Integrity and Respiration of Red Beet Mitochondria Isolated and Examined in Different Sucrose Concentrations

Respiration experiments with succinate as substrate were made with red beet mitochondria isolated in soluitions containing 0.25 to 1.25 M sucrose. The respiration was measured in reaction media adjusted to be 0.25, 0.50, 0.75 or 1.0 osmolar. With mitochondria isolated in 0.25 or 0.50 M sucrose the rate of succinate oxidation was completely dependent on the osmotic pressure of the reaction medium (decreasing with increasing osmotic pressures). Isolation in 0.75 M sucrose caused a slight after-effect of the osmotic pressure of the isolation medium, and by isolation in 1.0 M or 1.25, M sucrose the after-effect was complete. The rate of oxidation was low and independent of the osmotic pressure of the reaction medium. An electron microscopic examination of the state of the mitochondria before and after the respiration period showed that with the conditions used in the present experiments the structure of the mitochondria remained well preserved regardless of the osmotic pressures used.     

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.     

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.     

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.     

Sucrose Phosphate Is Not Transported into Vacuoles or Tonoplast Vesicles from Red Beet (Beta vulgaris) Hypocotyl

Tonoplast vesicles and vacuoles isolated from red beet (Beta vulgaris L.) hypocotyl accumulated externally supplied [¹⁴C]sucrose but not [¹⁴C]sucrose phosphate despite the occurrence of sucrose phosphate phosphohydrolytic activity in the vacuole. The activities of sucrose synthase and sucrose phosphate synthase in whole cell extracts were 960 and 30 nanomoles per milligram protein per minute, respectively; whereas, no sucrose synthesizing activity was measured in tonoplast preparations. The results obtained in this investigation are incompatible with the involvement of sucrose phosphate synthase in the process of sucrose synthesis and accumulation in the storage cells of red beet.     

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.     

免疫沉淀法纯化线粒体的研究

传统的线粒体分离技术,如差速离心和密度梯度离心不能有效应用于小规模高纯度的线粒体制备。为了提高线粒体纯化的速度和质量,我们尝试运用免疫沉淀法制备线粒体。通过用EGFP-4Flag融合蛋白来标记线粒体外膜,然后用单克隆Flag抗体和Protein G Agarose珠子做免疫沉淀,从培养的细胞中分离出高纯度的线粒体。这一简便高效的方法将有助于线粒体的生化研究。     

Effect of Ethylene on Sucrose Uptake in Root Discs of Sugar Beet

Exogenously-added ethylene stimulated active sucrose uptakein root discs of sugar beet (Beta vulgaris L.) in a log dose-linearresponse manner. The ethylene precursor, 1-aminocyclopropane-1-carboxylicacid (ACC) stimulated both endogenous ethylene production andsucrose uptake. Conversely, an inhibitor of ACC synthesis, aminoethoxyvinylglycine(AVG) inhibited both endogenous ethylene production and sucroseuptake. Exogenously-added ethylene can overcome the AVG effecton sucrose uptake. Root tissue from freshly-harvested sugarbeet plants contain gas-phase ethylene levels slightly belowthat required to stimulate active sucrose uptake. No differenceswere found in gas-phase ethylene levels in the root tissue ofsugar beet cultivars having different concentrations of sucrose.The root tissue has an inherent capacity to synthesize ACC andethylene at high rates. Like ethylene, propylene can stimulate active sucrose uptakein beet root discs, but it is not detected in the gas phaseof the tissue. Acetylene, propane, and ethane had no effecton sucrose uptake. Exogenously-added IAA and ABA each make ethylenesensitivetissue insensitive to ethylene stimulation of sucrose uptake.Other plant hormones have no apparent effect on the ethyleneresponse. The role that ethylene may play on sucrose uptakein root tissue of sugar beet is discussed. (Received February 12, 1986; Accepted April 22, 1986)     

Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet

The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na⁺ and K⁺) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m-chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets.     

Sucrose-Phosphate Synthase,Sucrose Synthase,and Invertase in Sugar Beet Leaves

The activity of sucrose-phosphate synthase (SPS) in sugar beet (Beta vulgaris L.) leaves was shown to exceed considerably the synthesizing activity of sucrose synthase (SS). The rise in SPS activity was related to the daylight period; i.e., it was associated with the rate of photosynthesis. The highest SPS activity was characteristic of fully expanded source leaves. In young developing leaves (leaves expanded to less than half of their final size), which represent the sink organs, the SPS activity was 2.5 times lower. At all stages of leaf development, the synthesizing SS activity was rather low. The diurnal change of SS activity was independent of photosynthesis and showed a slight rise from 6:00–8:00 p.m. Under field conditions, the highest SPS activity was found in leaves in the terminal stage of their development (105-day-old plants); the synthesizing activity of SS showed little changes during this period. The activity of soluble acid invertase was characteristic of young leaves. In mature leaves, the activity of this enzyme correlated with the daylight period. These changes occurred on the background of low sucrose content in leaves. The regulation of SPS, SS, and invertase activity is discussed. It is supposed that compartmentation of these enzymes in the photosynthesizing cell is important for transport, metabolism, and the osmotic function of sucrose in leaves.     

DPPC Bilayers in Solutions of High Sucrose Content

The properties of lipid bilayers in sucrose solutions have been intensely scrutinized over recent decades because of the importance of sugars in the field of biopreservation. However, a consensus has not yet been formed on the mechanisms of sugar-lipid interaction. Here, we present a study on the effect of sucrose on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers that combines calorimetry, spectral fluorimetry, and optical microscopy. Intriguingly, our results show a significant decrease in the transition enthalpy but only a minor shift in the transition temperature. Our observations can be quantitatively accounted for by a thermodynamic model that assumes partial delayed melting induced by sucrose adsorption at the membrane interface.     

Development and Characteristics of Sodium-selective Transport in Red Beet

Slices of storage tissue of red beet (Beta vulgaris L.) washed for only 1 day in distilled water readily absorb K⁺ but lack a mechanism for rapid Na⁺ uptake. A Na⁺ transport mechanism develops if the tissue is washed for several days, and the tissue then excludes K⁺ during Na⁺ uptake.     

Characteristics of Sucrose Transport and Sucrose-Induced H+ Transport on the Tonoplast of Red Beet (Beta vulgaris L.) Storage Tissue

Sucrose-induced changes of the energization state of the red beet root (Beta vulgaris L. ssp. conditiva) vacuolar membrane were observed with the fluorescent dyes 6-chloro-9-{[4-(diethylamino)- 1-methylbutyl]-amino}-2-methoxyacridine dihydrochloride, as a pH monitor, and 9-amino-6-chloro-2-methoxyacridine (ACMA). Consequently, transient acidification of the surrounding suspension medium could be measured with a pH electrode. This signal was specific for Suc and was not seen for sorbitol, mannitol, or maltose. Sucrose-induced medium acidification was sensitive to the same inhibitors that were efficient in inhibiting sucrose transport, including the monoclonal antibodies TNP56-12 and C50-5-3. It was seen with vacuoles and vesicles energized with MgATP before sucrose was added but also with vacuoles not artificially energized previously. Using bafilomycin A1 for the inhibition of the vacuolar ATPase of vacuoles previously energized by MgATP, apparent Km values for H+ export from the vacuoles to the medium could be calculated taking into account the passive proton leak. Apparent Km values for H+ export determined from data obtained with pH measurements in the medium and with ACMA corresponded to those obtained previously for sucrose uptake. Comparing sucrose uptake rates with corresponding H+ export rates at the respective sucrose concentrations and at Km, a stoichiometry of approximately one proton per transported sucrose was estimated.     

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.     

Effect of KCl Medium on Malate Oxidation in Mitochondria of Sugar Beet Taproot

Isolated mitochondria were obtained from growing and stored sugar beet (Beta vulgaris L.) taproots. These preparations were used to monitor the mitochondrial matrix volume and malate oxidation after the replacement of sucrose with KCl in the reaction medium. The transfer of mitochondria from sucrose-containing isolation medium to the isoosmotic KCl solution initiated spontaneous or energy-dependent (in the presence of respiratory substrate) swelling whose kinetic parameters (the initial rate and amplitude) were virtually independent of the plant age. At the same time, effects of KCl-induced swelling on oxidative and phosphorylating activities of mitochondria were age-dependent. In mitochondria from growing taproots, K⁺ ions stimulated nonphosphorylating malate oxidation, thereby decreasing the respiratory control ratio and the ADP/O coefficient. The incubation of mitochondria from stored taproots in KCl solution induced a short-term activation and subsequent progressive inhibition of malate oxidation but did not inhibit the oxidation of exogenous NADH. The inhibition of malate oxidation was not released by adding ADP or uncouplers and was enhanced in the presence of valinomycin. The swelling of mitochondria in KCl solutions did not impair the integrity of mitochondrial membranes and did not preclude stimulation of malate oxidation by exogenous NAD. It is supposed that the KCl-induced inhibition of respiration is related to a large increase in the matrix volume and a drastic decrease in the concentration of a coenzyme NAD. Previous studies with isolated mitochondria from stored taproots showed that the mitochondrial NAD level was a rate-limiting factor of malate oxidation assayed in the sucrose-containing media. A possible role of K⁺-transporting mechanisms in regulation of mitochondrial matrix volume and metabolic activity of plant mitochondria is discussed.