Diurnal fluctuations in cotton leaf carbon export, carbohydrate content, and sucrose synthesizing enzymes

In fully expanded leaves of greenhouse-grown cotton (Gossypium hirsutum L., cv Coker 100) plants, carbon export, starch accumulation rate, and carbon exchange rate exhibited different behavior during the light period. Starch accumulation rates were relatively constant during the light period, whereas carbon export rate was greater in the afternoon than in the morning even though the carbon exchange rate peaked about noon. Sucrose levels increased throughout the light period and dropped sharply with the onset of darkness; hexose levels were relatively constant except for a slight peak in the early morning. Sucrose synthase, usually thought to be a degradative enzyme, was found in unusually high activities in cotton leaf. Both sucrose synthase and sucrose phosphate synthetase activities were found to fluctuate diurnally in cotton leaves but with different rhythms. Diurnal fluctuations in the rate of sucrose export were generally aligned with sucrose phosphate synthase activity during the light period but not with sucrose synthase activity; neither enzyme activity correlated with carbon export during the dark. Cotton leaf sucrose phosphate synthase activity was sufficient to account for the observed carbon export rates; there is no need to invoke sucrose synthase as a synthetic enzyme in mature cotton leaves. During the dark a significant correlation was found between starch degradation rate and leaf carbon export. These results indicate that carbon partitioning in cotton leaf is somewhat independent of the carbon exchange rate and that leaf carbon export rate may be linked to sucrose formation and content during the light period and to starch breakdown in the dark.     

Possible control of maize leaf sucrose-phosphate synthase activity by light modulation

Sucrose phosphate synthase (SPS) activity was measured in extracts of maize (Zea mays L.) and soybean (Glycine max L. [Merr.]) leaves over a single day/night cycle. There was a 2- to 3-fold postillumination increase in extractable enzyme activity in maize leaves, whereas the activity of soybean SPS was only about 30% higher in extracts prepared from light- compared to dark-adapted leaves. Alterations in extractable maize leaf SPS activity correlated with light/dark transitions suggesting that the enzyme may be light modulated. Diurnal variations of extractable maize leaf SPS activity were also observed in a greenhouse experiment. A transition from high (light) to low (dark) extractable SPS activity occurred near the light compensation point for photosynthesis (about 20 micromole photons per square meter per second). Further increases in irradiance did not increase extractable SPS activity. Substrate affinities for uridine 5′-diphosphoglucose (Michaelis constant = 3.5 and 5.1 millimolar) and fructose-6 phosphate (half maximal concentration = 1.0 and 2.5 millimolar) were lower for partially purified SPS obtained from light compared to dark acclimated maize leaves. Light-induced changes in extractable SPS activity were stable for at least one column chromatography step. The above results indicate that light-induced changes in SPS activity may be important in controlling the photosynthetic production of sucrose.     

Ligh-dependent activity of glutamate synthase in vitro

Glutamate synthase from rice (Oryza sativa) green leaves was assayed in a chloroplast reconstituted system. The enzyme activity was totally dependent on externally supplied thylakoid membranes and ferredoxin in the light. Glutamate synthase activity was also detected from etiolated leaves with photoreduced ferredoxin as an electron donor.     

An investigation of the mechanism of activation of tryptophan by tryptophanyl-tRNA synthetase from beef pancreas

Exposure of dark-grown Euglena to white or red light, but not blue light, produced a twofold increase in the specific activity of citrate synthase. A 400-fold purification of mitochondrial citrate synthase (subunit Mr = 44000) was achieved from cells of Euglena gracilis by affinity chromatography on ATP-activated agarose. Antisera, raised against the homogeneously pure enzyme, were used to demonstrate that the increase in citrate synthase activity on exposure of dark-grown cells to light resulted from an increase in citrate synthase protein. Anti-(citrate synthase) was used to detect precursor citrate synthase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. Citrate synthase mRNA was found to be present in cells at all stages of regreening. However, extraction and translation of polyadenylated RNA from free polysomes isolated from darkgrown and regreening cells demonstrated that appreciable translation of citrate synthase mRNA was only occurring in regreening cells.     

Changes of Sucrose-Phosphate Synthase Activity in Barley Primary Leaves during Light/Dark Transitions

The activity of sucrose-phosphate synthase (SPS) in 9-day-old barley (Hordeum vulgare L.) primary leaves was measured over a 24-hour period. Extractable enzyme activity was constant in the light, decreased 50 to 60% during the first one-half hour of darkness, and then returned to full activity before the start of the normal light period. Decreases of SPS activity in the dark were fully reversed by less than 10 minutes of illumination. In contrast to results with barley, the measurable activity of SPS in soybean, spinach, and pea leaves was unchanged during the first hour of darkness. Changes of SPS activity in barley primary leaves were stable upon gel filtration. The exact biochemical mechanism responsible for the enzyme activity changes in barley leaf extracts is unknown. The above findings support the suggestion by de Fekete (1973 Eur J Biochem, 10: 73-80) that SPS is controlled by posttranslational protein modification. These results are discussed in relation to the regulation of photosynthetic sucrose metabolism.     

Characterization of Citrate Synthase from Geobacter sulfurreducens and Evidence for a Family of Citrate Synthases Similar to Those of Eukaryotes throughout the Geobacteraceae

Members of the family Geobacteraceae are commonly the predominant Fe(III)-reducing microorganisms in sedimentary environments, as well as on the surface of energy-harvesting electrodes, and are able to effectively couple the oxidation of acetate to the reduction of external electron acceptors. Citrate synthase activity of these organisms is of interest due to its key role in acetate metabolism. Prior sequencing of the genome of Geobacter sulfurreducens revealed a putative citrate synthase sequence related to the citrate synthases of eukaryotes. All citrate synthase activity in G. sulfurreducens could be resolved to a single 49-kDa protein via affinity chromatography. The enzyme was successfully expressed at high levels in Escherichia coli with similar properties as the native enzyme, and kinetic parameters were comparable to related citrate synthases (k_cat = 8.3 s⁻¹; K_m = 14.1 and 4.3 μM for acetyl coenzyme A and oxaloacetate, respectively). The enzyme was dimeric and was slightly inhibited by ATP (K_i = 1.9 mM for acetyl coenzyme A), which is a known inhibitor for many eukaryotic, dimeric citrate synthases. NADH, an allosteric inhibitor of prokaryotic hexameric citrate synthases, did not affect enzyme activity. Unlike most prokaryotic dimeric citrate synthases, the enzyme did not have any methylcitrate synthase activity. A unique feature of the enzyme, in contrast to citrate synthases from both eukaryotes and prokaryotes, was a lack of stimulation by K⁺ ions. Similar citrate synthase sequences were detected in a diversity of other Geobacteraceae members. This first characterization of a eukaryotic-like citrate synthase from a prokaryote provides new insight into acetate metabolism in Geobacteraceae members and suggests a molecular target for tracking the presence and activity of these organisms in the environment.     

Selective measurement of starch synthesizing enzymes in permeabilized potato tuber slices

Osmotically permeabilized potato (Solanum tuberosum L.) tuber slices were used to study the biosynthesis of starch under semi in vivo conditions. Criteria to distinguish the various enzymes involved in starch biosynthesis were developed based on the characteristics of the enzymes in in vitro experiments. Branching enzyme activity was inhibited at pH 8.5 or higher, while the starch synthases functioned optimally between pH 8.8 and 9.1. Unprimed soluble starch synthase activity was only apparent in the presence of sodium citrate (0.4 molar or higher). Granulebound and primed soluble starch synthase were active in the absence of sodium citrate. Primed soluble starch synthase activity was susceptible to inhibition by 10 millimolar zinc sulfate, while granule-bound starch synthase activity was not. The incorporation of the Glc moiety of ADP-Glc into starch in tissue slices by the various starch synthases was consistent with in vitro data with respect to the affinity of the enzymes for substrate, the pH profile, the stimulation by citrate, and the inhibition by zinc sulfate. These data were used to determine the activity of each of the starch synthases in tissue slices: granule-bound and soluble starch synthase transferred 37 and 55 picomoles ADP-Glc per hour per milligram fresh weight into starch of permeabilized tissue slices at 30°C and pH 9.1. In the presence of 0.5 molar sodium citrate, at least 40 picomoles ADP-Glc per hour per milligram fresh weight as transferred into starch by unprimed soluble starch synthase activity.     

Engineered citrate synthase alters Acetate Accumulation in Escherichia coli

Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such as Escherichia coli. A wide range of biochemicals are derived from the central carbon metabolite acetyl-CoA, and the largest native drain of acetyl-CoA in most microbes including E. coli is entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by the gltA gene). Since the pathway to any biochemical derived from acetyl-CoA must ultimately compete with citrate synthase, a reduction in citrate synthase activity should facilitate the increased formation of products derived from acetyl-CoA. To test this hypothesis, we integrated into E. coli C ΔpoxB twenty-eight citrate synthase variants having specific point mutations that were anticipated to reduce citrate synthase activity. These variants were assessed in shake flasks for growth and the production of acetate, a model product derived from acetyl-CoA. Mutations in citrate synthase at residues W260, A267 and V361 resulted in the greatest acetate yields (approximately 0.24 g/g glucose) compared to the native citrate synthase (0.05 g/g). These variants were further examined in controlled batch and continuous processes. The results provide important insights on improving the production of compounds derived from acetyl-CoA.     

Regulation of citrate synthase activity ofSaccharomyces cerevisiae

Citrate synthase activity ofSaccharomyces cerevisiae was determined by a radioactive assay procedure and the reaction product,¹⁴C-citric acid, was identified by chromatographic techniques. ATP, d-ATP, GTP and NADPH were most inhibitory to the citrate synthasein vitro. The activity was inhibited to a lesser extent by ADP, UTP, and NADP whereas, AMP and CTP were much less inhibitory. NADH, like NAD, glutamic acid, glutamine, arginine, ornithine, proline, aspartic acid and α-ketoglutarate exhibited no inhibition. These results have been discussed in the light of the role of citrate synthase for the energy metabolism and glutamic acid biosynthesis.     

Changes in Starch Formation and Activities of Sucrose Phosphate Synthase and Cytoplasmic Fructose-1,6-bisphosphatase in Response to Source-Sink Alterations

Short term experiments were conducted with vegetative soybean plants (Glycine max L. Merr. `Ransom' or `Arksoy') to determine whether sourcesink manipulations, which rapidly changed the `demand' for sucrose and partitioning of photosynthetically fixed carbon into starch, were associated with alterations in activities of sucrose-P synthase and/or cytoplasmic fructose-1,6-bisphosphatase in leaf extracts. When demand for sucrose from a particular source leaf was increased by defoliation of other source leaves, starch accumulation was restricted and activities of both enzymes were markedly enhanced. When demand for sucrose from source leaves was limited by excision, starch accumulation in the detached leaves was increased while activity of sucrose-P synthase declined sharply. The consistent responsiveness of sucrose-P synthase activity to changes in demand for sucrose supports the contention that regulation of sucrose-P synthase is an integral component of the system which controls sucrose biosynthesis and partitioning of carbon between starch and sucrose biosynthesis in the light.     

Role of malate synthase in citric Acid synthesis by maturing cotton embryos: a proposal

Cotton embryos from 34 to 54 days after anthesis were analyzed for organic acids, and enzymes associated with organic acid metabolism. During this developmental period, embryos accumulated citrate. Malate synthase activity appeared at 46 days after anthesis and increased rapidly to 54 days. Of other enzymes examined, only citrate synthase activity increased during this period. As isocitrate lyase activity was absent from cotton embryos during maturation, an alternative source of glyoxylate would be required for in vivo malate synthase activity. Of several metabolic sources tested, glycine was converted to glyoxylate via a transamination reaction.     

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 regulation of synthesis of mitochondrial enzymes in regreening and division-synchronized Euglena cultures

The effect of light and carbon nutrition on the synthesis of citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) in dark-grown resting (carbon deficient) and in phototrophic division-synchronized cultures of Euglena gracilis Klebs strain z were investigated. Exposure of dark-grown Euglena to white or red light produced a transient increase in the specific activities of citrate synthase and malate dehydrogenase but blue light (of equal energy) was ineffective. Citrate-synthase activity increased at the end of the light phase and in early dark phase in phototrophic cultures division-synchronized by a regime of 14 h light-10 h dark. The addition of ethanol or malate produced a twofold increase in citrate-synthase activity compared with phototrophic cultures. White and blue light, but not red light, produced a transient repression of the metabolite-induced increase in citrate-synthase activity in division-synchronized cultures. Since only red light could effect a transient increase in the specific activity of mitochondrial enzymes, and the blue-red plastid receptor should respond to both blue and red light, the synthesis of mitochondrial enzymes in regreening cultures may be under the control of a new photoreceptor responding only to red light. In division-synchronized phototrophic cells the primary effector of synthesis of mitochondrial enzymes is not light but carbon nutrition.     

Interaction of ligands with pig heart citrate synthase: conformational changes and catalysis.

The fluorescence polarization of 8-hydroxypyrene (1,3,6)trisulfonate (HPT) increases upon interaction with pig heart citrate synthase. Titration of HPT with increasing concentrations of citrate synthase exhibits a hyperbolic saturation behavior, from which the dissociation constant of the enzyme-HPT complex (3.64 +/- 0.3 microM) was determined. The enzyme-HPT interaction is competitively inhibited by oxaloacetate (but not affected by acetyl CoA) with a Ki of 4.3 +/- 1.8 microM. This value is similar to the dissociation constant (Kd = 4.5 +/- 1.6 microM) for the enzyme-oxalocetate complex (determined in the absence of any effector ligand), as well as to the Km for oxaloacetate (3.9 +/- 0.7 microM) in a steady-state citrate synthase catalyzed reaction at a saturating concentration of acetyl CoA. However, the dissociation constant for the citrate synthase-oxaloacetate complex determined by the urea denaturation method is at least 25-fold lower than those determined by the other methods. This suggests an effector role of urea in strengthening the enzyme-oxaloacetate interaction. At low nondenaturing concentrations, urea inhibits the citrate synthase catalyzed reaction in an uncompetitive manner with respect to oxaloacetate, i.e., the Km for oxaloacetate decreases with an increase in urea concentration. This further suggests that urea stabilizes the interaction between citrate synthase and oxaloacetate. The effect of urea is specific for the substrate oxaloacetate, and not for the substrate analogue, HPT, although both these ligands bind citrate synthase with equal affinities, and protect the enzyme against thermal denaturation with equal magnitudes. The results presented herein are discussed in the light of known conformational states of the enzyme.     

Properties of Citrate-stimulated Starch Synthesis Catalyzed by Starch Synthase I of Developing Maize Kernels

Chromatography of extracts of maize on diethylaminoethyl-cellulose resolves starch synthase activity into two fractions (Ozbun, Hawker, Preiss 1971 Plant Physiol 48: 785-769). Only starch synthase I is capable of synthesis in the absence of added primer and the presence of 0.5 molar citrate. This enzyme fraction has been purified about 1,000-fold from maize kernels homozygous for the endosperm mutant amylose-extender (ae). Because ae endosperm lacks the starch-branching enzyme which normally purifies with starch synthase I, the final enzyme fraction was free of detectable branching enzyme activity. This allowed a detailed characterization of the citrate-stimulated reaction. The citrate-stimulated reaction was dependent upon citrate concentrations of greater than 0.1 molar. However, the reaction is not specific for citrate and malate also stimulated the reaction. Branching enzyme increased the velocity of the reaction about 4-fold but did not replace the requirement for citrate. Citrate reduced the K_m for the primers amylopectin and glycogen from 122 and 595 micrograms per milliliter, respectively, to 6 and 50 micrograms per milliliter, respectively. The enzyme was found to contain 1.7 milligrams of anhydroglucose units per enzyme unit. Thus reaction mixtures contained 1 to 5 micrograms (5 to 25 micrograms per milliliter) of endogenous primer. The citrate-stimulated reaction could be explained by an increased affinity for this endogenous primer. The starch synthase reaction in the absence of primer is dependent upon several factors including endogenous primer concentration, citrate concentration as well as branching enzyme concentration.     

Purification and properties of a citrate synthase from Nitrosomonas sp. TK794 and a comparison with the enzymes of another nitrifying bacteria

Citrate(si)-synthase (citrate oxaloacetate-lyasem EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from an ammonia-oxidizing chemoautotrophic bacterium, Nitrosomonas sp. TK794. The molecular mass of the native enzyme was estimated to be about 287 kDa by gel filtration, whereas SDS-PAGE produced one band with M_r values of 44.7 kDa, suggesting that the enzyme is a hexamer consisting of identical subunits. The isoelectric point of the enzyme was 5.0. The pH and temperature optima for citrate synthase (CS) activity was about 7.5–8.0 and 40°C, respectively. The citrate synthase was stable over a pH range of 6.0–8.5 and up to 40°C. The apparent K_m values for oxaloacetate and acetyl-CoA were about 11 μM and 247 μM, respectively. The activity of the citrate synthase was not inhibited by ATP, NADH or 2-oxoglutarate at 5mM, and was activated by potassium chloride at 0.1–100 mM. The N-terminal amino acid sequence of the enzyme protein was PPQDVATLSPGENKKTIELPILG.     

Citrate Synthase Mutants of Sinorhizobium fredii USDA257 Form Ineffective Nodules with Aberrant Ultrastructure

The tricarboxylic acid (TCA) cycle plays an important role in generating the energy required by bacteroids to fix atmospheric nitrogen. Citrate synthase is the first enzyme that controls the entry of carbon into the TCA cycle. We cloned and determined the nucleotide sequence of the gltA gene that encodes citrate synthase in Sinorhizobium fredii USDA257, a symbiont of soybeans (Glycine max [L.] Merr.) and several other legumes. The deduced citrate synthase protein has a molecular weight of 48,198 and exhibits sequence similarity to citrate synthases from several bacterial species, including Sinorhizobium meliloti and Rhizobium tropici. Southern blot analysis revealed that the fast-growing S. fredii strains and Rhizobium sp. strain NGR234 contained a single copy of the gene located in the bacterial chromosome. S. fredii USDA257 gltA mutant HBK-CS1, which had no detectable citrate synthase activity, had diminished nodulation capacity and produced ineffective nodules on soybean. Light and electron microscopy observations revealed that the nodules initiated by HBK-CS1 contained very few bacteroids. The infected cells contained large vacuoles and prominent starch grains. Within the vacuoles, membrane structures that appeared to be reminiscent of disintegrating bacteroids were detected. The citrate synthase mutant had altered cell surface characteristics and produced three times more exopolysaccarides than the wild type produced. A plasmid carrying the USDA257 gltA gene, when introduced into HBK-CS1, was able to restore all of the defects mentioned above. Our results demonstrate that a functional citrate synthase gene of S. fredii USDA257 is essential for efficient soybean nodulation and nitrogen fixation.     

Simultaneous Overexpression of Citrate Synthase and Phosphoenolpyruvate Carboxylase in Leaves Augments Citrate Exclusion and Al Resistance in Transgenic Tobacco

Phosphoenolpyruvate carboxylase (PEPC) and citrate synthase (CS) are two key enzymes in organic acid synthesis metabolism. In the present study, a cytoplasmic form of CS from tobacco and a mutant (with reduced sensitivity to organic acid inhibition) PEPC from Synechococcus vulcanus were overexpressed simultaneously using a light-inducible promoter in tobacco leaves. The analysis for enzyme activity showed that CS and PEPC enzyme activities were increased by 235% to 257% and 218% to 236% in the selected cs and pepc (double-gene) overexpression lines, respectively, compared with those in the wild-type plants (WT). The measurement for the relative root elongation rate of the tobacco plants exposed to 30???M aluminum (Al) indicated that Al tolerance in the double-gene overexpression lines was stronger than that of the transgenic cs or pepc lines and WT plants. The ¹³C-NMR analysis with NaH¹³CO₃ showed that overexpression of CS and PEPC in the transgenic tobacco successfully constructed a new citrate synthesis pathway. Under the conditions with Al stress, the amount of citrate secreted from the double-transgenic tobacco roots was the largest among the tested plants. When grown on sandy soil supplied with a nutritional solution containing 500???M Al, the growth of the double-transgenic tobacco was better than that of the transgenic cs or pepc tobacco and WT, and their root biomass was the highest among the tested plants. These results demonstrated that construction of a new citrate synthesis pathway by simultaneous overexpression of CS and PEPC in the cytoplasm of transgenic plant leaves could enhance Al resistance in plants.     

Purification and some properties of citrate synthase from a nitrite-oxidizing chemoautotroph,Nitrobacter agilis ATCC 14123

Citrate (si)-synthase (citrate oxaloacetate-lyase, EC 4.1.3.7) was purified as an electrophoretically homogeneous protein from a nitrite-oxidizing chemoautotrophic bacterium, Nitrobacter agilis ATCC 14123. The molecular mass (M_r) of the native enzyme was estimated to be about 250,000 by gel filtration, whereas SDS-PAGE gave two bands with M_r values of 45,000 and 80,000, respectively, suggesting that the enzyme is a tetramer consisting of two different subunits (α: 45,000, β: 80,000). The isoelectric point of the enzyme was 5.4. The pH and temperature optima on the citrate synthase activity were about 7.5–8.0 and 30–35°C, respectively. The citrate synthase was stable in the pH range of 6.0–9.0 and up to 55°C. The apparent K_m values for oxaloacetate and acetyl-CoA were about 27 μM and 410 μM, respectively. The activity of citrate synthase was not inhibited by ATP (1 mM), NADH (1 mM) or 2-oxoglutarate (10 mM), but was strongly inhibited by SDS (1 mM). Activation by metal ions was not observed.