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.     

Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles

The substrate and ionic requirements of ATP and inorganic pyrophosphate (PPi) hydrolysis by tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were compared with the requirements of ATP-and PPi-dependent proton translocation by the same material. Both ATP hydrolysis and ATP-dependent proton translocation are most stimulated by Cl⁻ and inhibited by NO₃⁻. NaCl and KCl support similar rates of ATP hydrolysis and ATP-dependent proton translocation while K₂SO₄ supports lesser rates for both. PPi hydrolysis and PPi-dependent proton translocation are most stimulated by K⁺. KCl and K₂SO₄ support similar rates of PPi hydrolysis and PPi-dependent proton translocation but NaCl has only a small stimulatory effect on both. Since PPi does not inhibit ATP hydrolysis and ATP does not interfere with PPi hydrolysis, it is inferred that the two phosphohydrolase and proton translocation activities are mediated by different tonoplast-associated enzymes. The results indicate the presence of an energy-conserving proton-translocating pyrophosphatase in the tonoplast of red beet.     

Proton Transport in Plasma Membrane and Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : A Comparative Study of Ion Effects on DeltapH and DeltaPsi

The proton transport properties of plasma membrane and tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue were examined and compared. Membrane vesicles isolated with 250 millimolar KCl in the homogenization media and recovered at low density following sucrose density gradient centrifugation displayed characteristics of proton transport (nitrate inhibition, no inhibition by orthovanadate, pH optimum of 7.75, pyrophosphate-driven proton transport) which were consistent with a tonoplast origin. When the KCl in the homogenization medium was replaced by 250 millimolar KI, sealed membrane vesicles were recovered at higher densities in sucrose gradients and displayed properties (orthovanadate sensitivity, no inhibition by nitrate, pH optimum of 6.5) consistent with a plasma membrane origin. A comparison of anion effects (potassium salts) upon ΔpH and ΔΨ revealed a direct correspondence between the relative ability of anions to stimulate proton transport and reduce ΔΨ. For tonoplast vesicles, the relative order for this effect was KI > KBr ≥ KCl > KClO₃ > K₂SO₄ while for plasma membrane vesicles, a different order KI > KNO₃ ≥ KBr ≥ KClO₃ > KCl > K₂SO₄ was observed. Proton transport in plasma membrane and tonoplast vesicles was inhibited by fluoride; however, plasma membrane vesicles appeared to be more sensitive to this anion. In order to correlate anion effects in the two vesicle fractions with anion transport, the kinetics of anion stimulation of steady-state pH gradients established in the absence of monovalent ions was examined. Anions were added as potassium salts and the total potassium concentration (100 millimolar) was maintained through the addition of K⁺/Mes. For plasma membrane vesicles, chlorate and nitrate displayed saturation kinetics while chloride displayed stimulation of proton transport which followed a linear profile. For tonoplast vesicles, the kinetics of chloride stimulation of proton transport displayed a saturable component. The results of this study indicate differences in proton transport properties of these two vesicle types and provide information on conditions where proton transport in the two fractions can be optimized.     

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : II. Characterization of Ca Uptake into Plasma Membrane Vesicles

Calcium uptake was examined in sealed plasma membrane vesicles isolated from red beet (Beta vulgaris L.) storage tissue using (45)Ca(2+). Uptake of (45)Ca(2+) by the vesicles was ATP-dependent and radiotracer accumulated by the vesicles could be released by the addition of the calcium ionophore A23187. The uptake was stimulated by gramicidin D but slightly inhibited by carbonylcyanide m-chlorophenylhydrazone. Although the latter result might suggest some degree of indirect coupling of (45)Ca(2+) uptake to ATP utilization via deltamuH(+), no evidence for a secondary H(+)/Ca(2+) antiport in this vesicle system could be found. Following the imposition of an acid-interior pH gradient, proton efflux from the vesicle was not enhanced by the addition of Ca(2+) and an imposed pH gradient could not drive (45)Ca(2+) uptake. Optimal uptake of (45)Ca(2+) occurred broadly between pH 7.0 and 7.5 and the transport was inhibited by orthovanadate, N,N'-dicyclohexylcarbodiimide, and diethylstilbestrol but insensitive to nitrate and azide. The dependence of (45)Ca(2+) uptake on both calcium and Mg:ATP concentration demonstrated saturation kinetics with K(m) values of 6 micromolar and 0.37 millimolar, respectively. While ATP was the preferred substrate for driving (45)Ca(2+) uptake, GTP could drive transport at about 50% of the level observed for ATP. The results of this study demonstrate the presence of a unique primary calcium transport system associated with the plasma membrane which could drive calcium efflux from the plant cell.     

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.     

CHAPS Solubilization and Functional Reconstitution of beta-Glucan Synthase from Red Beet Root (Beta vulgaris L.) Storage Tissue

A method for the solubilization and reconstitution of red beet (1,3)-β-d-glucan synthase with the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) was developed. Glucan synthase was effectively solubilized from microsomal or plasma membranes by 0.6% CHAPS in the presence of EGTA and EDTA. Chelators were found essential for effective solubilization and divalent cations inhibitory. A preextraction of membranes with 0.3% CHAPS and 5 millimolar Mg²⁺ prior to the solubilization step was found to remove protein contaminants and increase the specific activity of the solubilized enzyme. Conditions for recovering activity from Sepharose 4B gel filtration columns were defined. Addition of phospholipids and low levels of CHAPS in column elution buffers resulted in complete functional reconstitution with 100% recovery of added activity. Specific activities were increased 20- to 22-fold over microsomes. Active vesicles were recovered by centrifugation. These results provide independent and direct confirmation of the enzyme's requirement for a phospholipid environment.     

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : I. Characterization of a Ca-Pumping ATPase Associated with the Endoplasmic Reticulum

Calcium transport was examined in microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue using chlorotetracycline as a fluorescent probe. This probe demonstrates an increase in fluorescence corresponding to calcium accumulation within the vesicles which can be collapsed by the addition of the calcium ionophore A23187. Calcium uptake in the microsomal vesicles was ATP dependent and completely inhibited by orthovanadate. Centrifugation of the microsomal membrane fraction on a linear 15 to 45% (w/w) sucrose density gradient revealed the presence of a single peak of calcium uptake which comigrated with the marker for endoplasmic reticulum. The calcium transport system associated with endoplasmic reticulum vesicles was then further characterized in fractions produced by centrifugation on discontinous sucrose density gradients. Calcium transport was insensitive to carbonylcyanide m-chlorophenylhydrazone indicating the presence of a primary transport system directly linked to ATP utilization. The endoplasmic reticulum vesicles contained an ATPase activity that was calcium dependent and further stimulated by A23187 (Ca(2+), A23187 stimulated-ATPase). Both calcium uptake and Ca(2+), A23187 stimulated ATPase demonstrated similar properties with respect to pH optimum, inhibitor sensitivity, substrate specificity, and substrate kinetics. Treatment of the red beet endoplasmic reticulum vesicles with [gamma-(32)P]-ATP over short time intervals revealed the presence of a rapidly turning over 96 kilodalton radioactive peptide possibly representing a phosphorylated intermediate of this endoplasmic reticulum associated ATPase. It is proposed that this ATPase activity may represent the enzymic machinery responsible for mediating primary calcium transport in the endoplasmic reticulum linked to ATP utilization.     

Kinetics of Ca/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris L

Artificial pH gradients across tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were used to study the kinetics of a Ca²⁺/H⁺ antiport across this membrane. Ca²⁺-dependent H⁺ fluxes were measured by the pH-dependent fluorescence quenching of acridine orange. ΔpH-dependent Ca²⁺ influx was measured radiometrically. Both H⁺ efflux and Ca²⁺ influx displayed saturation kinetics and an identical dependence on external calcium with apparent K_m values of 43.9 and 41.7 micromolar, respectively. Calcium influx was unaffected by an excess of Mg²⁺ but was inhibited by La³⁺ > Mn²⁺ > Cd²⁺. The apparent K_m for external calcium was greatly affected (5-fold) by internal pH in the range of 6.0 to 6.5 and a transmembrane effect of internal proton binding on the affinity for external calcium is suggested.     

Na/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris

The pH-dependent fluorescence quenching of acridine orange was used to study the Na⁺- and K⁺-dependent H⁺ fluxes in tonoplast vesicles isolated from storage tissue of red beet and sugar beet (Beta vulgaris L.). The Na⁺-dependent H⁺ flux across the tonoplast membrane could be resolved into two components: (a) a membrane potential-mediated flux through conductive pathways; and (b) an electroneutral flux which showed Michaelis-Menten kinetics relationship to Na⁺ concentration and was competitively inhibited by amiloride (K_i = 0.1 millimolar). The potential-dependent component of H⁺ flux showed an approximately linear dependence on Na⁺ concentration. In contrast, the K⁺-dependent H⁺ flux apparently consisted of a single component which showed an approximately linear dependence on K⁺ concentration, and was insensitive to amiloride. Based on the Na⁺- and K⁺-dependent H⁺ fluxes, the passive permeability of the vesicle preparation to Na⁺ was about half of that to K⁺.

The apparent K_m for Na⁺ of the electroneutral Na⁺/H⁺ exchange varied by more than 3-fold (7.5-26.5 millimolar) when the internal and external pH values were changed in parallel. The results suggest a simple kinetic model for the operation of the Na⁺/H⁺ antiport which can account for the estimated in vivo accumulation ratio for Na⁺ into the vacuole.

     

Determination of H/ATP Stoichiometry for the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue

The H⁺/ATP stoichiometry was determined for the plasma membrane H⁺-ATPase from red beet (Beta vulgaris L., var Detroit Dark Red) storage tissue associated with native vesicles. The determination of H⁺/ATP stoichiometry utilized a kinetic approach where rates of H⁺ influx, estimated by three different methods, were compared to rates of ATP hydrolysis measured by the coupled enzyme assay under identical conditions. These methods for estimating H⁺ influx were based upon either determining the initial rate of alkalinization of the external medium from pH 6.13, measuring the rate of vesicle H⁺ leakage from a steadystate pH gradient after stopping the H⁺-ATPase or utilizing a mathematical model which describes the net transport of H⁺ at any given point in time. When the rate of H⁺ influx estimated by each of these methods was compared to the rate of ATP hydrolysis, a H⁺/ATP stoichiometry of about 1 was observed. In consideration of the maximum free energy available from ATP hydrolysis (ΔG_atp), this value for H⁺/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasma membrane in vivo.     

Proton-Coupled Sucrose Transport in Plasmalemma Vesicles Isolated from Sugar Beet (Beta vulgaris L. cv Great Western) Leaves

Sucrose is the predominant form of photosynthetically reduced carbon transported in most plant species. In the experiments reported here, an active, proton-coupled sucrose transport system has been identified and partially characterized in plasmalemma vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves. The isolated vesicles concentrated sucrose fivefold in the presence of an imposed pH gradient (basic interior). The presence of carbonyl cyanide m-chlorophenylhydrazone, a protonophore, prevented sucrose accumulation within the vesicles. ΔpH-dependent sucrose transport exhibited saturation kinetics with an apparent K_m of 1.20 ± 0.40 millimolar, suggesting translocation was carrier-mediated. In support of that conclusion, two protein modifiers, diethyl pyrocarbonate and p-chloromercuribenzenesulfonic acid, were found to be potent inhibitors with 50% inactivation achieved at 750 and 30 micromolar, respectively. ΔpH-Dependent sucrose transport was not inhibited by glucose, fructose, raffinose, or maltose suggesting the transport system was specific for sucrose. Transport activity was associated with the plasmalemma because ΔpH-dependent sucrose transport equilibrated on a linear sucrose gradient at 1.17 grams per cubic centimeter and comigrated with a plasmalemma enzyme marker, vanadate-sensitive K⁺, Mg²⁺-ATPase. Taken together, these results provide the first In vitro evidence in support of a sucrose-proton symport in the plasmalemma of mature leaf tissue.     

Isolation of Vacuoles from Root Storage Tissue of Beta vulgaris L

Morphologically intact and osmotically active vacuoles were isolated from root storage tissue of the red beet Beta vulgaris L., and the factors influencing both yield and stability of the vacuoles were determined. Successful isolation depended upon slicing the tissue in an apparatus specifically designed to cut open plant cells without the use of high shear forces and to liberate cellular organelles into an undisturbed reservoir of osmoticum. The resulting brei was centrifuged at 2,000g for 10 min to yield a pellet which contained many vacuoles but which also contained tissue fragments, nuclei, mitochondria, and plastids. The vacuoles were further purified by accelerated flotation through a Metrizamide step gradient. Biochemical assays, light microscopy, and electron microscopy confirmed that there was only trace contamination of the final vacuole preparation by other organelles. Isolated vacuoles were intact and retained their in vivo coloration.     

Effects of Inorganic Phosphate on the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.)

The effects of inorganic phosphate on the plasma membrane H⁺-ATPase of red beet (Beta vulgaris L.) were studied. ATPase activity was inhibited weakly and noncompetitively by phosphate. This anion also relieved the inhibition caused by vanadate by displacing it from the enzyme. From this effect, a dissociation constant for phosphate of 25 millimolar and an extrapolated activity at infinite phosphate concentration of 84% of the activity without inhibitors were calculated. The partial inhibition by phosphate indicates the existence of a catalytically active enzyme-phosphate complex. In the presence of 24% dimethylsulfoxide, the inhibition of ATPase activity by phosphate is much greater than in its absence. This suggests that the active enzyme-phosphate complex could be converted into a covalent phosphoenzyme through a dehydration promoted by the low water activity of the medium. The inhibitory ability of phosphate in 24% dimethylsulfoxide was dependent on the presence of potassium. Potassium ions increased both the affinity for phosphate and the inhibition caused by an infinite phosphate concentration, suggesting that potassium stimulates both phosphate binding and phosphoenzyme formation.     

Phosphorylation by Inorganic Phosphate of the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.)

The phosphorylation of plasma membrane proteins from red beet (Beta vulgaris L.) by radioactive inorganic phosphate was studied. Only few proteins were phosphorylated, among them was one polypeptide with an apparent molecular weight of about 100,000. The phosphorylation of this protein was decreased when orthovanadate was present in the reaction mixture, or when the phosphorylated protein was treated with hydroxylamine. These facts suggest that this protein is a transport ATPase which is phosphorylated in a carboxyl group during the catalytic cycle. This protein was identified immunologically as the plasma membrane H⁺-ATPase. The phosphorylation level of this enzyme was enhanced by dimethyl sulfoxide, whereas potassium ions did not have a significant effect on this level unless ATP was present. ATP stimulated the phosphorylation by inorganic phosphate. This stimulation was more apparent in the presence of potassium ions.     

Enzymes Involved in the Postharvest Degradation of Sucrose in Beta vulgaris L. Root Tissue

The reducing sugar content of sugar beet (Beta vulgaris L.) roots increased during 30 days of storage at 21 C and 160 days at 5 C as a result of an increase in acid invertase activity. Sucrose synthetase and neutral invertase activities were high at harvest but declined during storage, thus showing no relationship with postharvest reducing sugar accumulation in sugar beet roots. Acid α-glucosidase activity was detected in fresh roots but showed no activity with sucrose as a substrate.     

UDP-Glucose-Dependent Sucrose Translocation in Tonoplast Vesicles from Stalk Tissue of Sugarcane

Tonoplast vesicles isolated from stalk parenchyma tissue of sugarcane plants transport sucrose via a uridine diphosphate glucose (UDPGlc)-dependent group translocator. No sucrose transport via an ATP-dependent system could be detected. The products of UDPGlc uptake in the vesicles were sucrose and sucrose phosphate which, upon hydrolysis with alkaline phosphatase and invertase, showed that both hexose moieties are derived from UDPGlc.     

DeltapH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet (Beta vulgaris L.) Leaves: II. Evidence for Multiple Aliphatic, Neutral Amino Acid Symports

Proton-coupled aliphatic, neutral amino acid transport was investigated in plasma membrane vesicles isolated from sugar beet (Beta vulgaris L., cv Great Western) leaves. Two neutral amino acid symport systems were resolved based on inter-amino acid transport competition and on large variations in the specific activity of each porter in different species. Competitive inhibition was observed for transport competition between alanine, methionine, glutamine, and leucine (the alanine group) and between isoleucine, valine, and threonine (the isoleucine group). The apparent K_m and K_i values were similar for transport competition among amino acids within the alanine group. In contrast, the kinetics of transport competition between these two groups of amino acids did not fit a simple competitive model. Furthermore, members of the isoleucine group were weak transport antagonists of the alanine group. These results are consistent with two independent neutral amino acid porters. In support of that conclusion, the ratio of the specific activity of alanine transport versus isoleucine transport varied from two- to 13-fold in plasma membrane vesicles isolated from different plant species. This ratio would be expected to remain relatively stable if these amino acids were moving through a single transport system and, indeed, the ratio of alanine to glutamine transport varied less than twofold. Analysis of the predicted structure of the aliphatic, neutral amino acids in solution shows that isoleucine, valine, and threonine contain a branched methyl or hydroxyl group at the β-carbon position that places a dense electron cloud close to the α-amino group. This does not occur for the unbranched amino acids or those that branch further away, e.g. leucine. We hypothesize that this structural feature of isoleucine, valine, and threonine results in unfavorable steric interactions with the alanine transport system that limits their flux through this porter. Hydrophobicity and hydrated volumes did not account for the observed differences in transport specificity.     

Purification of an Acid Invertase from Washed Discs of Storage Roots of Red Beet (Beta vulgaris L)

The purification of an acid invertase from washed discs of storageroots of red beet (Beta vulgaris L.) is described. An overallpurification of 1210-fold was obtained using a combination of(NH₄)₂SO₄ precipitation, size-exclusion chromatography, ion-exchangechromatography, conA-sepharose chromatography and two roundsof FPLC on Mono Q HR 5/5, the first at pH 7·5, the secondat pH 6·5. The purified enzyme had a specific activityof 206     

Agrobacterium tumefaciens-Mediated Genetic Transformation of Sugar Beet (Beta vulgaris L.)

Genetic transformation of sugar beet (Beta vulgaris L.) cv Bella (2n = 3x = 27) cv Dipple Ero (2n = 2x = 18) and accession SVP31-188 (2n = 2x = 18) was investigated by Infecting in vitro-derived shoot base segments with Agrobacterium tumefaciens strain LBA 4404 carrying either pBin 19 or pSI 121 plasmid. MS media supplemented with 0.5 mg.l^-1 naphthalene acetic acid, 0.25 mg.l^-1 6-benzylaminopurine and 100 βg.ml^-l kanamycin were effective for the production of callus and shoots from Agrobacterium Infected explants irrespective of the method of infection. Most of pSin 19- and all of pSI 121-medlated transformants failed to root on selective rooting media. Only 11.1% of pSin 19-mediated Sella transformants rooted on media containing 0.5 mg.l^-1 kinetin and 100 βg.ml^-1 kanamycin and all of them were the product of co-cultivation In the presence of acetosyringone. In the case of pSI 121-mediated transformants as many as 40–50 copies of the GUS gene was determined to be integrated with tandem repeats into at least six different sites of cv Sella genome. Fluorometrlc assay also demonstrated the transformed nature of a number of sugar beet shoots.     

甜菜胞质型谷氨酰胺合成酶基因组DNA的克隆

以甜菜叶片为材料,用CTAB法提取基因组DNA.以分段PCR法扩增得到了完整的甜菜胞质型谷氨酰胺合成酶(GS1)基因组DNA.采用RT-PCR法扩增此GS1基因(GS1)的cDNA序列应用于对照.获得了长度为9 606bp的完整的GS1 DNA序列和长度为1 068 bp的GSI cDNA序列.分析GS1基因组DNA序列表明,它包含13个外显子,被12个内含子分隔开.其外显子区与已公布的GS1 mRNA序列的相似性达99.5%.RT-PCR法获得的cDNA序列与已知的GS1 mRNA序列相似性达99.6%.而2次实验中GS1基因组DNA外显子区与GS1 cDNA序列的相似性达99.9%.GenBank登录号为EU370974.