Isolation and characterization of the cytosolic and chloroplastic 3-phosphoglycerate kinase from spinach leaves

The cytosol and chloroplast 3-phosphoglycerate kinases (3-PGK) from spinach (Spinacia oleracea L.) were purifled to apparent homogeneity. The procedure included a conventional anion-exchange chromatography on DEAE-cellulose and mainly a series of HPLC columns. The charge differences of the two isoenzymes were so small that separation was only successful by anion-exchange chromatography on a HPLC SynChropak AX 300 column. The portion of the two isoenzmyes in leaf tissue was estimated as 5% and 95%. The major 3-PGK was associated with isolated chloroplasts while the other 3-PGK was only found in the soluble cell fraction. The specific activity of the purified enzymes were in the order of 800 units (per milligram of protein). The molecular weight for the two 3-PGKs under nondenaturing (size exclusion chromatography) and denaturing (SDS-PAGE) conditions were in the order of 40 kilodaltons, with the cytosolic 3-PGK being slightly smaller than the chloroplastic 3-PGK. An antiserum against the chloroplastic 3-PGK showed only 4.6% cross-reaction of the chloroplastic 3-PGK with the cytosolic 3-PGK. The kinetics for glycerate-3-phosphate and MgATP²⁻ were biphasic. The presence of Na₂SO₄ changed the MgATP²⁻ dependence to linearity but not the glycerate-3-phosphate dependence.     

Photorespiratory glycine enhances glutathione accumulation in both the chloroplastic and cytosolic compartments

Transformed poplars overexpressing -glutamylcysteine synthetase (-ECS) in the chloroplast (Lggs) were used to investigate chloroplastic biosynthesis of glutathione (GSH). In Lggs leaves, GSH contents were enhanced by up to 3.7 fold. In general, the highest GSH contents were observed in lines with highest -glutamylcysteine (-EC) contents. These lines had relatively low glycine. In darkness, foliar GSH decreased and -EC increased. Illumination of pre-darkened Lggs in air resulted in a 5-fold decrease in the -EC : GSH ratio. This light-induced decrease was largely abolished if leaves were illuminated at high CO2. Consequently, the -EC : GSH ratio of illuminated leaves was much higher at high CO2 than in air. At high CO2 total foliar amino acids were higher, but glycine and serine were lower, than in air. These results suggest that photorespiratory glycine is used in chloroplastic GSH synthesis. Despite this net CO2 fixation was similar in Lggs to untransformed poplars. Pre-illuminated leaf discs from Lggs, and poplars overexpression -ECS in the cytosol (ggs), were incubated in darkness with a range of metabolites. After 15 h, discs for both types of transformant incubated on water had accumulated high levels of -EC and showed marked increases in the -EC : GSH ratio. Feeding glycine, serine, glycollate or phosphoserine, attenuated the dark-induced changes in the -EC : GSH ratio, whereas 3-phosphoglycerate (PGA), phosphoenolpyruvate, glycerate, and hydroxypyruvate did not. Glycine produced from glycollate was therefore required for maximal GSH accumulation in both the chloroplastic and cytosolic compartment. Production of glycine from PGA failed to meet the demand of increased GSH synthetic capacity.     

Sugar, Amino Acid and Inorganic Contents in Rice Phloem Sap

Rice phloem sap was obtained through severed stylets of brownplanthoppers and its chemical composition was determined. Sucrose,the only sugar detected, was present at 17–25% (w/v) inthe sap. Amino acids were present at a total of 3–8% (w/v)and were found to be mostly in a free form. The amount of boundamino acids was estimated to be small, if any were present.Among the free amino acids, asparagine (17–33% on a molarbasis), glutamate (6–14%), serine (10–13%), glutamine(7–15%), threonine (5–6%) and valine (6–7%)were dominant, while cystine and methionine (0–0.2%) werepresent in minor amounts and -aminobutyric acid was not detected.The sap had a slightly alkaline pH (ca. 8.0). The inorganicconstituents detected by electron probe x-ray microanalyzerwere Na, S, P and K, with the K content being the highest. Theosmotic pressure was estimated to be 13–15 atm. The aminoacid composition of the plant parts was determined and the differencesin the ease of phloem loading among amino acids were compared. (Received October 5, 1981; Accepted December 17, 1981)     

Amino acid sequence of copper,zinc-superoxide dismutase from spinach leaves

The complete amino acid sequence of Cu,Zn-superoxide dismutase (SOD) from spinach leaves has been determined on the basis of peptides obtained by cyanogen bromide (BrCN) cleavage and by enzymic hydrolyses with Achromobacter lyticus lysylendopeptidase, Staphylococcus aureus V8 protease, trypsin, and thermolysin. The spinach SOD consists of a total of 154 amino acid residues with alanine as the amino(N)-terminus and valine as the carboxy(C-)terminus. The present sequence, which has been established for the enzyme from a plant, is also highly homologous to those of the enzymes from other species. Especially, the residues essential for metal binding and enzyme activity have been extensively conserved among all of the Cu,Zn-SODs hitherto analyzed.     

Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves

The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K⁺. The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (ΔpH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K⁺-selective ionophore, valinomycin, stimulated uptake in the presence of a ΔpH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited ΔpH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H⁺/sucrose symporter in PM vesicles isolated from spinach leaves.     

A role for fructose 2,6-bisphosphate in the regulation of sucrose synthesis in spinach leaves

The subcellular distribution of fructose 2,6-bisphosphate in spinach (Spinacia oleracea) leaves was studied using nonaqueous fractionation, showing that all, or almost all, is located in the cytosol. The amount of fructose 2,6-bisphosphate present in leaves during the diurnal cycle was measured and compared to the accumulation of starch and sucrose, and the amounts of selected phosphorylated intermediates in the leaf. Upon illumination, the level of fructose 2,6-bisphosphate decreases, but prolonged illumination leads to an increase in the level to above that found in the dark, which accompanies the onset of rapid accumulation of starch in the leaf.     

Changes in adenosine phosphates and energy charge in chloroplastic and nonchloroplastic compartments of wheat leaves (author's transl)]

Changes in the adenine nucleotides and energy charge (= (ATP)+1/2(ADP)/(AMP)+(ADP)+(ATP)) levels were studied in chloroplastic and non-chloroplastic compartments using non-aqueously isolated wheat leaves chloroplasts. The two adenine nucleotides pools (of chloroplasts and non-chloroplastic part of the cell), though distinct, are linked. This linkage substantiates an energy-rich bond exchange between the two compartments. When both photphosphorylation and oxidative phosphorylation occur simultaneously, energy charge takes high values, generally higher than 0.80. When neither oxidative phosphorylation nor photophosphorylation occur, energy charge is very low and takes values generally lower than 0.45. When one compartment alone produces approximately P, energy charge in the two compartments takes intermediate values which remain relatively high. Dark-light transition in nitrogen resulted in changes of the AMP, ADP and ATP levels which quickly reach a steady state. Chloroplast energy charge shifts rapidly from 0.45 to 0.75 in 10 s; after 1 min it reaches 0.86, a value that corresponds to a steady level. In the cytoplasm, energy charge changes from 0.44 to 0.71 in 1 min. Energy charge increase in the non-chloroplastic compartment substantiates an energy transfer from chloroplasts to the cytoplasm. On nitrogen-air transition in the dark, the cytoplasm energy charge reaches a steady level in 30 s. In chloroplasts, it also increases but slowly. There is indeed a transfer of energy from cytoplasm to chloroplasts. Darkening of the leaves in air causes a drastic and lasting drop of energy charge in the chloroplasts where it has a low value after 5 min in the dark. Then it increases again but slowly and is still lower than 0.70 after 10 min in the dark. Meanwhile, energy charge in cytoplasm keeps values higher than 0.75. Metabolic regulation by energy charge and control of adenine nucleotides level by adenylate kinase (EC 2.7.4.3) are discussed.     

Light/Dark profiles of sucrose phosphate synthase, sucrose synthase, and Acid invertase in leaves of sugar beets

The activity of sucrose phosphate synthase, sucrose synthase, and acid invertase was monitored in 1- to 2-month-old sugar beet (Beta vulgaris L.) leaves. Sugar beet leaves achieve full laminar length in 13 days. Therefore, leaves were harvested at 2-day intervals for 15 days. Sucrose phosphate synthase activity was not detectable for 6 days in the dark-grown leaves. Once activity was measurable, sucrose phosphate synthase activity never exceeded half that observed in the light-grown leaves. After 8 days in the dark, leaves which were illuminated for 30 minutes showed no significant change in sucrose phosphate synthase activity. Leaves illuminated for 24 hours after 8 days in darkness, however, recovered sucrose phosphate synthase activity to 80% of that of normally grown leaves. Sucrose synthase and acid invertase activity in the light-grown leaves both increased for the first 7 days and then decreased as the leaves matured. In contrast, the activity of sucrose synthase oscillated throughout the growth period in the dark-grown leaves. Acid invertase activity in the dark-grown leaves seemed to be the same as the activity found in the light-grown leaves.     

The Relationship between Sugar and Amino Acid Export to the Phloem in Young Wheat Plants

The relationship between amino acid and sugar export to thephloem was studied in young wheat plants (Triticum aestivumL. ‘Pro-INTA, Isla Verde’) using the EDTA-phloemcollection technique. Plants grown with a 16 h photoperiod showeda rapid decrease in the concentration of sugars and amino acidsin the phloem exudate from the beginning of the dark period.When plants grown with a 16 h photoperiod were kept in the darkfor longer than 8 h the free amino acid content in leaves andexudate (on a dry weight basis) increased continually throughoutthe 72 h of darkness. During the first 24 h of darkness thesugars in the phloem exudate decreased to 30% of the initialvalue, and returned to the control level when plants were returnedto light. When plants grown under low light intensity for 10d were transferred to high light intensity, they showed an increasein leaf sugar content (dry weight basis) after 3 d but therewere no differences in leaf free amino acid content (dry weightbasis) compared to low-light plants. The sugar concentrationin the phloem exudate was increased by higher light intensities,but there was no difference in the amino acid concentrationof the phloem exudate, and thus the amino acid:sugar ratio inthe phloem decreased in the high-light plants. The present resultssuggest that amino acids can be exported to the phloem independentlyof the export of sugars. Copyright 1999 Annals of Botany Company Sugar exudation, amino acid transport, nitrogen, phloem, transport, wheat, Triticum aestivum L.     

Free Amino Acid Composition of Phloem Sap and Growing Fruit of Lycopersicon esculentum

Pure phloem sap of tomato leaves was collected by stylectomy.Glutamine and glutamate were the predominant free amino acidstranslocated by the phloem stream. In developing fruits glutaminecontent increased significantly, reaching 35% of the total freeamino acids. Comparison in the amino acid composition betweenthe two tissues are discussed. (Received October 6, 1997; Accepted January 27, 1998)     

Free amino Acid composition of leaf exudates and Phloem sap : a comparative study in oats and barley

Comparisons were made between the free amino acid composition in leaf exudates and that in pure phloem sap, using twin samples taken from a single leaf of two oat (Avena sativa L.) and three barley (Hordeum vulgare L.) varieties. Leaf exudate was collected in a 5 mm EDTA-solution (pH 7.0) from cut leaf blades and phloem sap was obtained through excised aphid (Rhopalosiphum padi L.) stylets. Fluorescent derivatives of amino acids were obtained using 9-fluorenylmethyl chloroformate and were separated by means of high performance liquid chromatography. The total concentration of free amino acids varied considerably in the exudate samples. There was no correlation between the total amino acid content in the exudate samples and that of the corresponding phloem sap samples, but the amino acid composition of the corresponding samples was highly correlated (median R²-value 0.848). There was only limited between-plant variation in phloem sap amino acid composition. Nevertheless, in comparisons involving all samples, many of the amino acids showed significant correlations between their relative amounts in exudate and phloem sap. The results presented here indicate that the exudate technique holds great promise as an interesting alternative to the laborious and time-consuming stylet-cutting technique of obtaining samples for comparative studies of phloem sap.     

Seasonal dynamics of nutrients in leaves and xylem sap of coffee plants as related to different soil compartments

The northwestern province of Costa Rica is a marginal coffee growing area. At the onset of the rainy season low redox potentials probably induce the mobilization of soil Mn resulting in enhanced plant uptake of Mn. To test this hypothesis we monitored from April to the end of June 1995 the mobile Mn in the soil and nutrient and Mn concentrations in leaves and xylem sap of coffee plants. Every 2 weeks we took aggregate and bulk soil samples. The aggregates were mechanically separated into interior and exterior, air-dried and all soil samples were extracted with 1 M NH₄NO₃. We also extracted the field moist soil with distilled water. In addition, the 3rd and the youngest pair of coffee leaves and xylem sap were sampled and analyzed. According to the results of leaf analyses the nutrient supply of the coffee plants in general seemed to be balanced. However, Mn concentrations of 223 mg kg^-1 in the 3rd leaf pair at 18 April were above the optimum and the youngest leaves indicated Fe deficiency, but senescent leaves accumulated Fe and overcame the deficiency. Manganese concentrations in the xylem sap showed a pronounced maximum 2 weeks prior to a similar maximum of mobile Mn in the aggregate exterior. But in general the temporal variation of nutrient concentrations (especially Ca and Mg) in the plants are well correlated with the easily extractable nutrient concentrations in bulk soil. Probably due to its specific absorption and high rates of redistribution within the plant, K in the soil extracts did not correlate with plant concentrations. Element concentrations of youngest leaves could not be correlated with soil concentrations and are not considered to be an adequate tool for monitoring current nutrient uptake. Since plant element concentrations did not correlate with the aggregate interior, plants probably cannot use that nutrient source efficiently.     

Photosynthesis and ion content of leaves and isolated chloroplasts of salt-stressed spinach

Spinach (Spinacia oleracea) plants were subjected to salt stress by adding NaCl to the nutrient solution in increments of 25 millimolar per day to a final concentration of 200 millimolar. Plants were harvested 3 weeks after starting NaCl treatment. Fresh and dry weight of both shoots and roots was decreased more than 50% compared to control plants but the salt-stressed plants appeared healthy and were still actively growing. The salt-stressed plants had much thicker leaves. The salt-treated plants osmotically adjusted to maintain leaf turgor. Leaf K⁺ was decreased but Na⁺ and Cl⁻ were greatly increased.

The potential photosynthetic capacity of the leaves was measured at saturating CO₂ to overcome any stomatal limitation. Photosynthesis of salt-stressed plants varied only by about 10% from the controls when expressed on a leaf area or chlorophyll basis. The yield of variable chlorophyll a fluorescence from leaves was not affected by salt stress. Stomatal conductance decreased 70% in response to salt treatment.

Uncoupled rates of electron transport by isolated intact chloroplasts and by thylakoids were only 10 to 20% below those for control plants. CO₂-dependent O₂ evolution was decreased by 20% in chloroplasts isolated from salt-stressed plants. The concentration of K⁺ in the chloroplast decreased by 50% in the salt-stressed plants, Na⁺ increased by 70%, and Cl⁻ increased by less than 20% despite large increases in leaf Na⁺ and Cl⁻.

It is concluded that, for spinach, salt stress does not result in any major decrease in the photosynthetic potential of the leaf. Actual photosynthesis by the plant may be reduced by other factors such as decreased stomatal conductance and decreased leaf area. Effective compartmentation of ions within the cell may prevent the accumulation of inhibitory levels of Na⁺ and Cl⁻ in the chloroplast.

     

Accumulation of phosphate, sulfate and sucrose by excised Phloem tissues

Excised petiolar vascular bundles and excised phloem tissues have been shown to take up phosphate, sulfate and sucrose by a true accumulation process and against high concentration ratios. Phosphate was accumulated principally as inorganic phosphate, and sucrose principally as sucrose. The rates of accumulation of the 3 solutes into the phloem-containing tissues were from 4 to 35 times higher than into comparable parenchyma tissue. It is suggested that this active accumulation mechanism plays an important role in the phenomenon of phloem transport.

The excised vascular, phloem and parenchyma tissues show an aging phenomenon: aerating the excised tissues for 18 hours prior to their use causes marked changes in the accumulatory behavior of the tissue. The data suggest that 1 phosphate accumulation system of low affinity but high capacity exists in fresh tissue, and that aging allows the development of a second, additional phosphate accumulation mechanism of high affinity and low capacity. A possible role in the control of phosphate movement is suggested.

     

Phloem transport,solute flux and the kinetics of sap exudation in Ricinus communis L.

The osmotic characteristics of phloem-sap exudation were examined in soil-grown and watercultured plants of Ricinus communis L. Prolonged exudation occurred from bark incisions in water-cultured plants. Fresh incisions caused large alterations in solute flux, but phloem-sap solute potential _s changed by less than ±8% over a period of 7 h. This was associated with a constancy in the levels of sucrose and K⁺, the principal solutes in the sap. Studies with foliar-applied tracers and leaf-excision experiments suggested that exudation was maintained by solute loading from mature leaves. A wide range of mass transfer values through the phloem was found, these being a function of exudation rate. We consider that the exudation process possesses essentially similar characteristics to phloem transport in the intact plant. The way in which bark incisions bring about large changes in solute flux is discussed in terms of the physical properties of the sieve-tube system.Abbreviations water potential - _s solute potential - _p pressure potential     

Ionic composition and pH of the vacuolar sap in marine dinoflagellate Noctiluca

Ionic composition of the vacuolar sap of Noctiluca miliariswas as follows: [Na⁺] = 487.3 mM, [K⁺]=24.1 mM, [Ca²⁺]=6.6 mM,[Mg²⁺]=2.8 mM, [Cl^–]=500mM, [NH₄⁺]=15–25 mM, and[SO₄^2–]=undetectable. To measure the vacuolar pH of singleliving cells, a pH-sensitive glass microelectrode was used.The vacuolar pH value was 3.50 ±0.18. When the cellswere transferred from normal sea water into osmotically adjusted50% sea water for one day, the vacuolar ion concentrations remainedalmost constant. Upon immersing the cells in osmotically unadjustedsea water of various concentrations for one day, the observedincrements or decrements of the vacuolar ion concentrationscould be accounted for largely by the migration of water outof or into the cells. The intrinsic ionic composition of thevacuole seems to be constant against changes in ion concentrationsof the bathing medium. (Received October 20, 1975; )     

Phloem loading in Vicia faba leaves: Effect of N-ethylmaleimide and parachloromercuribenzenesulfonic acid on H+ extrusion,K+ and sucrose uptake

The effects of a penetrating (NEM) and a non-penetrating (PCMBS) sulfhydryl-specific reagent on proton extrusion, ⁸⁶Rb and [U-¹⁴C]sucrose uptake by Vicia faba leaves have been studied. Proton extrusion was strongly or completely inhibited by 0.1 mM NEM. ⁸⁶Rb and [U-¹⁴C]sucrose uptake were markedly reduced by NEM concentrations equal to or higher than 0.5 mM. Under our experimental conditions, PCMBS (1 mM) exerted a strong inhibition on [¹⁴C]sucrose uptake but did not inhibit proton extrusion and ⁸⁶Rb uptake. The sensitivity of phloem loading to PCMBS is thought to be a consequence of sugar-carrier blockage and not of inhibition of the proton pump.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - DES diethylstilbestrol - DCCD dicyclohexylcarbodiimide - FC Fusicoccin - NEM N-ethylmaleimide - PCMBS p-chloromercuribenzenesulfonic acid     

Resolution and characterization of multiple cytosolic phosphatases capable of hydrolyzing fructose-1,6-bisphosphate in spinach and soybean leaves

The apparent activity of cytoplasmic fructose bisphosphatase (EC 3.1.3.11) in crude extracts of spinach ( Spinacia oleracea L.) and soybean ( Glycine max [L.] Merr.) leaves was only partially dependent on Mg²⁺. At least two major non-chloroplastic fructose bisphosphatases that differed in dependence on Mg²⁺ were chromatographically resolved from spinach leaves. The Mg²⁺-dependent enzyme had an apparent Michaelis constant of 4 μM for fructose-1,6-P₂, was highly specific, and was strongly inhibited by fructose-2,6-P₂. Enzyme activity was inhibited by physiological levels of fructose-6-P.
Both species also contained at least one major enzyme, the activity of which was independent of Mg²⁺. These enzymes had pH optima near neutrality, Michaelis constants of 25 to 30 μM for fructose-1,6-P₂, and were inhibited by AMP. Although hexose monophosphates were not metabolized, the enzymes were not specific for fructose-1,6-P₂: phosphate was released from phosphoenolpyruvate and ribulose-1, 5-P₂, and with fructose-1,6-P₂, as substrate, Pi release was about 1.5-fold greater than fructose-6-P production. It is concluded that only the Mg²⁺-dependent fructose bisphosphatase, previously characterized, functions in the photosynthetic sucrose formation pathway. Inhibition of the Mg²⁺-dependent enzyme by fructose-6-P may be involved in regulation of sucrose formation.     

Both chloroplastic and cytosolic phosphofructoaldolase isozymes are present in the pea leaf nucleus

Summary. In higher plants, fructose bisphosphate aldolase (EC 4.1.2.13) occurs in chloroplast, cytosol, and nucleus. Immunocytolocalization experiments with isozyme-directed antibodies indicate that both chloroplastic and cytosolic aldolase isoforms are present in the pea (Pisum sativum L.) leaf nucleus. Correspondence and reprints: Department of Biological Sciences m/c 066, University of Illinois-Chicago, 845 West Taylor, Chicago, Illinois 60607-7060, U.S.A.