Evidence for a significant contribution by peroxidase-mediated O2 uptake to root respiration of Brachypodium pinnatum

This study describes the O₂ uptake characteristics of intact roots of Brachypodium pinnatum. In the presence of 25 mM salicylhydroxamic acid (SHAM), concentrations of KCN below 3.5 νM had no effect on the rate of root respiration, whereas in the absence of 25 mM SHAM a significant inhibition of approx. 18% was observed. This indicates that an O₂-consuming reaction, not associated with the cytochrome pathway, the alternative pathway or the “residual component”, operates in the absence of any inhibitors in roots of B. pinnatum. We demonstrate here that this fourth O₂-consuming reaction is mediated by a peroxidase. A peroxidase which catalyzed O₂ reduction in the presence of NADH was readily washed from the roots of B. pinnatum. This peroxidase was stimulated by 5 mM SHAM, whereas ascorbic acid, catalase, catechol, gentisic acid, low concentrations potassium cyanide (3.5 μM), sodium azide, sodium sulfide, superoxide dismutase and high concentrations SHAM (25 mM) inhibited this reaction. Except for high concentrations of SHAM and concentrations of KCN higher than approx. 3.5 μM, these effectors could not be used to inhibit the peroxidase-mediated O₂ uptake in intact roots of B. pinnatum. Concentrations of SHAM below 10 mM stimulated O₂ uptake up to 15% of the control rate, depending on concentration, whereas 25 mM SHAM inhibited O₂ uptake by 35%. The stimulation at low concentrations resulted from a SHAM-stimulated peroxidase activity, whereas 25 mM SHAM completely inhibited both the peroxidase-mediated O₂ uptake and the activity of the alternative pathway. A method is presented for determining the relative contributions of each of the four O₂-consuming reactions, i.e. the cytochrome pathway, the alternative pathway, the “residual component” and the peroxidase-mediated O₂ uptake. The peroxidase-mediated O₂ uptake contributed 21% to the total rate of oxygen uptake in roots of B. pinnatum, the cytochrome pathway contributed 41%, the alternative pathway 14% and the “residual component” 24%.     

Sulfide-Resistant Respiration in Leaves of Elodea canadensis Michx: Comparison with Cyanide-Resistant Respiration

The rate of dark O₂ uptake of Elodea canadensis leaves was titrated with either cyanide or sulfide in the presence and in the absence of 5 millimolar salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase. The inhibition of O₂ uptake by SHAM alone was very small (3-6%), suggesting that actual respiration mainly occurred through the cytochrome pathway. O₂ uptake was slightly stimulated by cyanide at concentrations of 50 micromolar or higher, but in the presence of SHAM respiration was strongly suppressed. The effects of sulfide on O₂ uptake were similar to those of cyanide, except that the percent stimulation of O₂ uptake by sulfide alone was somewhat higher than that of cyanide. However, the estimates of the capacity of the alternative pathway were similar with both inhibitors. Another difference is that maximal inhibition of respiration in the presence of SHAM was observed with lower concentrations of sulfide (50 micromolar) than cyanide (250 micromolar). The results suggest that sulfide can be used as a suitable inhibitor of cytochrome c oxidase in studies with intact plant tissues, and that sulfide does not apparently inhibit the alternative oxidase.     

Salicylhydroxamic Acid (SHAM) Inhibition of the Dissolved Inorganic Carbon Concentrating Process in Unicellular Green Algae

Rates of photosynthetic O₂ evolution, for measuring K_0.5(CO₂ + HCO₃⁻) at pH 7, upon addition of 50 micromolar HCO₃⁻ to air-adapted Chlamydomonas, Dunaliella, or Scenedesmus cells, were inhibited up to 90% by the addition of 1.5 to 4.0 millimolar salicylhydroxamic acid (SHAM) to the aqueous medium. The apparent K₁(SHAM) for Chlamydomonas cells was about 2.5 millimolar, but due to low solubility in water effective concentrations would be lower. Salicylhydroxamic acid did not inhibit oxygen evolution or accumulation of bicarbonate by Scenedesmus cells between pH 8 to 11 or by isolated intact chloroplasts from Dunaliella. Thus, salicylhydroxamic acid appears to inhibit CO₂ uptake, whereas previous results indicate that vanadate inhibits bicarbonate uptake. These conclusions were confirmed by three test procedures with three air-adapted algae at pH 7. Salicylhydroxamic acid inhibited the cellular accumulation of dissolved inorganic carbon, the rate of photosynthetic O₂ evolution dependent on low levels of dissolved inorganic carbon (50 micromolar Na-HCO₃), and the rate of ¹⁴CO₂ fixation with 100 micromolar [¹⁴C] HCO₃⁻. Salicylhydroxamic acid inhibition of O₂ evolution and ¹⁴CO₂-fixation was reversed by higher levels of NaHCO₃. Thus, salicylhydroxamic acid inhibition was apparently not affecting steps of photosynthesis other than CO₂ accumulation. Although salicylhydroxamic acid is an inhibitor of alternative respiration in algae, it is not known whether the two processes are related.     

Redox activity and peroxidase activity associated with the plasma membrane of guard-cell protoplasts

Redox systems have been reported in the plasma membrane of numerous cell types and in cells from various species of higher plant. A search for a redox system in the plasma membrane of guard cells was therefore made in efforts to explain how blue light stimulates stomatal opening, a process which is coupled to guard cell H⁺ efflux and K⁺ uptake. The rates of O₂ uptake by intact guard-cell protoplasts (GCP) of Commelina communis L., in the dark, were monitored in the presence of NAD(P)H since the stimulation of O₂ consumption by reduced pyridine nucleotides is used as an indicator of the presence of a redox system in the plasma membrane. Oxygen consumption by intact GCP increased two- to threefold in the presence of NAD(P)H. The NAD(P)H-stimulation of O₂ uptake was dependent on Mn²⁺ and was stimulated 10- to 15-fold by salicylhydroxamic acid (SHAM). Catalase, cyanide and ascorbate, a superoxide scavenger, all individually inhibited the SHAM-stimulated O₂ uptake. These are all characteristics of peroxidase activity although some of these features have been used to imply the presence of a redox system located in the plasma membrane. High levels of peroxidase activity (using guaiacol as a substrate) were also detected in the GCP and in the supernatant. The activity in the supernatant increased with time indicating that peroxidase was being excreted by the protoplasts. The properties of O₂ uptake by the incubation medium after separation from the protoplasts were similar to those of the protoplast suspension. It is concluded that our observations can be more readily explained by peroxidase activity associated with the plasma membrane and secreted by the GCP than by the presence of a redox system in the plasma membrane of the protoplasts.Abbreviations EDTA ethylenediaminetetraacetic acid - GCP guard cell protoplast - Mes 2-(N-morpholino)ethanesulphonic acid - SHAM salicylhydroxamic acid     

Effects of KCN and Salicylhydroxamic Acid on Respiration of Soybean Leaves at Different Ages

Measurements of respiration were made on leaf discs from glasshouse-grown soybean (Glycine max [L.] Merr. cv `Corsoy') plants in the presence and absence of cyanide (KCN) and salicylhydroxamic acid (SHAM). O₂ uptake by mature leaves measured at 25°C was stimulated by 1 millimolar KCN (63%) and also by 5 millimolar azide (79%). SHAM, an inhibitor of the alternative oxidase and a selection of other enzymes, also stimulated O₂ uptake by itself at concentration of 10 millimolar. However, in combination, KCN and SHAM were inhibitory. The rate of O₂ uptake declined consistently with leaf age. The stimulation of O₂ uptake by KCN and by SHAM occurred only after a certain stage of leaf development had been reached and was more pronounced in fully expanded leaves. In young leaves, O₂ uptake was inhibited by both KCN and SHAM individually. The uncoupler, p-trifluoromethoxy carbonylcyanide phenylhydrazone, stimulated leaf respiration at all ages studied, the stimulation being more pronounced in fully expanded leaves. The uncoupled rate was inhibited by KCN and SHAM individually. The capacity of the cytochrome path declined with leaf age, paralleling the decline in total respiration. However, the capacity of the alternative path peaked at about full leaf expansion, exceeding the cytochrome capacity and remaining relatively constant. These results are consistent with the presence in soybean leaves of an alternative path capacity that seems to increase with age, and they suggest that the stimulation of O₂ uptake by KCN and NaN₃ in mature leaves was mainly by the SHAM-sensitive alternative path. The stimulation of O₂ uptake by SHAM was not expected, and the reason for it is not clear.     

Effects of KCN and Salicylhydroxamic Acid on the Root Respiration of Pea Seedlings

Polarography, using cylindrical platinum electrodes, proved suitable for measuring changes in the internal apical O₂ concentration of the primary root of pea (Pisum sativum L. cv Meteor) effected by KCN and/or salicylhydroxamic acid (SHAM) in the bathing medium. An electrical rootaeration analog was used to help evaluate some of the results. Concentrations of KCN ≤0.05 millimolar had no significant effect. In response to 0.1 millimolar KCN, the O₂ concentration rose substantially for approximately 2 hours, then declined, and after 10 hours had frequently fallen below the pretreatment level. Such changes suggest an initial inhibition of cytochrome oxidase-mediated O₂ uptake followed by an induction of the alternative, cyanide-resistant respiratory pathway. These treatments proved nonlethal. Changes in O₂ concentration similar to those described for 0.1 millimolar KCN were observed in response to 1 and 10 millimolar KCN but these treatments were lethal and the root apex became soft and often appeared flooded. Roots survived and showed no significant responses when treated with SHAM at concentrations ≤5 millimolar. However, when the alternative pathway had been (apparently) induced by 0.1 millimolar KCN, the addition of 5 millimolar SHAM to the bathing medium caused a substantial and persistent rise in the root apical O₂ concentration, suggesting that this (nonlethal) concentration of SHAM could indeed inhibit O₂ uptake via the cyanide-resistant pathway.

It is concluded that while O₂ uptake normally occurs by the cytochrome pathway in the primary pea root, the alternative, cyanide-resistant pathway can be induced by 0.1 millimolar KCN.

     

Cyanide-insensitive, Salicylhydroxamic Acid-sensitive Processes in Potentiation of Light-requiring Lettuce Seeds

Phytochrome-mediated germination of Lactuca sativa L. cv. Waldmann's Green seeds was inhibited strongly by 10 millimolar salicylhydroxamic acid (SHAM), but only slightly delayed by the same level of KCN. SHAM was most effective if applied within the 8-hour potentiation period (release from dormancy) following red light treatment, but much less effective with completely potentiated seeds. SHAM at 3 millimolar actually hastened completion of potentiation, whereas concentrations of 6.6 millimolar or higher retarded the process. A temporary upsurge of O₂ consumption was particularly evident during the period of most rapid potentiation (3 hours after red light), especially in the seed sections containing the embryonic axis. The embryonic axis obtained from dormant seeds also contained most of the SHAM-sensitive O₂ uptake. However, 8 hours of potentiation caused loss of SHAM sensitivity from axes and a simultaneous gain of SHAM sensitivity by cotyledons. Concomitant with this increased sensitivity to SHAM, O₂ uptake by cotyledonary tissues lost some sensitivity to KCN. Red light-stimulated metabolic processes leading to germination were blocked more effectively by SHAM than by KCN, but O₂ consumption by both dormant and nondormant seeds was much less sensitive to 10 millimolar SHAM than to the same concentration of KCN. This apparent contradiction between effects of SHAM on potentiation and O₂ uptake may be a result of: (a) compensatory electron flow through the cytochrome pathway at the expense of the alternate pathway; (b) a functional site of action of SHAM that differs from the organized, energy-coupled respiratory system; or (c) a combination of these possibilities.     

Variations in the Alternative Oxidase in Chlamydomonas Grown in Air or High CO(2)

Chlamydomonas in the resting phase of growth has an equal capacity of about 15 micromole O₂ uptake per hour per milligram of chlorophyll for both the cytochrome c, CN-sensitive respiration, and for the alternative, salicylhydroxamic acid-sensitive respiration. Alternative respiration capacity was measured as salicylhydroxamic acid inhibited O₂ uptake in the presence of CN, and cytochrome c respiration capacity as CN inhibition of O₂ uptake in the presence of salicylhydroxamic acid. Measured total respiration was considerably less than the combined capacities for respiration. During the log phase of growth on high (2-5%) CO₂, the alternative respiration capacity decreased about 90% but returned as the culture entered the lag phase. When the alternative oxidase capacity was low, addition of salicylic acid or cyanide induced its reappearance. When cells were grown on low (air-level) CO₂, which induced a CO₂ concentrating mechanism, the alternative oxidase capacity did not decrease during the growth phase. Attempts to measure in vivo distribution of respiration between the two pathways with either CN or salicylhydroxamic acid alone were inconclusive.     

Zinc-alleviating effects on iron-induced phytotoxicity in roots of <Emphasis Type="Italic">Triticum aestivum</Emphasis>

The mechanisms of growth inhibition and antioxidative response were investigated in wheat roots exposed to 300 μM iron together with different zinc concentrations (0, 50, and 250 μM). All Zn concentrations decreased Fe content but increased Zn content in the roots and leaves of Fe-treated seedlings. Compared with Fe stress alone, 50 or 250 μM Zn + Fe treatment stimulated root growth, and increased cell viability but decreased malondialdehyde content, which were correlated with the decreases of total and apoplastic hydrogen peroxide and superoxide anion radical (O₂ ^·?) content along with apoplastic hydroxyl radical content. Generation of O₂ ^·? in response to 10 μM diphenylene iodonium suggested that NADPH oxidase activity was lower in Zn + Fe-treated roots than in other roots. In addition, cell wallbound peroxidase, diamine oxidase, and polyamine oxidase in Fe-treated roots were insensitive to Zn addition. Further study showed the stimulation of total superoxide dismutase and glutathione reductase (GR) activities as well as apoplastic catalase, ascorbate peroxidase, and GR in Zn + Fe-stressed roots in comparison with Fe-alone-treated ones. Taken together, Zn could alleviate iron-inhibitory effect on root growth, which might be associated with the decrease of lipid peroxidation, the increase of cell viability and the reductions of reactive oxygen species generation.     

Temperature shift-induced changes in the antioxidant enzyme system of cyanobacteriumSynechocystis PCC 6803

The 24 h effect of low (20°C) and high (43°C) temperature on the antioxidant enzyme activities and lipid peroxidation was investigated in intact cells of the cyanobacteriumSynechocystis PCC 6803 grown at 36°C. At low temperature treated cells, the superoxide dismutase, catalase and glutathione peroxidase activities were significantly higher and the protein content lower than in high temperature treated cells. The increase of hydroxyl free radical level and malonyldialdehyde formation, when algal cells were exposed to low temperature, were due to the stimulated production of superoxide radicals O₂ ⁻ and hydrogen peroxide (H₂O₂).     

Is the Cytosolic Pi Concentration a Limiting Factor for Plant Cell Respiration?

The substrate-dependent O₂ uptake by sycamore (Acer pseudoplatanus L.) cell mitochondria in the presence of ADP and limiting Pi concentrations has been measured. The Pi concentration for half-maximum O₂ uptake rate was found to be in the range 20 to 50 micromolar for all the substrates tested. ³¹P NMR of intact sycamore cells indicated that the Pi concentration in the cytoplasm was in the range 5 to 6 millimolar, approximately 100-fold higher than the Pi concentration required for maximum O₂ uptake rates by isolated mitochondria. When sycamore cells were transferred to a culture medium devoid of Pi, the cytoplasmic Pi concentration decreased from 6 to less than 3 millimolar, but the intact cell respiration remained practically constant for at least 4 days. These results strongly suggest that, in vivo, the respiration rate of sycamore cells is not limited by the quantity of Pi supplied to the mitochondria.     

Regulation of Respiration in the Leaves and Roots of Two Lolium perenne Populations with Contrasting Mature Leaf Respiration Rates and Crop Yields

Measurements of O₂ uptake were made on leaves and roots of two populations of Lolium perenne L. cv S23 (GL66 and GL72), previously shown to have contrasting rates of CO₂ evolution and yields of dry matter. O₂ uptake was faster in the mature leaves of GL66 than those of GL72, but no difference was observed in the respiratory rates of meristematic leaf bases or mature roots. The growth rate of GL72 was faster than that of GL66. Cyanide resistance was substantial in mature leaves but the alternative path did not contribute to O₂ uptake in the dark. In both populations, adding malate and glycine stimulated O₂ uptake, but exogenous sucrose only stimulated when uncoupler was also present. The difference between the respiratory rates of the two populations was maintained under all investigated conditions. We conclude that the rate of mature leaf respiration in the dark in L. perenne is limited by adenylate control of glycolysis. The difference between the fast (GL66) and slow (GL72) respiring populations reflected a greater respiratory capacity and higher turnover of ATP in GL66. Alternative path capacity was also high in the roots of both and contributed substantially to O₂ uptake, as indicated by inhibition by salicylhydroxamic acid in the absence of KCN. The alternative path capacity of meristematic leaf bases was considerably less than that in mature leaves.

Transverse and cross-sections were made of mature leaves of both populations to study anatomical features which might explain the differences in ATP turnover, suggested by the biochemical experiments. Leaves of GL72 were thicker but did not show a different anatomy when compared with GL66. The increased thickness was not due to more or larger cells but entirely to a larger intercellular volume.

     

Role of extracellular peroxidase in the superoxide production by wheat root cells

Summary Extracellular peroxidase has been shown to contribute to superoxide production in wounded wheat (Triticum aestivum L. cv. Ljuba) root cells. The superoxide-synthesizing system of root cells was considerably inhibited by KCN and NaN₃ and activated by MnCl₂ and H₂O₂. Treatment of roots with salicylic acid and a range of di- and tri-carbonic acids (malic, citric, malonic, fumaric, and succinic acids) stimulated superoxide production in both root cells and extracellular solution. The H₂O₂-stimulated superoxide production in the extracellular solution was much higher when roots were preincubated with salicylic or succinic acid. Exogenous acids enhanced peroxidase activity in the extracellular solution. Pretreatment of root cells with the detergents trypsin and sodium dodecyl sulfate had similar effects on the peroxidase activity. Significant inhibition of both superoxide production and peroxidase activity by diphenylene iodonium suggests that the specificity of the latter as an inhibitor of NADPH oxidase is doubtful. Results obtained indicate that extracellular peroxidase is involved in the superoxide production in wheat root cells. The mobile form of peroxidase can be readily secreted to the apoplastic solution and serve as an emergency enzyme involved in plant wound response.Abbreviations DPI diphenylene iodonium - ECS extracellular solution - ROS reactive oxygen species - SA salicylic acid     

Evidence of singlet oxygen participation in the chlorophyll-sensitized photooxidation of indoleacetic Acid

Chlorophyll-sensitized photooxidation of indoleacetic acid (IAA)—with chlorophyll extracted from Pisum sativum L. cv. Alaska W.R.—was determined in the presence of deuterium oxide and known quenchers of singlet oxygen (¹O₂) to explore the involvement of ¹O₂ in the reaction. O₂ uptake was measured in light in a buffered aqueous micellar system containing Triton X-100, KCl, chlorophyll, and IAA. The rate of O₂ uptake was zero in darkness. The reaction was stimulated by deuterium oxide and inhibited by sodium azide indicating that ¹O₂ participated in IAA photooxidation. Both mannitol and superoxide dismutase failed to inhibit O₂ uptake suggesting that neither the hydroxyl radical nor the superoxide anion played a significant role in the reaction.     

Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid Metabolism Plant Sedum praealtum

Intact chloroplasts were isolated from protoplasts of the Crassulacean acid metabolism plant Sedum praealtum D.C. Typical rates of CO₂ fixation or CO₂-dependent O₂ evolution ranged from 20 to 30 micromoles per milligram chlorophyll per hour and could be stimulated 30 to 50% by several Calvin cycle intermediates. The pH optimum for CO₂ fixation was 7.0 to 7.6 with considerable activity as low as pH 6.4. Low concentrations of orthophosphate (Pi) (optimum 0.4 millimolar) stimulated photosynthesis while high concentrations (5 millimolar) caused some inhibition. Both CO₂ fixation and CO₂-dependent O₂ evolution exhibited a relatively long lag phase (4 to 6 minutes) which remained constant between 0.4 to 5 millimolar Pi. The lag phase could be decreased by addition of dihydroxyacetone-phosphate or ribose 5-phosphate. Further results are presented which suggest these chloroplasts have a functional phosphate translocator.     

Regulation of Photosynthetic Electron Transport in Intact Spinach Chloroplasts: II. MECHANISM OF SALT-INDUCED INCREASE IN OXALOACETATE PHOTOREDUCTION

The main focus of this study was to determine the mechanism by which certain exogenous monovalent salts stimulate rates of net O₂ evolution linked to oxaloacetate reduction in intact spinach chloroplasts. The influence of salts on the dicarboxylate translocator involved in the transport of oxaloacetate and on the activity and activation of the chloroplast enzyme NADP-malate dehydrogenase, which mediates electron transport to oxaloacetate, was examined. High concentrations of KCl (155 millimolar) increased the apparent K_m for oxaloacetate but did not significantly alter the maximal velocity of uptake. Likewise, external salts (KCl, MgCl₂, or KH₂PO₄) had minimal effects on the magnitude of light activation of NADP-malate dehydrogenase. In contrast, measurements of chloroplast NADP-malate dehydrogenase activity (after release by osmotic shock) showed a marked dependence on salt concentration. Rates were stimulated approximately 2-fold by both monovalent (optimally 75 millimolar) and divalent (optimally 20 millimolar) salts. It was inferred that the salt-induced increase in net rates of O₂ evolution linked to oxaloacetate reduction is due, at least in part, to stimulation of NADP-malate dehydrogenase caused by monovalent cation permeability of the chloroplast inner envelope membrane.     

Alternative Respiratory Pathway: ITS ROLE IN SEED RESPIRATION AND ITS INHIBITION BY PROPYL GALLATE

Oxygen uptake during the first hours of imbibition in intact soybean and mung bean seeds showed a marked sensitivity to potassium cyanide but was unaffected by addition of either salicylhydroxamic acid or propyl gallate. However O₂ uptake by finely ground seed particles was very sensitive to the addition of either compound. The results indicated that O₂ uptake in intact, imbibing seeds was associated with a cyanide-sensitive process, most probably mitochondrial mediated respiration, and not the result of the cyanide-insensitive lipoxygenase activity which was readily detectable in ground seed particles.     

Effect of inorganic cations and metabolic inhibitors on putrescine transport in roots of intact maize seedlings

The specificity and regulation of putrescine transport was investigated in roots of intact maize (Zea mays L.) seedlings. In concentration-dependent transport studies, the kinetics for putrescine uptake could be resolved into a single saturable component that was noncompetitively inhibited by increasing concentrations of Ca²⁺ (50 micromolar to 5 millimolar). Similarly, other polyvalent cations, including Mg²⁺ (1.8 millimolar) and La³⁺ (200 micromolar), almost completely abolished the saturable component for putrescine uptake. This suggests that putrescine does not share a common transport system with other divalent or polyvalent inorganic cations. Further characterization of the putrescine transport system indicated that 0.3 millimolar N-ethyl-maleimide had no effect on putrescine uptake, and 2 millimolar p-chloromercuribenzene sulfonic acid only partially inhibited transport of the diamine (39% inhibition). Metabolic inhibitors, including carbonylcyanide-m-chlorphenylhydrazone (20 micromolar) and KCN (0.5 millimolar), also partially inhibited the saturable component for putrescine uptake (V_max reduced 48-60%). Increasing the time of exposure to carbonylcyanide-m-chlorphenylhydrazone from 30 minutes to 2 hours did not significantly increase the inhibition of putrescine uptake. Electrophysiological evidence indicates that the inhibitory effect on putrescine uptake by these inhibitors is correlated to a depolarization of the membrane potential, suggesting that the driving force for putrescine uptake is the transmembrane electrical potential across the plasmalemma.     

ROS generation and proline metabolism in calli of halophyte Nitraria tangutorum Bobr. to sodium nitroprusside treatment

Nitric oxide (NO) is a stress factor or a signal molecule involved in various plant physiological and developmental processes. In the present study, the generation of reactive oxygen species and the metabolism of proline due to different sodium nitroprusside (SNP, an NO donor) concentrations were investigated in callus from halophyte Nitraria tangutorum Bobr. Treatment with SNP led to significant increases of hydrogen peroxide (H₂O₂) content and cell viability but notable reductions in hydrogen radical level and lipid peroxidation degree, and superoxide onion (O₂ ^?) content also enhanced in 100 μM SNP-treated calli. Using a chemical inhibitor for plasma membrane (PM) NADPH oxidase diphenylene iodonium (DPI), we found low O₂ ^? generation in untreated and 25 μM SNP-treated calli, whereas in those treated with 100 μM SNP O₂ ^? level exhibited a very little alteration, comparable to the absence of DPI. These suggest a high activity of PM NADPH oxidase in untreated calli. H₂O₂ scavenging enzymes (catalase, peroxidase [POD] and ascorbate peroxidase) and H₂O₂ forming enzymes (superoxide dismutase [SOD], cell wall-POD and diamine oxidase [DAO]) stimulated significantly in calli treated with different SNP concentrations while glutathione reductase activity decreased. In addition, a reduction in proline content was observed in SNP-treated calli. Moreover, different SNP concentrations stimulated proline dehydrogenase (PDH) and ornithine δ-aminotransferase but inhibited r-glutamyl kinase (GK). In conclusion, our results suggest that the increasing H₂O₂ generation was associated with the stimulation of SOD, cell wall-POD and DAO, and that the reduction of proline content might be the consequence of increased PDH activity and decreased GK activity in N. tangutorum Bobr. calli under SNP treatment.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.