Concentrations Of Sugar Phosphates Research Articles

Phosphorus toxicity disrupts Rubisco activation and reactive oxygen species defence systems by phytic acid accumulation in leaves.

Phosphorus (P) is an essential mineral nutrient for plants. Nevertheless, excessive P accumulation in leaf mesophyll cells causes necrotic symptoms in land plants; this phenomenon is termed P toxicity. However, the detailed mechanisms underlying P toxicity in plants have not yet been elucidated. This study aimed to investigate the molecular mechanism of P toxicity in rice. We found that under excessive inorganic P (Pi) application, Rubisco activation decreased and photosynthesis was inhibited, leading to lipid peroxidation. Although the defence systems against reactive oxygen species accumulation were activated under excessive Pi application conditions, the Cu/Zn-type superoxide dismutase activities were inhibited. A metabolic analysis revealed that excessive Pi application led to an increase in the cytosolic sugar phosphate concentration and the activation of phytic acid synthesis. These conditions induced mRNA expression of genes that are activated under metal-deficient conditions, although metals did accumulate. These results suggest that P toxicity is triggered by the attenuation of both photosynthesis and metal availability within cells mediated by phytic acid accumulation. Here, we discuss the whole phenomenon of P toxicity, beginning from the accumulation of Pi within cells to death in land plants.

Sugar-Phosphate Metabolism Regulates Stationary-Phase Entry and Stalk Elongation in Caulobacter crescentus.

Bacteria have a variety of mechanisms for adapting to environmental perturbations. Changes in oxygen availability result in a switch between aerobic and anaerobic respiration, whereas iron limitation may lead to siderophore secretion. In addition to metabolic adaptations, many organisms respond by altering their cell shape. Caulobacter crescentus, when grown under phosphate-limiting conditions, dramatically elongates its polar stalk appendage. The stalk is hypothesized to facilitate phosphate uptake; however, the mechanistic details of stalk synthesis are not well characterized. We used a chemical mutagenesis approach to isolate and characterize stalk-deficient mutants, one of which had two mutations in the phosphomannose isomerase gene (manA) that were necessary and sufficient to inhibit stalk elongation. Transcription of the pho regulon was unaffected in the manA mutant; therefore, ManA plays a unique regulatory role in stalk synthesis. The mutant ManA had reduced enzymatic activity, resulting in a 5-fold increase in the intracellular fructose 6-phosphate/mannose 6-phosphate ratio. This metabolic imbalance impaired the synthesis of cellular envelope components derived from mannose 6-phosphate, namely, lipopolysaccharide O-antigen and exopolysaccharide. Furthermore, the manA mutations prevented C. crescentus cells from efficiently entering stationary phase. Deletion of the stationary-phase response regulator gene spdR inhibited stalk elongation in wild-type cells, while overproduction of the alarmone ppGpp, which triggers growth arrest and stationary-phase entry, increased stalk length in the manA mutant strain. These results demonstrate that sugar-phosphate metabolism regulates stalk elongation independently of phosphate starvation.IMPORTANCE Metabolic control of bacterial cell shape is an important mechanism for adapting to environmental perturbations. Caulobacter crescentus dramatically elongates its polar stalk appendage in response to phosphate starvation. To investigate the mechanism of this morphological adaptation, we isolated stalk-deficient mutants, one of which had mutations in the phosphomannose isomerase gene (manA) that blocked stalk elongation, despite normal activation of the phosphate starvation response. The mutant ManA resulted in an imbalance in sugar-phosphate concentrations, which had effects on the synthesis of cellular envelope components and entry into stationary phase. Due to the interconnectivity of metabolic pathways, our findings may suggest more generally that the modulation of bacterial cell shape involves the regulation of growth phase and the synthesis of cellular building blocks.

A concerted systems biology analysis of phenol metabolism in Rhodococcus opacus PD630

Rhodococcus opacus PD630 metabolizes aromatic substrates and naturally produces branched-chain lipids, which are advantageous traits for lignin valorization. To provide insights into its lignocellulose hydrolysate utilization, we performed 13C-pathway tracing, 13C-pulse-tracing, transcriptional profiling, biomass composition analysis, and metabolite profiling in conjunction with 13C-metabolic flux analysis (13C-MFA) of phenol metabolism. We found that 1) phenol is metabolized mainly through the ortho–cleavage pathway; 2) phenol utilization requires a highly active TCA cycle; 3) NADPH is generated mainly via NADPH-dependent isocitrate dehydrogenase; 4) active cataplerotic fluxes increase plasticity in the TCA cycle; and 5) gluconeogenesis occurs partially through the reversed Entner–Doudoroff pathway (EDP). We also found that phenol-fed R. opacus PD630 generally has lower sugar phosphate concentrations (e.g., fructose 1,6-bisphosphatase) compared to metabolite pools in 13C-glucose-fed Escherichia coli (set as internal standards), while its TCA metabolites (e.g., malate, succinate, and α-ketoglutarate) accumulate intracellularly with measurable succinate secretion. In addition, we found that phenol utilization was inhibited by benzoate, while catabolite repressions by other tested carbon substrates (e.g., glucose and acetate) were absent in R. opacus PD630. Three adaptively-evolved strains display very different growth rates when fed with phenol as a sole carbon source, but they maintain a conserved flux network. These findings improve our understanding of R. opacus’ metabolism for future lignin valorization.

Development of a core Clostridium thermocellum kinetic metabolic model consistent with multiple genetic perturbations

BackgroundClostridium thermocellum is a Gram-positive anaerobe with the ability to hydrolyze and metabolize cellulose into biofuels such as ethanol, making it an attractive candidate for consolidated bioprocessing (CBP). At present, metabolic engineering in C. thermocellum is hindered due to the incomplete description of its metabolic repertoire and regulation within a predictive metabolic model. Genome-scale metabolic (GSM) models augmented with kinetic models of metabolism have been shown to be effective at recapitulating perturbed metabolic phenotypes.ResultsIn this effort, we first update a second-generation genome-scale metabolic model (iCth446) for C. thermocellum by correcting cofactor dependencies, restoring elemental and charge balances, and updating GAM and NGAM values to improve phenotype predictions. The iCth446 model is next used as a scaffold to develop a core kinetic model (k-ctherm118) of the C. thermocellum central metabolism using the Ensemble Modeling (EM) paradigm. Model parameterization is carried out by simultaneously imposing fermentation yield data in lactate, malate, acetate, and hydrogen production pathways for 19 measured metabolites spanning a library of 19 distinct single and multiple gene knockout mutants along with 18 intracellular metabolite concentration data for a Δgldh mutant and ten experimentally measured Michaelis–Menten kinetic parameters.ConclusionsThe k-ctherm118 model captures significant metabolic changes caused by (1) nitrogen limitation leading to increased yields for lactate, pyruvate, and amino acids, and (2) ethanol stress causing an increase in intracellular sugar phosphate concentrations (~1.5-fold) due to upregulation of cofactor pools. Robustness analysis of k-ctherm118 alludes to the presence of a secondary activity of ketol-acid reductoisomerase and possible regulation by valine and/or leucine pool levels. In addition, cross-validation and robustness analysis allude to missing elements in k-ctherm118 and suggest additional experiments to improve kinetic model prediction fidelity. Overall, the study quantitatively assesses the advantages of EM-based kinetic modeling towards improved prediction of C. thermocellum metabolism and develops a predictive kinetic model which can be used to design biofuel-overproducing strains.

Elucidation of the regulatory role of the fructose operon reveals a novel target for enhancing the NADPH supply in Corynebacterium glutamicum

The performance of Corynebacterium glutamicum cell factories producing compounds which rely heavily on NADPH has been reported to depend on the sugar being metabolized. While some aspects of this phenomenon have been elucidated, there are still many unresolved questions as to how sugar metabolism is linked to redox and to the general metabolism. We here provide new insights into the regulation of the metabolism of this important platform organism by systematically characterizing mutants carrying various lesions in the fructose operon. Initially, we found that a strain where the dedicated fructose uptake system had been inactivated (KO-ptsF) was hampered in growth on sucrose minimal medium, and suppressor mutants appeared readily. Comparative genomic analysis in conjunction with enzymatic assays revealed that suppression was linked to inactivation of the pfkB gene, encoding a fructose-1-phosphate kinase. Detailed characterization of KO-ptsF, KO-pfkB and double knock-out (DKO) derivatives revealed a strong role for sugar-phosphates, especially fructose-1-phosphate (F1P), in governing sugar as well as redox metabolism due to effects on transcriptional regulation of key genes. These findings allowed us to propose a simple model explaining the correlation between sugar phosphate concentration, gene expression and ultimately the observed phenotype. To guide us in our analysis and help us identify bottlenecks in metabolism we debugged an existing genome-scale model onto which we overlaid the transcriptome data. Based on the results obtained we managed to enhance the NADPH supply and transform the wild-type strain into delivering the highest yield of lysine ever obtained on sucrose and fructose, thus providing a good example of how regulatory mechanisms can be harnessed for bioproduction.

Dynamic metabolomics differentiates between carbon and energy starvation in recombinant Saccharomyces cerevisiae fermenting xylose

BackgroundThe concerted effects of changes in gene expression due to changes in the environment are ultimately reflected in the metabolome. Dynamics of metabolite concentrations under a certain condition can therefore give a description of the cellular state with a high degree of functional information. We used this potential to evaluate the metabolic status of two recombinant strains of Saccharomyces cerevisiae during anaerobic batch fermentation of a glucose/xylose mixture. Two isogenic strains were studied, differing only in the pathways used for xylose assimilation: the oxidoreductive pathway with xylose reductase (XR) and xylitol dehydrogenase (XDH) or the isomerization pathway with xylose isomerase (XI). The isogenic relationship between the two strains ascertains that the observed responses are a result of the particular xylose pathway and not due to unknown changes in regulatory systems. An increased understanding of the physiological state of these strains is important for further development of efficient pentose-utilizing strains for bioethanol production.ResultsUsing LC-MS/MS we determined the dynamics in the concentrations of intracellular metabolites in central carbon metabolism, nine amino acids, the purine nucleotides and redox cofactors. The general response to the transition from glucose to xylose was increased concentrations of amino acids and TCA-cycle intermediates, and decreased concentrations of sugar phosphates and redox cofactors. The two strains investigated had significantly different uptake rates of xylose which led to an enhanced response in the XI-strain. Despite the difference in xylose uptake rate, the adenylate energy charge remained high and stable around 0.8 in both strains. In contrast to the adenylate pool, large changes were observed in the guanylate pool.ConclusionsThe low uptake of xylose by the XI-strain led to several distinguished responses: depletion of key metabolites in glycolysis and NADPH, a reduced GTP/GDP ratio and accumulation of PEP and aromatic amino acids. These changes are strong indicators of carbon starvation. The XR/XDH-strain displayed few such traits. The coexistence of these traits and a stable adenylate charge indicates that xylose supplies energy to the cells but does not suppress a response similar to carbon starvation. Particular signals may play a role in the latter, of which the GTP/GMP ratio could be a candidate as it decreased significantly in both strains.

Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

Engineering the level of metabolic cofactors to manipulate metabolic flux is emerging as an attractive strategy for bioprocess applications. We present the metabolic consequences of increasing NADH in the cytosol and the mitochondria of Saccharomyces cerevisiae. In a strain that was disabled in formate metabolism, we either overexpressed the native NAD(+)-dependent formate dehydrogenase in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence encoded by the CYB2 gene. Upon exposure to formate, the mutant strains readily consumed formate and induced fermentative metabolism even under conditions of glucose derepression. Cytosolic overexpression of formate dehydrogenase resulted in the production of glycerol, while when this enzyme was directed into the mitochondria, we observed glycerol and ethanol production. Clearly, these results point toward different patterns of compartmental regulation of redox homeostasis. When pulsed with formate, S. cerevisiae cells growing in a steady state on glucose immediately consumed formate. However, formate consumption ceased after 20 min. Our analysis revealed that metabolites at key branch points of metabolic pathways were affected the most by the genetic perturbations and that the intracellular concentrations of sugar phosphates were specifically affected by time. In conclusion, the results have implications for the design of metabolic networks in yeast for industrial applications.

Antibiotic Overproduction in Streptomyces coelicolor A3(2) Mediated by Phosphofructokinase Deletion

Streptomycetes are exploited for production of a wide range of secondary metabolites, and there is much interest in enhancing the level of production of these metabolites. Secondary metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of the primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by (13)C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely because of accumulation of glucose 6-phosphate and fructose 6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations, we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate. Integrated analysis of gene expression data using a genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate biosynthesis.

Diffusional limitations of immobilized Escherichia coli in hollow‐fiber reactors: Influence on 31P NMR spectroscopy

Escherichia coli cells were immobilized and grown in hollow-fiber reactors allowing simultaneous NMR spectroscopy and perfusion with nutrient medium. The extent to which the cells were starved due to inadequate mass transfer was predicted using a mathematical model of reaction and diffusion. Reactors were experimentally characterized using (35)S autoradiography to visualize spatial variations in protein synthesis rates and transmission electron microscopy to indicate spatial variations in cell morphology. Mass transfer limitations in reactors operated at 37 degrees C were shown to be severe, with regions of starved cells occupying up to 80% of the cell-containing region. Phosphorus-31 nuclear magnetic resonance (NMR) spectra of the immobilized, perfused cells revealed abnormally low volume-averaged concentrations of sugar phosphates, NTP, and ratios of NTP/NDP in these reactors. Intracellular pH was also depressed in the cells. In order to overcome mass transfer limitations in the cell layer, the reactor growth temperature was decreased. Sulfur-35 autoradiographs of a reactor operated at 16 degrees C did not indicate the presence of starved cells. The NMR spectra obtained from this reactor showed near-normal intracellular pH, metabolite concentrations, and NTP/NDP ratios. The presence of significant mass transfer limitations in a perfused cell sample during NMR spectroscopy is generally undesirable since the resulting spectra can be ambiguous and difficult to interpret. The strategy adopted in this work, namely estimation of the relative rates of reaction and diffusion in the cell mass and appropriate changes in reactor design and operating parameters, should prove generally applicable for the design of perfused cell samples for NMR spectroscopic experiments.

Comparative Studies of Glucose Catabolism by Escherichia coli Grown in a Complex Medium under Aerobic and Anaerobic Conditions

AbstractEscherichia coli HB101 was grown in complex medium under anaerobic and aerobic conditions. Cells prepared under these two different conditions were characterized by two‐dimensional protein gel electrophoresis, by NMR measurements under identical (anaerobic) conditions, and by measuring the kinetics of glucose uptake and catabolite end‐product appearance in the medium under identical anaerobic conditions. Specific rates of glucose uptake and end‐product formation were significantly greater for the anaerobically grown cells, which also exhibited lower intracellular concentrations of sugar phosphates, nucleoside di‐and triphosphates, UDPG, and NAD(H). Two‐dimensional gel electrophoretic analyses reveal changes in the intracellular levels of proteins involved in pyruvate catabolism that have been observed previously for E. coli grown in minimal medium under aerobic and anaerobic conditions. Enzymes involved in the TCA cycle were not detected in cells grown aerobically or anaerobically in complex medium.

Estimation of intracellular sugar phosphate concentrations in Saccharomyces cerevisiae using 31P nuclear magnetic resonance spectroscopy

A systematic procedure has been formulated for estimating the relative intracellular concentrations of sugar phosphates in Saccharomyces cerevisiae based upon (31)P nuclear magnetic resonance (NMR) measurements. The sugar phosphate region of the (31)P NMR spectrum is first decomposed by computer analysis, and the decomposition consistency and identification of individual sugar phosphate resonances are established based on in vitro chemical shift calibrations determined in separate experiments. Numerous evaluations of intracellular S. cerevisiae compositions for different strains and different cell environments provide the basis for in vivocorrelations of inorganic phosphate chemical shift with the chemical shifts of 3-phosphoglycerate, beta;-fructose 1,6-diphosphate, fructose 6-phosphate, and glucose 6 phosphate. Relative intracellular sugar phosphate concentrations are obtained by correcting peak areas for partial saturation during transient in vivo experiments. In vivo concentrations estimated by this method agree well with estimates for similar systems based on other techniques. This approach does not require costly la belled compounds, and has the advantage that other important metabolic state variables such-as internal and external pH and intracellular levels of phosphate, ATP, ADP, NAD(H), and polyphosphate may be determined from the same (31)P spectrum. Extension of this strategy to other cellular systems should be straightforward.

Assessment of human liver metabolism by phosphorus-31 magnetic resonance spectroscopy.

Phosphorus 31 magnetic resonance (MR) spectroscopy was used for the study of liver metabolism in vivo in seven healthy subjects. Subjects were examined in a 1.6 T whole-body magnet using surface coils for data acquisition. The region of the liver from which MR signals were collected was selected by magnetic-field profiling. The concentration ratios of adenosine triphosphate (ATP), inorganic phosphate (Pi) and sugar phosphates contained in liver cells could be reproducibly assessed. Cytosolic pH and the free magnesium concentration were determined to be 7.18 and 300 microM, respectively. During intravenous fructose tolerance tests the hepatic concentrations of sugar phosphates, ATP and Pi altered markedly. During the first 5 min following bolus injection of 250 mg fructose/kg body weight the concentration of sugar phosphates increased sevenfold whereas Pi and ATP decreased by three- to fourfold. Metabolism of sugar phosphates was complete within 20 min and could be followed by 31P MR with a time resolution of 5 min. Thus, 31P MR spectroscopy yields insight into liver metabolism which has not been accessible so far using conventional non-invasive methods. In conjunction with intravenous fructose loading, 31P MR spectroscopy may provide a means for the functional assessment of the liver.

Sugar accumulation in Gram-positive bacteria: exclusion and expulsion mechanisms

Bacteria use a diverse range of mechanisms to regulate sugar accumulation in response to internal and external energy availability. Uptake of sugars is regulated by exclusion mechanisms while the intracellular sugar phosphate concentration is modulated in certain Gram-positive bacteria by an expulsion mechanism. Recent evidence suggests that a regulatory protein of the phosphotransferase system catalyses ATP-dependent phosphorylation of the phosphocarrier protein, HPr, thereby controlling sugar accumulation by exclusion and/or expulsion processes.

Regulation of lactate dehydrogenase inBrochothrix thermosphacta

Purified lactate dehydrogenase from Brochothrix thermosphacta is stimulated by Fru-1,6-P2 and G6P although saturating concentrations are high (> 20 mM). Neither is essential for activity. AMP, ADP and ATP inhibit enzyme activity consistent with either non-competitive (with Fru-1,6-P2 present) or uncompetitive (G6P present) inhibition. Activity is not dependent on Pi (< 200 mM). Based on 31P-NMR of cells, sugar phosphate concentration can reach 30 mM with excess glucose present; NDP and NTP also accumulate to levels that inhibit the isolated enzyme. The effector levels in vitro are therefore appropriate to in vivo metabolism and support a regulatory role for sugar phosphates during pyruvate metabolism in this organism.

Insulin action in intact mouse diaphragm I.

The incubation of intact mouse diaphragms with insulin caused a dose and time dependent increase in the independent activity of glycogen synthase in tissue extracts. 2-deoxyglucose (2-10 mM) alone markedly stimulated the conversion of glycogen synthase to the independent activity under conditions in which tissue ATP concentrations were not affected. The incubation of diaphragms with both insulin and 2-deoxyglucose resulted in a greater than additive effect. Insulin stimulated the uptake of 2-deoxyglucose into mouse diaphragms, accumulating as 2-deoxyglucose-6-phosphate. The accumulation of 2-deoxyglucose-6-phosphate correlated well with the increase in the independent activity of glycogen synthase and with the activation of glycogen synthase phosphatase in tissue extracts. The uptake of 3-0 methyl glucose was also markedly stimulated by insulin, without affecting the activity of glycogen synthase. Both glucose-6-phosphate and 2-deoxyglucose-6-phosphate stimulated the activation of endogenous glycogen synthase phosphatase activity in muscle homogenates. We conclude that insulin, in addition to its effects in the absence of exogenous sugars, increases the independent activity of glycogen synthase through increased sugar transport resulting in increased concentrations of sugar-phosphates which promote the activity of glycogen synthase phosphatase.

Characteristics of a Galactose-adapted Sugarcane Cell Line Grown in Suspension Culture

Although d-galactose is normally toxic to sugarcane (Saccharum sp.) cells, a cell line that grows on 100 mm galactose has been propagated. Nonadapted cells in a medium containing galactose instead of sucrose accumulate UDP-galactose; these cells also have much lower UDP-galactose 4-epimerase (EC 5.1.3.2) activity than do adapted cells. This enzyme may determine whether or not galactose will cause toxicity symptoms to develop. The growth rate of galactose-adapted cells is similar to most cell lines on several other carbohydrates. The galactose-adapted cells are also similar to sucrose stock cells in cell wall composition and sugar phosphate concentrations, but, like the nonadapted cells, accumulate free galactose.

Interactive Effects of Salinity and Phosphorus Nutrition of the Concentrations of Phosphate and Phosphate Esters in Mature Photosynthesizing Corn Leaves

The effects of salinity on corn plants (Zea mays L.) are influenced by the concentration of nutrient orthophosphate. Salinity (-2 bars each of NaCl and CaCl(2)) was more injurious in combination with a high concentration of orthophosphate (2 mm) (that gave optimum yields in the absence of salinity) than it was with a lower concentration (0.1 mm). With 2 mm orthophosphate, salinity seemed to damage the plant mechanisms that normally regulate the internal concentration of orthophosphate resulting in excessive accumulation and P toxicity. On the other hand, with 0.1 mm orthophosphate, salinity decreased orthophosphate concentration in mature leaves. This effect was paralleled by decreases in the concentration of adenosine 5'-triphosphate and in the energy charge of the adenylate system, indicating an orthophosphate deficit. Even so, plants survived salinity better under these conditions than in the presence of 2 mm orthophosphate. The data indicated that salinity affected the phosphorylated state of the adenine nucleotides only indirectly through its effect on the concentration of orthophosphate in the cells.Salinity, especially in the presence of 2 mm orthophosphate, resulted in an increase in the concentrations of sugar phosphates in mature photosynthesizing leaves, suggesting that translocation rather than photosynthesis was a limiting process. Decreased translocation could be a secondary effect of decreased growth. However, a decreased translocation rate could cause decreased growth by limiting the supply of essential metabolites reaching growing tissues.

Correlations between fluorescence, X-ray diffraction, and physiological properties in cytoplasmic membrane vesicles isolated from Escherichia coli

The catalytic properties ( i.e. transport) of cytoplasmic membrane vesicles isolated from Escherichia coli ML strain can be dissociated from their barrier properties. Initial rates of sugar and amino acid transport exhibit sharp activity transitions with respect to temperature at 15 to 20 °C, and optima at 45 to 50 °C. On the other hand, accumulation of maximal intramembranal concentrations of sugar phosphates via the phosphoenolpyruvate-phosphotransferase system exhibits temperature optima from 30 to 46 °C depending on the carbon source used for the growth of the parent cells. Moreover, the temperature optimum for accumulation of sugar phosphate in a particular membrane preparation is correlated with an abrupt increase in passive membrane permeability that occurs at that same temperature. By means of X-ray diffraction and fluorescence techniques applied to membrane vesicles into which dansyl phosphatidylethanolamine has been incorporated, two transitions are detected as functions of temperature. One transition is correlated with the transition in transport activity that occurs at around 20 °C, and coincides with the “melting” of paraffin chains of the membrane phospholipids. The second transition is correlated with the abrupt increases in passive membrane permeability.

Adenosine Diphosphate-Glucose Pyrophosphorylase Control of Starch Accumulation in Rust-infected Wheat Leaves

The variation in starch content in healthy and Puccinia striiformsi-infected wheat leaves was measured from 5 to 15 days after inoculation. The starch content of diseased leaves relative to healthy leaves decreased from 5 to 9 days, increased from 9 to 12 days to twice that of healthy leaves, and decreased from 12 to 15 days after inoculation. Electron micrographs of plant tissues indicated that the starch accumulated in the chloroplasts of host cells adjacent to fungal hyphae. Variations in sugar phosphates, ATP, and inorganic phosphate were measured during the infection process. ADP-glucose pyrophosphorylase was extracted and partially purified from healthy and diseased leaves. When proportionate concentrations of sugar phosphates and inorganic phosphate found in healthy and diseased leaves during the infection process were placed in the assay mixture, ADP-glucose pyrophosphorylase activity was similar to the pattern of starch accumulation and was almost the inverse of the variation observed in inorganic phosphate in diseased leaves during the infection process. A mechanism to explain the accumulation of starch is presented and discussed. This mechanism is based on the regulation of ADP-glucose pyrophosphorylase by changes in effector molecule concentrations during the infection process. Reasons for these changes are presented.