Characterisation of a salt-stimulated ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg²⁺ and in the presence of Mg²⁺ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl^->HCO ₃ ^- >Br^->malate>acetate>SO ₄ ^2- . For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH ₄ ⁺ Na⁺>K⁺). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na⁺ and K⁺. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in V_max rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO₃ but other inhibitors tested (azide, oligomycin, orthovanadate, K₃[Cr(oxalate)₆] and ethyl-3-[3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.Abbreviations ATP_F free ATP - ATP_T total ATP - BSA bovine serum albumen - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DNP 2,4-dinitrophenol - EDAC ethyl-3-(3-dimethylaminopropyl)carbodiimide - K_m apparent Michaelis constant - MgATP complex of Mg²⁺ and ATP - Mg _F ²⁺ free Mg²⁺ - Mg _T ² total Mg²⁺ - MES 2-(N-Morpholino)ethanesulphonic acid - Na₂EDTA disodium ethylenediaminetetraacetic acid - NEM N-ethylmaleimide - P_i inorganic phosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine - V_max maximum velocity     

Mg2+-Dependent,cation-stimulated inorganic pyrophosphatase associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Vacuoles isolated from storage roots of red beet (Beta vulgaris L.) posess a Mg²⁺-dependent, alkaline pyrophosphatase (PPase) activity which is further stimulated by salts of monovalent cations. The requirement for Mg²⁺ is specific. Mn²⁺ and Zn²⁺ permitted only 20% and 12%, respectively, of the PPase activity obtained in the presence of Mg²⁺ while Ca²⁺, Co²⁺ and Cu²⁺ were ineffective. Stimulation of Mg²⁺-PPase activity by salts of certain monovalent cations was due to the cation and the order of effectiveness of the cations tested was K⁺=Rb⁺=NH ₄ ⁺ >Cs⁺. Salts of Li⁺ and Na⁺ inhibited Mg²⁺-PPase activity by 44% and 24%, respectively. KCl-stimulation of Mg²⁺-PPase activity was maximal with 60–100 mM KCl. There was a sigmoidal relationship between PPase activity and Mg²⁺ concentrations which resulted in markedly non-linear Lineweaver-Burk plots. At pH 8.0, the optimal [Mg²⁺]:[PP_i] ratio for both Mg²⁺-PPase and (Mg²⁺+KCl)-PPase activities was approximately 1:1, which probably indicates MgP₂O₇ ^2- is the true substrate.Abbreviations BSA bovine serum albumen - EDTA ethylenediamine tetra-acetic acid, disodium salt - MES 2-(N-morpholino)ethanesulphonic acid - Mg _T ²⁺ total magnesium - P_i inorganic phosphate - PPase inorganic pyrophosphatase - PP_i inorganic pyrophosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine     

ATPase and acid phosphatase activities associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Phosphatase activities were measured in preparations of vacuoles isolated from storage roots of red beet (Beta vulgaris L.). The vacuoles possessed both acid phosphatase and ATPase activities which could be distinguished by their susceptibility to inhibition by low concentrations of ammonium molybdate [(NH₄)₆Mo₇O₂₄·4H₂O]. The acid phosphatase was completely inhibited by 100 M ammonium molybdate but the ATPase was unaffected. The acid phosphatase was a soluble enzyme which hydrolysed a large number of phosphate esters and had a pH optimum of 5.5. In contrast, the ATPase was partially membrane-bound, had a pH optimum of 8.0 and hydrolysed ATP preferentially, although it was also active agianst PP_i, GTP and GDP. At pH 8.0 both the ATPase and PPase activities were Mg²⁺-dependent and were further stimulated by KCl. The ATPase and PPase activities at pH 8.0 may be different enzymes. The recovery and purification of the ATPase during vacuole isolation were determined. The results indicate that the Mg²⁺-dependent, KCl-stimulated ATPase activity is not exclusively associated with vacuoles.Abbreviations BSA bovine serum albumen - MES 2-(N-Morpholino)ethanesulphonic acid - MOPS 3-(N-Morpholino)propanesulphonic acid - Na₂EDTA ethylenediaminetetra-acetic acid, disodium salt - P_i inorganic phosphate - PP_i inorganic pyrophosphate - PPase inorganic pyrophosphatase - TCA trichloroacetic acid - TES N-tris(hydroxymethyl)methyl-2-amino-ethanesulphonic acid - Tris tris(hydroxymethyl)methylamine     

Determination of the membrane potential of vacuoles isolated from red-beet storage tissue

The membrane potential of isolated vacuoles of red beet (Beta vulgaris L.) was estimated using several methods. The quenching of the fluorescence of the cyanine dyes 3,3-diethylthiodicarbocyanine iodide (DiS-C₂–(5)) and 3,3-dipropylthiodicarbocyanine iodide (DiS-C₃–(5)) in vacuoles indicated a transmembrane potential difference, negative inside at low external potassium concentrations. The was found to be-55 mV with two other methods, the distribution of ²⁰⁴T1⁺ in the presence of valinomycin and the distribution of the lipophilic cation triphenylmethylphosphonium. Uncouplers reduced this value to-35 mV. High external potassium concentrations, comparable to cytosolic values, abolished the membrane potential almost completely. The addition of 1 mM Tris-Mg²⁺-ATP markedly hyperpolarized the membrane to-75 mV. This effect was prevented by inhibitors of the ATPase activity located in isolated vacuole membranes.Abbreviations ANS aminonaphthalene sulfonate - DiS-C₂–(5) 3,3-diethylthiodicarbocyanine iodide - DiS-C₃–(5) 3,3-dipropylthiodicarbocyanine iodide - EDAC 1-ethyl-3-C-3dimethylaminopropylcarbodiimide - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - MES morpholinoethylsulfonic acid - TPP⁺ tetraphenylphoshonium - TPMP triphenylmethylphosphonium - Tris tris(hydroxymethyl)aminomethane     

The function of tonoplast ATPase in intact vacuoles of red beet is governed by direct and indirect ion effects

The pH gradient and the electric potential across the tonoplast in mechanically isolated beetroot vacuoles has been studied by following the uptake of [¹⁴C]methylamine and [¹⁴C]triphenyl-methylphosphoniumchloride. In response to Mg-ATP, the vacuolar interior is acidified by 0.8 units. This strong acidification is accompanied by a slight hyperpolarization of the membrane potential, which is probably caused by a proton diffusion potential. In preparations where only a small acidification (0.4 units) occurred, the membrane potential was depolarized by the addition of Mg-ATP. Different monovalent cations and anions were tested concerning their effect on the pH gradient and ATPase activity in proton-conducting tonoplasts. Chloride stimulation and NO ₃ ^- inhibition were clearly present. The observed decline of the pH gradient upon the addition of Na⁺ salts is probably caused by an Na⁺/H⁺ antiport system.Abbreviations and symbol CCCP carbonylcyanide-m-chlorophenylhydrazone - Mes 2(N-morpholino)ethanesulfonic acid - TPMP⁺ triphenylmethylphosphoniumchloride - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - membrane potential Dedicated to Professor A. Betz on the occasion of his 65th birthday     

Accumulation of sucrose in vacuoles isolated from red beet tissue

Vacuoles were isolated from red beets (Beta vulgaris L.) by slicing the tissue and separated using a discontinuous dextran gradient centrifugation. The uptake of sucrose against a concentration gradient into the dextran-impermeable [³H]-H₂O space of these organelles was studied using silicone layer filtering centrifugation on both fluorometric and ¹⁴C-measurement of sucrose. The rate is 24 nmol sucrose (unit betacyanin)^-1 h^-1 and appears to be stimulated by ATP to an uptake rate of 34 nmol. Control experiments with slices cut from red beet tissue and incubated with [¹⁴C]sucrose gave comparable results. An ATPase activity dependent on both Mg²⁺ and K⁺ seems to be localized at the inner surface of the tonoplast. This activity is strongly inhibited by EDAC and tartrate and there is no effect of oligomycin, whereas a slight stimulation was caused by DCCD.Abbreviation CCCP carbonylcyanide-m-chlorophenylhydrazone - EDAC ethyl-3(3-dimethylaminopropylcarbodiimide) - EDTA ethylenediamine tetraacetic acid - fr.wt. fresh weight - HEPES n-2-hydroxyethylepiperazine-n-2-ethanesulfonic acid - MES 2(n-morpholino)ethane sulfonic acid - P_i inorganic phosphate - Tris tris-(hydroxymethyl)-aminomethan Dedicated to A.L. Kursanov, Moscow, on his 75th birhday     

Surcose transport in isolated plasma-membrane vesicles from sugar beet (Beta vulgaris L.) Evidence for an electrogenic sucrose-proton symport

An analysis of the molecular mechanism of sucrose transport across the plasmalemma was conducted with isolated plasma-membrane (PM) vesicles. Plasma membrane was isolated by aqueous two-phase partitioning from fully expanded sugar beet (Beta vulgaris L.) leaves. The isolated fraction was predominantly PM vesicles as determined by marker-enzyme analysis, and the vesicles were oriented right-side-out as determined by structurally linked latency of the PM enzyme, vanadate-sensitive Mg²⁺-ATPase. Sucrose uptake was investigated by equilibrating PM vesicles in pH 7.6 buffer and diluting them 20-fold into pH 6.0 buffer. Using this pH-jump technique, vesicles accumulated acetate in a pH-dependent, protonophore-sensitive manner, which demonstrated the presence of a pH gradient (pH) across the vesicle membrane. Addition of sucrose to pH-jumped PM vesicles resulted in a pH-dependent, protonophoresensitive uptake of sucrose into the vesicles. Uptake was sucrose-specific in that a 10-fold excess of mannose, glucose, fructose, mannitol, melibiose, lactose or maltose did not inhibit sucrose accumulation. The rate of pH-dependent uptake was saturable with respect of sucrose concentration and had an apparent K _m, of 0.45 mM. Sucrose uptake was stimulated approximately twofold by the addition of valinomycin and K⁺, which indicated an electrogenic sucrose-H⁺ symport. Membrane potentials () were imposed across the vesicle membrane using valinomycin and K⁺. A membrane potential, negative inside, stimulated pH-dependent sucrose uptake while a , positive inside, inhibited uptake. Conditions that produce a negative in the absence of a pH gradient supported, although weakly, sucrose uptake. These data support an electrogenic sucrose-H⁺ symport as the mechanism of sucrose transport across the PM in Beta leaves.Abbreviations and symbols CCCP carbonyl cyanide m-chlorophenylhydrazone - cyt cytochrome - PM plasma-membrane(s) - electrical potential difference     

Evidence for the involvement of a UDP-glucose-dependent group translocator in sucrose uptake into vacuoles of storage roots of red beet

Vacuoles isolated from the storage roots of red beet (Beta vulgaris L.) accumulate sucrose via two different mechanisms. One mechanism transports sucrose directly, and its rate is increased by the addition of MgATP. The other mechanism utilizes uridine diphosphate glucose (UDP-glucose) to synthesize and simultaneously transport sucrose phosphate and sucrose into the vacuole. This group translocation mechanism has also been found in sugarcane vacuoles. As in sugarcane, the beet group translocator does not require fructose 6-phosphate, nor is the latter substance transported into the vacuole. The uptake of UDP[¹⁴C]glucose in inhibited by high concentrations of osmoticum.Abbreviations EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - UDP uridine 5-diphosphate     

Characteristics of the sucrose uptake system of vacuoles isolated from red beet tissue. Kinetics and specifity of the sucrose uptake system

The uptake of sucrose against a concentration gradient into the dextran-impermeable [³H]H₂O space of red beet (Beta vulgaris L.) vacuoles has been studied using silicone-layer-filtering centrifugation on both fluorometric and ¹⁴C-measurement of sucrose. Sucrose transport into vacuoles proceeds partly by an active transport system and partly by passive permeation. The K _M(20°C) for active sucrose uptake was found to be about 22 mM and the V _Max(20°C) was about 174 nmol sucrose x (unit betacyanin)^-1 x h^-1. The temperature dependency of sucrose transport appears to have an activation energy of 35,0 KJ×mol^-1. Among various mono-, di-, and trisaccharides tested, raffinose acts as a competitive inhibitor of sucrose uptake.Abbreviations EDTA ethylenediamine tetraacetic acid - fr. wt. fresh weight - Tris tris-(hydroxymethyl)-aminomethan     

Sucrose-dependent H+ transport in plasma-membrane vesicles isolated from sugarbeet leaves (Beta vulgaris L.)

The mechanism of sucrose transport across the plasma membrane (PM) was investigated in membrane vesicles isolated from sugarbeet (Beta vulgaris L.) leaves. In the presence of a membrane potential () generated as a K⁺-diffusion potential, negative inside, sucrose induced a rapid and transient alkalization of the medium. Alkalization was inhibited by carbonyl cyanide m-chlorophenylhydrazone, was specific for the sucrose sugar and was dependent on the sucrose concentration with a K_m of approx. 1 mM. Sucrose-induced alkalization and sucrose transport were inhibited by the sulfhydryl-reactive reagent, p-chloromercuribenzene sulfonic acid, and by the histidine-reactive reagent, diethyl pyrocarbonate. Parallel analysis of sucrose uptake and alkalization indicated that the stoichiometry of sucrose uptake to proton consumed was 11. These results provide clear evidence that the saturable mechanism of sucrose transport across the PM in plants is a coupled H⁺-sucrose symport.Abbreviations and Symbols CCCP carbonyl cyanide m-chlorophenylhydrazone - DEPC diethyl pyrocarbonate - PCMBS p-chloromercuribenzene sulfonic acid - pH pH gradient - membrane potential difference - PM plasma membrane The financial support for a portion of thus study was provided by the Deutsche Forschungsgemeinschaft. We thank Kimberly A. Mitchell for her excellent technical assistance and dedicate this report to the memory of Mr. William A. Dungey.     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

Basic peroxidases in isolated vacuoles of nicotiana tabacum L.

Vacuoles of tobacco mesophyll and of suspension-cultured cells were isolated in order to study the localization of peroxidase isoenzymes. Only basic peroxidases were detectable by electrophoretic separation of the vacuolar sap. Some of the basic peroxidases have formerly been described as an ionically bound cell-wall fraction. This fraction, however, was found to be an artifact produced by incomplete cell breakage. Reinvestigation of isolated cell walls confirmed that mainly acidic peroxidases are localized in the cell walls where they move freely or are bound. As a consequence of former and present results we think it probable that all of the peroxidase isoenzymes are secretory proteins because they have to be transported from the sites of synthesis in the cytoplasm to the sites of function, the extracytoplasmic spaces, cell wall (acidic peroxidases), and vacuole (basic peroxidases).Abbreviation ER endoplasmic reticulum - PAGE polyacrylamide gel electrophoresis     

Transport of phenylalanine into vacuoles isolated from barley mesophyll protoplasts

The energy-dependent transport of phenylalanine into isolated vacuoles of barley (Hordeum vulgare L.) mesophyll protoplasts has been studied by silicone-layer floatation filtering. The uptake of this aromatic amino acid into the vacuolar compartment is markedly increased by MgATP, showing saturation kinetics; the K _m values were 0.5 mM for MgATP and 1.2 mM for phenylalanine. V _max for phenylalanine transport was estimated to 140 nmol phenylalanine·(mg·Chl)^-1·h^-1. The transport shows a distinct pH optimum at 7.3 and is markedly inhibited by 40 mM nitrate. Azide (1 mM) and vanadate (400 M) had no or little effect on rates of transport while p-fluorophenylalanine seemed to be an effective inhibitor, indicating a possible competition at an amino-acid carrier. Ionophores such as valinomycin, nigericin or gramicidin were strong inhibitors of phenylalanine transport, indicating that this process is coupled to both the transmembrane pH gradient (pH) and the transmembrane potential ().Abbreviations and symbols BSA bovine serum albumin - Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - pH transmembrane pH gradient - transmembrane potential     

Short-term effects of Al3+ on the osmotic behavior of red beet (Beta vulgaris L.) protoplasts

The short-term (less than 10 min) effects of Al³⁺ on the biophysical properties of plasma membranes were investigated by time-series image analysis of osmotically-induced volumetric and morphologic changes of red beet (Beta vulgaris L.) protoplasts. Exposure to Al³⁺ under hypotonic conditions reduced the volumetric expansion of protoplasts and their resultant burst: i.e. lysis of protoplasts in a concentration-dependent manner. Under hypertonic conditions, protoplasts exposed to Al³⁺ underwent an enhanced volumetric contraction in cross-sectional area, while maintaining higher protoplast roundness. The residual effects of Al³⁺ pre-treatment on subsequent osmotic behavior were also examined, and protoplasts pre-treated with Al³⁺ also exhibited less lysis during subsequent exposure to hypotonic conditions and enhanced volumetric contractions and higher roundness under subsequent hypertonic conditions. Under our experimental conditions, Al³⁺ consistently minimized protoplast surface area by inhibiting osmotic expansion or by enhancing osmotic contraction, as well as by maintaining higher protoplast roundness. These results suggested that the electrostatic property of Al³⁺ might have induced the binding and possible cross-linking of negatively-charged sites on the plasma membrane surface. This may be an important factor in understanding the mechanism of Al³⁺ phytotoxicity.     

An attempt to use isolated vacuoles to determine the distribution of sodium and potassium in cells of storage roots of red beet (Beta vulgaris L.)

Vacuoles isolated from red beet (Beta vulgaris L.) storage roots contain Na⁺ and K⁺ but their analysis does not give reliable information about the size of vacuolar pools of these ions in vivo. Analyses of isolated vacuoles indicated that between 53% and 90% of the Na⁺ was located in the vacuole and that the vacuolar concentrations of Na⁺ ranged between 4 and 45 mol m^-3. Calculated concentrations of K⁺ in the vacuoles varied between 32 and 72 mol m^-3 but, in contrast to Na⁺, only about 50% of the K⁺ was located in the vacuole. Considerations of the likely cytoplasmic concentrations of Na⁺ and K⁺ suggest that if these results indicate conditions in vivo a large proportion of these ions must be located in the extracellular space, where they would exert considerable osmotic pressure. To test this, the effect of washing on cell turgor (measured directly with a pressure probe) and on loss of Na⁺ and K⁺ was determined. Washing caused an increase in turgor of 5 bar but losses of Na⁺ and K⁺ were less than predicted by the experiments with isolated vacuoles. It is concluded that beet vacuoles leak Na⁺ and K⁺ when isolated resulting in an underestimation of the size of vacuolar pools of these cations in vivo. Nonetheless, the turgor measurements provide evidence for the presence of osmotically active solute in the extracellular space. The possible contribution of extracellular Na⁺ and K⁺ to the observed turgor reduction is calculated and the physiological importance of the accumulation of extracellular solutes is discussed.     

A linkage map of sugar beet (Beta vulgaris L.)

Summary We have established a first linkage map for beets based on RFLP, isozyme and morphological markers. The population studied consisted of 96 F₂ individuals derived from an intraspecific cross. As was expected for outbreeding species, a relatively high degree of polymorphism was found within sugar beet; 47% of the DNA markers were polymorphic for the chosen population. The map consists of 115 independent chromosomal loci designated by 108 genomic DNA probes, 6 isozyme and one morphological marker. The loci cover 789 cM with an average spacing of 6.9 cM. They are dispersed over nine linkage groups corresponding to the haploid chromosome number of Beta species. Eighteen markers (15.4%) showed distorted segregation which, in most instances, can be explained by gametic selection of linked lethal loci. The application of the linkage map in sugar beet breeding is discussed.     

Activity of the tetramer and octamer of glutamine synthetase isoforms during primary leaf ontogeny of sugar beet (Beta vulgaris L.)

In extracts from the primary leaf blade of sugar beet (Beta vulgaris L.) we separated a chloroplastic isoform (GS 2) of glutamine synthetase (GS, EC 6.3.1.2) and one or two (depending on leaf age) cytosolic isoforms (GS 1a and GS 1b). The latter were prominent in the early (GS 1a) and late stages of leaf ontogeny (GS 1a and GS 1b), whereas during leaf maturation GS 2 was the predominantly active GS isoform. The GS 1 isoforms were active exclusively in the octameric state although tetrameric GS 1 protein was detected immunologically. Their activity stayed at a relatively constant level during leaf ontogeny; an increase was observed only in the senescent leaf. The activity of GS 2, however, changed drastically during primary leaf ontogeny and was modulated by changes in the oligomeric state of the active enzyme. In the early and late stages of leaf ontogeny when GS 2 activity was low (lower than that of the GS 1 isoforms), GS 2 was active only in the octameric state. In the maturing leaf, when GS 2 activity had reached its maximum level (much higher than that of the GS 1 isoforms), 80 of total GS 2 activity was due the activity of the tetrameric form of the enzyme and 20 was due to octameric GS 2. Tetrameric GS 2 was a hetero-tetramer and thus not the unspecific dissociation product of homo-octameric GS 2. In addition, GS 2 activity was modulated by an activation/inactivation of the tetrameric GS 2 protein. Due to an activation of the GS 2 tetramer, the activity of tetrameric GS 2 increased during leaf maturation from zero level 23-fold compared with that of GS 1a and 18-fold compared with that of GS 1b. Possible activators of tetrameric GS 2 are thiol-reactive substances. During leaf senescence, GS 2 activity decreased to zero; this decrease was due to an inactivation of the tetrameric GS 2 protein probably caused by oxidation.Abbreviations FLL final lamina length - FPLC fast protein liquid chromatography - GS glutamine synthetase - GHA -glutamyl hydroxamate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Phytohormone effects on hydrolytic activity of phosphohydrolases in red beet (Beta vulgaris L.) tonoplasts

The effects of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA₃) and kinetin on the hydrolytic activity of proton pumps (adenosine triphosphatase, H⁺-ATPase, pyrophosphatase, H⁺-PPase) of tonoplasts isolated from stored red beet (Beta vulgaris L. cv. Bordo) roots were studied. Results suggest that the phytohormones can regulate the hydrolytic activities of H⁺-ATPase and H⁺-PPase of the vacuolar membrane. Each of the proton pumps of the tonoplast has its own regulators in spite of similar localization and functions. IAA and kinetin seem to be regulators of the hydrolytic activity for H⁺-PPase whereas for H⁺-ATPase it may be GA₃. Stimulation of enzyme activity by all hormones occurred at concentrations of 10^–6 to 10^–7 M.Abbreviations IAA indole-3-acetic acid - ABA abscisic acid - GA₃ gibberellic acid - H⁺-ATPase adenosine triphosphatase - H⁺-PPase pyrophosphatase - ATP adenosine triphosphate - Tris Tris (hydroxymethyl)-aminomethane - MES (2[N-Morpholino]) ethane sulfonic acid - EDTA ethylene diamine tetraacetic acid - P_i inorganic phosphate     

Influence of long-term drought stress on osmolyte accumulation in sugar beet (Beta vulgaris L.) plants

Accumulation of various osmolytes was examined in plants of sugar beet cv. Janus grown under two soil water treatments: control (60% of the field water capacity; FWC) and drought (30–35% FWC). The water shortage started on the 61st day after emergence (DAE), at the stage of the beginning of tap-roots development and was imposed for 35 days. Osmotic potential of sugar beet plant organs, particularly tap-roots, was decreased significantly as a consequence of a long-term drought. Water shortage reduced univalent (K⁺, Na⁺) cations concentrations in the petioles and divalent (Ca²⁺, Mg²⁺) ions level in the mature and old leaves. Cation concentrations in the tap-roots were not affected by water shortage. The ratio of univalent to divalent cations was significantly increased in young leaves and petioles as a consequence of drought. Long-term water deficit caused a significant reduction of inorganic phosphorus (P_i) concentration in young and old leaves. Under the water stress condition, the concentration of proline was increased in all individual plant organs, except proline concentration in the youngest leaves. Drought treatment caused a significant increase of glycine betaine content in shoot without any change in tap-roots. Glucose concentrations were significantly increased only in tap-roots as the effect of drought. In response to water shortage the accumulation of sucrose was observed in all the examined leaves and tap-roots. Overall, a long-term drought activated an effective mechanism for osmotic adjustment both in the shoot and in the root tissues which may be critical to survival rather than to maintain plant growth but sugar beet organs accumulate different solutes as a response to water cessation.