% of the initially present being assimilated during this period. LN plantsassimilated 10% more of their total uptake than did HN plants. The in vitro nitrate reductase activity(NRA) was 10- to 50-fold higher in the youngest fully-expandedleaves than in roots and declined in the leaves during N deprivation.Between 2–6 d after defoliation, there was a large increasein NRA in leaves of HN (but not LN) plants. After defoliationof HN plants, net uptake from 10 mmol m^–3 declined to negligible levels within 15 h, but in defoliatedLN plants it increased to levels similar to those of entireHN plants (15–20 µmol h^–1 g^–1 fr. wt.root) within 8 h. When was re-supplied to entire LN plants, uptake of increased to levels similar to those of entire HN plants within 2.3 h, butdid not markedly exceed that of HN plants for at least 10 h.Net uptake of by LN plants during depletion of stirred static nutrient solutions containing 1.0 mol m^–3 lagged behind that by HN plants by 70–100 min, but the maximum unit absorption rate was similar for LNand HN plants (5–7 µmol h^–1 g^–1 fr.wt. root). The nature of the short-term demand for uptake following recovery from the stresses of defoliation andN starvation is discussed. Key words: Lolium perenne, Lolium multiflorum, N-deficiency, defoliation, nitrate uptake, nitrate reductase, N-assimilation     

Polyamine Titre in Relation to Chill-Sensitivity in Phaseolus sp.

Guye, M G., Vigh, L. and Wilson, J. M. 1986. Polyamine titrein relation to chill-sensitivity in Phaseolus sp.—J. exp.Bot. 37: 1036–1043. Endogenous levels of the polyamines putrescine, spermidine andspermine were quantified in the primary leaves of five cultivarsof bean (Phaseolus sp.) differing in their ‘wilting response’to a chilling exposure of 5 ?C for 24 h. Levels of polyamines prior to chilling treatment did not appearto be correlated with chill-tolerance as levels in the non-chilledcontrols were highest in cultivars of medium chill-sensitivity.Plants grown under a vapour saturation deficit (VSD) of 8?4gm^–3 day/6?1 g m^–3 night exhibited a mild hardeningas compared to plants grown under a VSD of 5?7 gm^–3 day/4?1gm^–3 night, as the former showed less wilting on chilling.Hardening at high VSD had the effect of slightly lowering theputrescine content of non-chilled tissue but total polyaminecontent remained unchanged. However, on chilling, the largestrelative increase in polyamine levels, in particular that ofputrescine, occurred in hardened plants. There was also a significantrelative increase in putrescine titre in response to chillingin non-hardened genotypes of high chill-tolerance, whereas morechill-sensitive genotypes remained unchanged or slightly declinedin putrescine content on chilling. Relative changes in putrescine content rather than absolutelevels appears to be correlated with chill-tolerance. Theseresults are discussed in view of present knowledge on the adaptivesignificance of stress-induced changes in polyamines, especiallywith regard to membrane stability Key words: Chilling, polyamines, Phaseolus sp.     

Difference in light-induced increase in ploidy level and cell size between adaxial and abaxial epidermal pavement cells of Phaseolus vulgaris primary leaves

Changes in nuclear DNA content and cell size of adaxial andabaxial epidermal pavement cells were investigated using brightlight-induced leaf expansion of Phaseolus vulgaris plants. Inprimary leaves of bean plants grown under high (sunlight) ormoderate (ML; photon flux density, 163 µmol m^–2s^–1) light, most adaxial epidermal pavement cells hada nucleus with the 4C amount of DNA, whereas most abaxial pavementcells had a 2C nucleus. In contrast, plants grown under lowintensity white light (LL; 15 µmol m^–2 s^–1)for 13 d, when cell proliferation of epidermal pavement cellshad already finished, had a 2C nuclear DNA content in most adaxialpavement cells. When these LL-grown plants were transferredto ML, the increase in irradiance raised the frequency of 4Cnuclei in adaxial but not in abaxial pavement cells within 4d. On the other hand, the size of abaxial pavement cells increasedby 53% within 4 d of transfer to ML and remained unchanged thereafter,whereas adaxial pavement cells continuously enlarged for 12d. This suggests that the increase in adaxial cell size after4 d is supported by the nuclear DNA doubling. The differentresponses between adaxial and abaxial epidermal cells were notinduced by the different light intensity at both surfaces. Itwas shown that adaxial epidermal cells have a different propertythan abaxial ones. Key words: Cell enlargement, endopolyploidization, epidermal pavement cells, incident light intensity, leaf expansion, nuclear DNA content, Phaseolus vulgaris     

Metabolic requirements associated with GSH synthesis during in vitro maturation of cattle oocytes

Glutathione (GSH) concentration increases in bovine oocytes during in vitro maturation (IVM). The constitutive amino acids involved in GSH synthesis are glycine (Gly), glutamate (Glu) and cysteine (Cys). The present study was conducted to investigate the effect of the availability of glucose, Cys, Gly and Glu on GSH synthesis during IVM. The effect of the amino acid serine (Ser) on intracellular reduced/oxidized glutathione (GSH/GSSG) content in both oocytes and cumulus cells was also studied. Cumulus-oocyte complexes (COC) of cattle obtained from ovaries collected from an abattoir were matured in synthetic oviduct fluid (SOF) medium containing 8 mg/ml bovine serum albumin-fatty acid-free (BSA-FAF), 10 microg/ml LH, 1 microg/ml porcine FSH (pFSH) and 1 microg/ml 17 beta-estradiol (17beta-E2). GSH/GSSG content was measured using a double-beam spectrophotometer. The COC were cultured in SOF supplemented with 1.5mM or 5.6mM glucose (Exp. 1); with or without Cys+Glu+Gly (Exp. 2); with the omission of one constitutive GSH amino acid (Exp. 3); with 0.6mM Cys or Cys+Ser (Exp. 4). The developmental capacity of oocytes matured in IVM medium supplemented with Cys and the cell number per blastocyst were determined (Exp. 5). The results reported here indicate (1) no differences in the intracellular GSH/GSSG content at any glucose concentrations. Also, cumulus cell number per COC did not differ either before or after IVM (Exp. 1). (2) Glutathione content in oocytes matured in SOF alone were significantly different from oocytes incubated with SOF supplemented with Cys+Glu+Gly (Exp. 2). (3) Addition of Cys to maturation medium, either with or without Gly and Glu supplementation resulted in an increase of GSH/GSSG content. However, when Cys was omitted from the IVM medium intracellular GSH in oocytes or cumulus cells was less but not significantly altered compared to SOF alone (Exp. 3). (4) Glutathione content in both oocytes and cumulus cells was significantly reduced by incubation with 5mM Ser (Exp.4). (5) There was a significant increase in cleavage and blastocyst rates when Cys was added to maturation medium. In contrast, the cleavage, morula and blastocyst rates were significantly different when 5mM Ser was added to maturation media. There was also a significant difference in mean cell number per blastocyst, obtained from oocytes matured with 5mM Ser (Exp. 5). This study provides evidence that optimal embryo development in vitro is partially dependent on the presence of precursor amino acids for intracellular GSH production. Moreover, the availability of Cys might be a critical factor for GSH synthesis during IVM in cattle oocytes. Greater Ser concentration in IVM medium altered "normal" intracellular GSH in both oocytes and cumulus cells with negative consequences for subsequent developmental capacity.     

Photosynthetic Units and Carbon Assimilation in Leaves of Grain Sorghum under Different Light Intensities

The Cyt f and P700 contents in leaves of three Sorghum, varietieswere measured, in relation to their carbon assimilation, underdifferent light intensities during growth. At the maximum irradiationused (1,800 µE m^–2 s^–1) the ratio of P700to Cyt f was close to unity, whereas under low irradiation (450µE m^–2 s^–1) the ratio of P700 to Cyt f rangedfrom two to three. A strikingly positive correlation existedbetween the P700 contents of the leaves and their rates of carbondioxide fixation, dry matter production and Cyt f contents,only when the plants were grown under high light intensities.The P700 content of the leaves in plants grown under low irradiationwas unrelated to the contents of Cyt f. Thus, at a high lightintensity there is a close relationship between the Cyt f andP700 levels, but at low intensities the amounts of electroncarriers and the reaction centre are independent. (Received March 7, 1983; Accepted August 24, 1983)     

Responses of nitrate assimilation and N translocation in tomato (Lycopersicon esculentum Mill) to reduced ambient air humidity

The impact of low humidity in ambient air on water relations,nitrate uptake, and translocation of recently absorbed nitrogen,was investigated in 5-week-old tomato (Lycopersicon esculentumMill cv. Ailsa Craig) plants grown hydroponically in a completenutrient solution. Plants were subjected to dry air (relativehumidity 2–4% for 6 h. The transpiration rate increasedseveral-fold and the shoot water content decreased by almost20%, whereas root water content was unaffected. No effect onin vitro nitrate reductase (NR) activity was detected when usingan EDTA-contraining assay buffer. Replacement of EDTA with Mg²⁺revealed a significant decline in shoot NR activity, which suggestsphosphorylation of the enzyme during the stress treatment. Plantswere grown in a split-root system, in which one root half wasfed ¹⁵N-nitrate during the treatment, in order to determinenitrate uptake and translocation of recently absorbed nitrogenin the plants. Uptake of nitrate was substantially inhibited,but the proportion of absorbed ¹⁵N that was translocated tothe shoots was only slightly affected. In untreated plants,71% of the ¹⁵N recovered in roots had been retranslocated fromthe shoots, whereas in plants subjected to stress the deliveryof ¹⁵N from shoots to roots appeared to be completely inhibited.The data show that lowered humidity in air has significant effectson both uptake of nitrate as well as translocation of nitrogenwithin the plants. Some of these effects appear to be commonwith those observed in plants subjected to reduced water potentialsin the root environment and point to the possibility of theshoot water relations being highly influential on nitrogen uptakeand translocation. Key words: Air humidity, nitrate assimilation, nitrate reductase activity, nitrogen translocation, tomato, water stress     

Carbon Economy of Sour Orange in Relation to Mycorrhizal Colonization and Phosphorus Status

The effects of plant phosphorus (P) status and the mycorrhizal(M) fungus, Glomus intraradices Schenck & Smith, on thecarbon (C) economy of sour orange (Citrus aurantium L.) weredetermined during and following active M colonization. Therewere four treatments: M seedlings grown at standard-strength(1 mM) P (M1) and nonmycorrhizal (NM) plants grown at one, twoand five times standard-strength P (NM1, NM2 and NM5). Mycorrhizalcolonization, tissue dry mass, P content, root length and leafarea were determined in five harvests from 6 to 15 weeks ofage. Rate of C assimilation (A) was determined at 7, 8 and 12weeks by gas exchange. Partitioning of ¹⁴ C was determined from7 to 15 weeks using a 10-min pulse followed by a 24-h chaseperiod. For a given attribute, M1 plants were compared to thecurve defining the NM response as a function of tissue P concentration.In contrast to the large effects of P nutrition on C economyof sour orange, M effects were generally subtle. Mycorrhizaeincreased the root biomass fraction, the root length/leaf arearatio and the percentage of ¹⁴C recovered from below-groundcomponents. A higher percentage of below-ground ¹⁴ C was inthe respiration and soil fractions in M than NM plants of equivalentP status. Mycorrhizal plants tended to enhance A only for abrief period. Mycorrhizal plants had lower relative growth ratesthan NM plants of equivalent P status, suggesting that the temporarilyenhance A of M plants did not fully compensate for their greaterbelow-ground carbon expenditure. Problems of interpreting thedynamic effects of mycorrhizae on C economy that are independentof P nutrition are discussed.Copyright 1993, 1999 Academic Press Citrus aurantium L., sour orange, carbon economy, ¹⁴carbon, CO₂ assimilation, vesicular-arbuscular mycorrhizae, phosphorus fertilization, phosphorus nutrition     

Hydroxyl radical activation of a Ca(2+)-sensitive nonselective cation channel involved in epithelial cell necrosis

In a previous work the involvement of a fenamate-sensitive Ca²⁺-activated nonselective cation channel (NSCC) in free radical-induced rat liver cell necrosis was demonstrated (5). Therefore, we studied the effect of radical oxygen species and oxidizing agents on the gating behavior of a NSCC in a liver-derived epithelial cell line (HTC). Single-channel currents were recorded in HTC cells by the excised inside-out configuration of the patch-clamp technique. In this cell line, we characterize a 19-pS Ca²⁺-activated, ATP- and fenamate-sensitive NSCC nearly equally permeable to monovalent cations. In the presence of Fe²⁺, exposure of the intracellular side of NSCC to H₂O₂ increased their open probability (P_o) by 40% without affecting the unitary conductance. Desferrioxamine as well as the hydroxyl radical (·OH) scavenger MCI-186 inhibited the effect of H₂O₂, indicating that the increase in P_o was mediated by ·OH. Exposure of the patch membrane to the oxidizing agent 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB) had a similar effect to ·OH. The increase in P_o induced by ·OH or DTNB was not reverted by preventing formation or by DTNB washout, respectively. However, the reducing agent dithiothreitol completely reversed the effects on P_o of both ·OH and DTNB. A similar increase in P_o was observed by applying the physiological oxidizing molecule GSSG. Moreover, GSSG-oxidized channels showed enhanced sensitivity to Ca²⁺. The effect of GSSG was fully reversed by GSH. These results suggest an intracellular site(s) of action of oxidizing agents on cysteine targets on the fenamate-sensitive NSCC protein implicated in epithelial cell necrosis. Ca²⁺-activated channels; radical oxygen species; oxidative stress     

Assessment of oxidative stress in the planktonic diatom Thalassiosira pseudonana in response to UVA and UVB radiation

Induction of oxidative stress by UVA and UVB in the diatom Thalassiosirapseudonana was experimentally studied. Cells, pre-grown in alight-limited continuous culture, were incubated for 4 h at175 µmol m^-2s^-1photosynthetically active radiation, withoptional supplementary UVA at an unweighted dose rate of 0.70W m^-2, or 2.79 W m^-2UVA plus 0.45 W m^-2UVB (unweighted). A fluorescence-basedmeasure of photosynthetic efficiency (F_v/F_m) decreased from0.69 to 0.58 in the presence of UVB, whereas UVA caused a minordecrease of F_v/F_m. Quantitative analysis of confocal imagesshowed a minor increase of active oxygen production associatedwith supplemental UVA alone, and a 100% increase with additionalUVB. Cellular malondialdehyde, an indicator of lipid peroxidationby active oxygen, almost doubled under UVA and increased three-foldwith additional UVB. Activities of superoxide dismutase (scavengingactive oxygen) and glutathione reductase (reducing GSSG to GSH)increased in response to UVB exposure, whereas ascorbate peroxidaseactivities did not. UVB caused a minor decrease in the glutathioneratio GSH : (GSH + 0.5GSSG), which indicates a moderate oxidativestress.     

Daily Fluctuations of Antioxidants in Bean (Phaseolus vulgaris L.) Leaves As Affected by the Presence of Ambient Air Pollutants

During the period 0800–1700 h (GMT) of a summer day, youngleaves were collected every 20 min from Phaseolus vulgaris L.cv. Horticultural plants grown in open-top field chambers locatedat an urban site in northern Italy and exposed either to ambientlevels of gaseous air pollutants or to filtered ambient air.Ascorbic and dehydroascorbic acids, GSH and GSSG, superoxidedismutase, ascorbate peroxidase, dehydroascorbate reductase,GSSG reductase, GSH peroxidase, catalase, guaiacol peroxidase,chlorophylls, carotenoids, soluble protein and dry weight weremeasured in these leaves. The main differences between treatedand control leaves were observed during the period 1100–1530h and concerned superoxide dismutase, catalase, ascorbate peroxidase,ascorbate/dehydroascorbate and GSH/GSSG ratios, chlorophylls,carotenoids and dry weight. On the basis of the pollution climateobserved at the experimental site on the day of leaves sampling,ozone appeared to be the causative agent of the observed divergencesamong the time patterns of antioxidants in treated and controlplants. It was deduced that ozone can induce oxidative stressvia the production of superoxide radical anion and hydrogenperoxide. (Received June 1, 1992; Accepted December 9, 1992)     

Pinitol occurrence in soybean plants as affected by temperature and plant growth regulators

Unsuitable temperatures are frequently encountered by soybean(Glycine max L. Merr.) plants grown in the field. Certain polyolshave been reported to protect plants from high temperature orfrost damage. Controlled environment studies were conductedto investigate the effect of stressful temperature regimes onthe content of pinitol (3-O-methyl-D-chiro-inositol) in soybeanplants. Hydroponically-grown soybean plants were subjected tohigh (35/30 C) or low (15/10 C) day/night temperature stresses,and pinitol content in different plant parts was determinedusing high performance liquid chromatography (HPLC). A syntheticplant growth regulator, PGR-IV, was foliarly applied to theplants to evaluate its effect on pinitol content in differentplant components. Uniformly-labelled ¹⁴C-glucose was fed intothe leaves via the transpiration stream, and the effects ofhigh temperature and EXP-S1089, another synthetic plant growthregulator, on the incorporation of ¹⁴C-glucose into pinitolwas evaluated using HPLC separation and scintillation spectrometry.High-temperature stress significantly increased plant pinitolcontent and the incorporation of ¹⁴C-glucose into pinitol, butdecreased the content of sucrose, glucose and fructose. Underlow-temperature stress, there was hardly any change in pinitolcontent, but a drastic increase in soluble sugars. PGR-IV enhancedpinitol translocation from leaves to stems and roots, whileEXP-S1089 increased pinitol/sucrose ratio. Accumulation of pinitolmay be an adjustment mechanism of the plant to reduce high-temperaturedamage, but not low-temperature injuries. Key words: Pinitol, soybean, temperature, plant growth regulator