Sucrose Leakage from Isolated Parenchyma of Sugar Beet Roots

The kinetics of sugar efflux from slices of sugar beet rootswas investigated using washing solutions of different osmoticpressure and calcium concentration. The leakage of sucrose isstrongly reduced in solutions of high osmotic pressure (>0·8MPa) or high calcium concentration (10 mM). Turgor-dependentnecrosis of parenchyma cells (plasmoptysis) is the main causeof sucrose efflux from the tissue in hypotonic media with lowcalcium activity. This was shown by good correlation betweenthe percentage of leaked sucrose and the percentage of tissuewater, which was in the free space after the washing procedure.The kinetics of sugar leakage from beet root parenchyma is nobasis for the estimation of the sugar contents of the free spaceor the cytoplasm in situ.     

Respiration of Mitochondria Isolated from Leaves and Protoplasts of Avena sativa

Mitochondria isolated from mesophyll protoplasts differed from mitochondria isolated directly from leaves of Avena sativa in that protoplast mitochondria (a) had a lower overall respiratory capacity, (b) were less able to use low concentrations of exogenous NADH, (c) did not respond rapidly or strongly to added NAD, (d) appeared to accumulate more oxaloacetate, and (e) oxidized both succinate and tetramethyl-p-phenylene-diamine (an electron donor for cytochrome oxidase) more slowly than did leaf mitochondria. It is concluded that cytochrome oxidase activity was inhibited, the external NADH dehydrogenase had a reduced affinity for NADH, succinate oxidation was inhibited, NAD and oxaloacetate porters were probably inhibited, and accessibility to respiratory paths may have been reduced in protoplast mitochondria. The results also suggest that there was a reduced affinity of a succinate porter for this substrate in oat mitochondria. In addition, all oat mitochondria required salicylhydroxamic acid (SHAM) as well as cyanide to block malate and succinate oxidation. Malate oxidation that did not appear to saturate the cytochrome pathway was sensitive to SHAM in the absence of cyanide, suggesting that the oat mitochondria studied had concomitant alternative and subsaturating cytochrome oxidase pathway activity.     

脱落酸对玉米胚芽和大豆子叶线粒体呼吸的影响

在提取玉米和大豆线粒体后,以ABA处理线粒体,发现在不同底物存在下,ABA均可增加线粒体的耗氧速率,呼吸速率随ABA浓度变化的结合常数（Kd）值为1．43μmol／L。ABA对4态呼吸的促进作用更为显著,因而导致呼吸控制下降,P／O比降低。蛋白质合成抑制剂环己酰亚胺不影响ABA的效应。以ABA预处理大豆子叶,虽然也促进了呼吸作用,但不改变呼吸控制和P／O.     

The Effects of Aqueous Extracts of Red Beet Root on Salt Accumulation and Respiration of Discs of Red Beet Root

An account is given of the preparation of aqueous extracts ofred beet root which are shown to stimulate potassium uptakein beet discs washed for a short period, but inhibit potassiumuptake in discs washed for four days or more. Analysis of extractsshowed them to contain organic anions (especially citrate andmalate) which affect both the metabolic phase of potassium uptakeand respiration of the tissue. The effects of extracts and organicacids on uptake of manganese by beet discs is described andcompared with effects on potassium absorption. The results arediscussed with respect to current theories of salt accumulationand in relation to the hypothesis relating an inhibitor of saltaccumulation to the lag phase of ion uptake by beet discs.     

Glutathione in Intact Vacuoles: Comparison of Glutathione Pools in Isolated Vacuoles,Plastids, and Mitochondria from Roots of Red Beet

Proportions between oxidized and reduced glutathione forms were determined in vacuoles isolated from red beet (Beta vulgaris L.) taproots. The pool of vacuolar glutathione was compared with glutathione pools in isolated plastids and mitochondria. The ratio of glutathione forms was assessed by approved methods, such as fluorescence microscopy with the fluorescent probe monochlorobimane (MCB), high-performance liquid chromatography (HPLC), and spectrophotometry with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). The fluorescence microscopy revealed comparatively low concentrations of reduced glutathione (GSH) in vacuoles. The GSH content was 104 μM on average, which was lower than the GSH levels in mitochondria (448 μM) and plastids (379 μM). The content of reduced (GSH) and oxidized (GSSG) glutathione forms was quantified by means of HPLC and spectrophotometric assays with DTNB. The glutathione concentrations determined by HPLC in the vacuoles were 182 nmol GSH and 25 nmol GSSG per milligram protein. The respective concentrations of GSH and GSSG in the plastids were 112 and 6 nmol/mg protein and they were 228 and 10 nmol/mg protein in the mitochondria. The levels of GSH determined with DTNB were 1.5 times lower, whereas the amounts of GSSG were, by contrast, 1.5–2 times higher than in the HPLC assays. Although the glutathione redox ratios depended to some extent on the method used, the GSH/GSSG ratios were always lower for vacuoles than for plastids and mitochondria. In vacuoles, the pool of oxidized glutathione was higher than in other organelles.     

Diurnal Changes in the Sugar and Organic Anion Concentrations in Red Beet Leaves

Marked diurnal fluctuations in the concentrations of ethanolsoluble sugars and organic anions occurred in red beet leaves.These fluctuations were related to solar radiation and air temperature.Leaves from four plots which had received different manurialtreatments contained significantly different mean sugar andanion concentrations, but the patterns of the diurnal fluctuationswere not affected by the manurial treatments. The results indicated a daily production of photosynthate equalto about 19 per cent per day (leaf-weight basis), compared tothe net assimilation rate of 17 per cent per day as estimatedfor similar plants from dry-weight increase. From the results no evidence can be adduced to support the hypothesisthat sugar accumulated in the leaves to an extent which inhibitedphotosynthesis.     

The Control of Iron-Induced Oxidative Damage in Isolated Rat-Liver Mitochondria by Respiration State and Ascorbate

The reaction of iron (II) with H₂O₂ is believed to generate highly reactive species (e.g., OH) capable of initiating biological damage. This study investigates the possibility that the severity of oxidative damage induced by iron in hepatic mitochondria is determined by the level of mitochondrial-H₂O₂ generation, which is believed to be particularly prominent in state-4 respiration.

Iron-induced damage is found to be greater in state-4 than in state-3 respiration. Experiments using uncoupling agents and Ca⁺⁺ to mimic state-3 conditions indicate that this effect reflects differences in the steady-state oxidation-level of the electron carriers of the respiratory chain (and hence the level of H₂O₂ -generation). rather than changes in redox potential or transportation of the metal-ion. Evidence is also presented for a mechanism in which Fe(II) and H₂O₂ react inside the mitochondrial matrix.

Ascorbate (vitamin C) is shown to be pro-oxidant in this system. except when present at very high concentration when it becomes antioxidant in nature.     

Effects of Anions on Swelling, Respiration, and Phosphorylation of Isolated Corn Mitochondria

The effects of a series of anions on swelling, respiration, and oxidative phosphorylation of corn mitochondria were studied. Active mitochondrial swelling similar to that found with HPO₄⁻² was demonstrated in the presence of IO₃⁻, NO₂⁻, MoO₄⁻², SO₄⁻², HAsO₄⁻², acetate, S₂O₃⁻², SeO₄⁻², CrO₄⁻², and WoO₄⁻². In general, those anions which caused active swelling also released respiration and reduced the efficiency of oxidative phosphorylation with exogenous NADH as substrate. The degree of passive swelling in the presence of certain of the monovalent anions was found to approximate the order of the lyotropic series (SCN⁻ > CIO₄⁻ > I⁻ > NO₃⁻ > CI⁻).     

The Control of Iron-Induced Oxidative Damage in Isolated Rat-Liver Mitochondria by Respiration State and Ascorbate

The reaction of iron (II) with H₂O₂ is believed to generate highly reactive species (e.g., OH) capable of initiating biological damage. This study investigates the possibility that the severity of oxidative damage induced by iron in hepatic mitochondria is determined by the level of mitochondrial-H₂O₂ generation, which is believed to be particularly prominent in state-4 respiration.

Iron-induced damage is found to be greater in state-4 than in state-3 respiration. Experiments using uncoupling agents and Ca⁺⁺ to mimic state-3 conditions indicate that this effect reflects differences in the steady-state oxidation-level of the electron carriers of the respiratory chain (and hence the level of H₂O₂ -generation). rather than changes in redox potential or transportation of the metal-ion. Evidence is also presented for a mechanism in which Fe(II) and H₂O₂ react inside the mitochondrial matrix.

Ascorbate (vitamin C) is shown to be pro-oxidant in this system. except when present at very high concentration when it becomes antioxidant in nature.     

Cyanide-insensitive Respiration in Plant Mitochondria

Pathways of electron transport have been studied in mitochondria isolated from hypocotyls of etiolated mung bean seedlings and skunk cabbage spadices that show cyanide-resistant respiratory activity. The residual flux through cytochrome c oxidase is shown to be small in comparison with the flux through an unidentified alternative oxidase that is known to have a high affinity for oxygen. This alternative oxidase is not a cytochrome. Skunk cabbage and mung bean mitochondria contain cytochromes a and a₃ that have absorption peaks differing slightly from those of animal preparations. A slow oxidation-reduction of cytochrome a₃-CN has been demonstrated. Cytochromes b undergo oxidation and reduction in the presence of cyanide but play no essential role in the cyanide-resistant pathway. Antimycin inhibits to an extent similar to that of cyanide; the respiratory chain bifurcates on the substrate side of the antimycin-sensitive site. Evidence is presented for the selective inhibition by thiocyanate, α, α′-dipyridyl, and 8-hydroxyquinoline of the alternative oxidase pathway, which may therefore contain a non-heme iron protein.     

Hormonal Control of Sucrose Phosphate Synthase and Sucrose Synthase in Sugar Beet

We studied the effects of synthetic analogs of phytohormones (benzyladenine, IAA, and GA) on the activities of the enzymes catalyzing sucrose synthesis and metabolism, sucrose phosphate synthase (SPS, EC 2.4.1.14) and sucrose synthase (SS, EC 2.4.1.13), and on the content of chlorophyll and protein during the sugar-beet (Beta vulgaris L.) ontogeny. Plant spraying with phytohormonal preparations activated SPS in leaves; direct interaction between phytohormones and the enzyme also increased its activity. The degree of this activation differed during the ontogeny and in dependence on the compound used for treatment. Analogs of phytohormones maintained high protein level in leaves, retarded chlorophyll breakdown, and, thus, prolonged leaf functional activity during development. Phytohormonal preparations practically did not affect the SS activity both after plant treatment and at their direct interaction with the enzyme. It is supposed that the SS activity in sugar-beet roots is controlled by sucrose synthesized in leaves rather than by phytohormones. The effects of hormones on leaf metabolism were mainly manifested in growth activation.     

Respiration of Mitochondria in Developing Sunflower Seeds

We studied the oxidative and phosphorylating activity of mitochondria in the seeds of three sunflower cultivars (Polevik, Peredovik, and Yubileinyi) during development of the seed embryo within 1 to 54 days after flowering. The rates of succinate oxidation by the mitochondria were 1.5–2 times those of malate or -ketoglutarate oxidation. The ratio of substrate oxidation rates underwent changes during the seed growth. The differences were recorded between cultivars as concerns the times when the maximum oxidation rates were reached. Oxidation was coupled with phosphorylation during the entire period of seed development: the value of respiratory control after Chance changed from 1.4 to 7. By the time of transition to maturation, the rates of oxidation of both substrates and the values of respiratory control and ADP/O decreased. The results we obtained suggest that by days 13–15 of seed embryo growth, the rate of ATP production decreases upon oxidation of Krebs cycle products.     

Selective Impairment of Respiration in Mitochondria Isolated from Brain Subregions Following Transient Forebrain Ischemia in the Rat

Using Percoll density gradient centrifugation, free (nonsynaptosomal) mitochondria were isolated from the dorsal-lateral striatum and paramedian neocortex of rats during complete forebrain ischemia and reperfusion. Mitochondria prepared from either region after 30 min of ischemia showed decreased state 3 (ADP and substrate present) and uncoupled respiration rates (19-45% reductions) with pyruvate plus malate as substrates, whereas state 4 respiration (no ADP present) was preserved. At 6 h of recirculation, state 3 and uncoupled respiration rates for mitochondria from the paramedian neocortex (a region resistant to ischemic damage) were similar to or even increased compared with control values. By contrast, in mitochondria from the dorsal-lateral striatum (a region containing neurons susceptible to global ischemia), decreases in state 3 and uncoupled respiration rates (25 and 30% less than control values) were again observed after 6 h of recirculation. With succinate as respiratory substrate, however, no significant differences from control values were found in either region at this time point. By 24 h of recirculation, respiratory activity with either pyruvate plus malate or succinate was greatly reduced in samples from the dorsal-lateral striatum, probably reflecting complete loss of function in some organelles. In contrast with these marked changes in free mitochondria, the respiratory properties of synaptosomal mitochondria, assessed from measurements in unfractionated homogenates, were unchanged from controls in the dorsal-lateral striatum at each of the time points studied, but showed reductions (19-22%) during ischemia and after 24 h of recirculation in the paramedian neocortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Influence of Internal Ion Concentration on Potassium Accumulation and Salt Respiration of Red Beet Root Tissue

It is shown that during a period of washing in serated distilledwater disks of Red Beet root tissue acquire the capacity toabsorb potassium rapidly. Subsequently the rate of accumulationof this ion is closely related to the internal salt-contentof the material. On the other hand, whilst the level of saltrespiration increases during the preliminary washing, it isnot influenced significantly by the internal potassium concentration.The implications of these observations are discussed in relationto a possible mechanism of mineral salt absorption.     

Cyanide-resistant Respiration of Sweet Potato Mitochondria

The oxidation of malate and succinate by sweet potato mitochondria (Ipomoea batatas [L.] Lam.) was blocked only partly by inhibitors of complexes III (2-heptyl-4-hydroxyquinoline-N-oxide) and IV (cyanide and azide). The respiration insensitive to inhibitors of complexes III and IV was inhibited by salicylhydroxamic acid. Essentially complete inhibition was obtained with inhibitors of complex I (rotenone, amytal, and thenoyltrifluoroacetone) and complex II (thenoyltrifluoroacetone). The observations indicated that electrons were transferred to the cyanide-resistant pathway from ubiquinone or from nonheme iron (iron-sulfur) proteins of complexes I and II before reaching the b cytochromes. In contrast, the oxidation of exogenous NADH did not involve the alternate pathway, as indicated by complete inhibition by inhibitors of complexes III and IV and the absence of an effect of inhibitors of complexes I and II. Hence, electrons from exogenous NADH appear to pass directly to complex III in sweet potato mitochondria.     

Phosphate-induced Stimulation of Acceptorless Respiration in Corn Mitochondria

By use of the organic mercurial mersalyl to block phosphate transport, it has been shown that only a small fraction of the respiratory increase of corn mitochondria in response to additions of inorganic phosphate is due to energy expended in phosphate accumulation. Most of the respiratory release occurs from accelerated turnover of the coupling mechanism with internal phosphate in an oligomycin-sensitive reaction. Addition of ADP to mersalyl-blocked mitochondria depletes internal phosphate in ATP formation and respiration declines. Arsenate produces the same responses as phosphate but is more effective in respiratory release.     

Translocation of C Sucrose in Sugar Beet during Darkness

The time-course of arrival of ¹⁴C translocate in a sink leaf was studied in sugar beet (Beta vulgaris L. cultivar Klein Wanzleben) for up to 480 minutes of darkness. Following darkening of the source leaf, translocation rapidly declined, reaching a rate approximately 25% of the light period rate by 150 minutes. Comparison of data from plants that were girdled 1 cm below the crown with data from ungirdled plants indicates that after about 150 minutes darkness the beet root becomes a source of translocate to the sink leaf. After about 90 minutes darkness, starch-like reserve polysaccharide from the source leaf begins to contribute ¹⁴C to ethanol soluble pools in that leaf. Because of a 15% isotope mass effect, sucrose, at isotopic saturation, reaches a specific activity which is about 85% of the level of the supplied CO₂. The source leaf sucrose specific activity remains at the isotopic saturation level for about 150 minutes of darkness, after which time input from polysaccharide reserves causes the specific activity to drop to about 55% of that of the supplied CO₂. Sucrose specific activity determinations, polysaccharide dissolution measurements, and pulse labeling experiments indicate that following partial depletion of the sucrose pool, source leaf polysaccharide contributes to dark translocation. Respired CO₂ from the source leaf appears to be derived from a pool which, unlike sucrose, remains at a uniform specific activity.