Analysis of metabolic and physiological responses to gnd knockout in Escherichia coli by using C-13 tracer experiment and enzyme activity measurement

The physiological and metabolic responses to gnd knockout in Escherichia coli K-12 was quantitatively investigated by using the (13)C tracer experiment (GC-MS/NMR) together with the enzyme activity analysis. It was shown that the general response to the gene knockout was the local flux rerouting via Entner-Doudoroff pathway and the direction reversing via non-oxidative pentose phosphate pathway (PPP). The mutant was found to direct higher flux to phosphoglucose isomerase reaction as compared to the wild-type, but the respiratory metabolism was comparable in both strains. The anaplerotic pathway catalyzed by malic enzyme was identified in the mutant, which was accompanied with an up-regulation of phosphoenolpyruvate carboxylase and down-regulation of phosphoenolpyruvate carboxykinase. The presented results provide first evidence that compensatory mechanism existed in PPP and anaplerotic pathway in response to the gnd deletion.     

Enzymatic Regulation of Glycoprotein Synthesis in Peripheral Nervous System Myelin

The enzyme UDP-N-acetylglucosamine: dolichyl phosphate, N-acetylglucosamine-1-phosphate transferase initiates the synthesis of the oligosaccharide chain of complex-type glycoproteins. In view of the high content of glycoprotein in peripheral nerve myelin, the properties of this enzyme, its changes with age, and the effect of the specific inhibitor tunicamycin were investigated. The enzyme activity in rat peripheral nerve homogenate was completely dependent on the presence of exogenous dolichyl phosphate as well as Mg2+ and a detergent (Triton X-100) and was also greatly stimulated by a high salt concentration (0.4 M KCl) and AMP. The highest specific activity was present in the postmitochondrial membranes. The specific activity in postmitochondrial membranes in the presence of exogenous dolichyl phosphate reached a maximum at 17 days and remained relatively high throughout development, up to 2 years of age, but the activity was much lower when dolichyl phosphate was not added. This indicates that the enzyme level does not decrease with age, but that the content of the lipid cofactor may limit glycoprotein synthesis in vivo. Tunicamycin (5 micrograms) was injected intraneurally into 24-day-old rat sciatic nerve, and the enzyme was assayed from 1 to 24 days after injection. The specific activity of the transferase remained at low levels (5-40% of the level in control nerve) in most injected nerves assayed throughout this postinjection period. A protein previously identified as the unglycosylated P0 protein was synthesized in vitro by the tunicamycin-injected nerve and could be demonstrated to be incorporated into myelin in large amounts at 2 days and in small amounts at 6 days after injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo photosynthetic electron transport does not limit photosynthetic capacity in phosphate-deficient sunflower and maize leaves

The effects of extreme phosphate (Pi) deficiency during growth on the contents of adenylates and pyridine nucleotides and the in vivo photochemical activity of photosystem II (PSII) were determined in leaves of Helianthus annuus and Zea mays grown under controlled environmental conditions. Phosphate deficiency decreased the amounts of ATP and ADP per unit leaf area and the adenylate energy charge of leaves. The amounts of oxidized pyridine nucleotides per unit leaf area decreased with Pi deficiency, but not those of reduced pyridine nucleotides. This resulted in an increase in the ratio of reduced to oxidized pyridine nucleotides in Pi-deficient leaves. Analysis of chlorophyll a fluorescence at room temperature showed that Pi deficiency decreased the efficiency of excitation capture by open PSII reaction centres (φ_e), the in vivo quantum yield of PSII photochemistry (φ_PSII) and the photochemical quenching co-efficient (q_P), and increased the non-photochemical quenching co-efficient (q_N) indicating possible photoinhibitory damage to PSII. Supplying Pi to Pi-deficient sunflower leaves reversed the long-term effects of Pi-deficiency on PSII photochemistry. Feeding Pi-sufficient sunflower leaves with mannose or FCCP rapidly produced effects on chlorophyll a fluorescence similar to long-term Pi-deficiency. Our results suggest a direct role of Pi and photophosphorylation on PSII photochemistry in both long-and short-term responses of photosynthetic machinery to Pi deficiency. The relationship between φ_PSII and the apparent quantum yield of CO₂ assimilation determined at varying light intensity and 21 kPa O₂ and 35 Pa CO₂ partial pressures in the ambient air was linear in Pi-sufficient and Pi-deficient leaves of sunflower and maize. Calculations show that there was relatively more PSII activity per mole of CO₂ assimilated by the Pi-deficient leaves. This indicates that in these leaves a greater proportion of photosynthetic electrons transported across PSII was used for processes other than CO₂ reduction. Therefore, we conclude that in vivo photosynthetic electron transport through PSII did not limit photosynthesis in Pi-deficient leaves of sunflower and maize and that the decreased CO₂ assimilation was a consequence of a smaller ATP content and lower energy charge which restricted production of ribulose, 1-5, bisphosphate, the acceptor for CO₂.     

Deposition of condensed phosphate as an effect of varying sulfur deficiency in the cyanobacterium Synechococcus sp. (Anacystis nidulans)

Uptake of orthophosphate and deposition of condensed phosphate were investigated in cells of Synechococcus sp. (Anacystis nidulans) deficient in phosphorus or sulfur. When phosphorus was restored to phosphorus-starved cells, uptake was rapid and immediate, with the greatest accumulation occurring within the first hour. Uptake was optimum in the pH 7.5–8.5 range. Long-term (6-day) studies of uptake and deposition with cells exposed to a wide range of sulfur deficiency showed that both processes were greatest when the level of exogenous sulfur was reduced to zero. The increase in cellular phosphorus as determined chemically was in agreement with the increased number and size of polyphosphate bodies at the ultrastructural level. Possible mechanisms for the control of phosphorus uptake and condensed phosphate formation by exogenous sulfur are discussed.     

Aluminium interactions with K+(86Rb+) and 45Ca2+ fluxes in three cultivars of sugar beet (Beta vulgaris)

Three cultivars of sugar beet (Beta vulgaris L.), which are sensitive to aluminium (Al) in the order Primahill > Monohill > Regina, were grown in water culture for 2 weeks. Nutrients were supplied at 15% increase of amounts daily, corresponding to the nutrient demand for maximal growth. The 2.4-dinitrophenol (DNP)-sensitive (metabolic) and DNP-insensitive (non-metabolic) uptake of aluminium, phosphate. ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in roots were measured as well as transport to shoots of intact plants. All 3 cultivars absorbed more aluminium if DNP was present during the aluminium treatment than in its absence. It is suggested that sugar beets are able to extrude aluminium activity or that they possess an active mechanism to keep Al outside the cell. The presence of Al in the medium during the 1-h experiment affected the metabolic and non-metabolic fluxes of ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in different ways. In the presence of DNP, the influx of both ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) and the efflux of ⁴⁵Ca²⁺ were inhibited by Al in a competitive way. At inhibition of ⁴⁵Ca²⁺ influx, 2 Al ions are probably bound per Ca²⁺ uptake site in cv. Regina (Al-tolerant), but in cvs Primahill and Monohill only one Al ion is bound (more Al sensitive). Aluminium competitively inhibited the active efflux of ⁴⁵Ca²⁺ (absence of DNP) in almost the same way in the 3 cultivars. In contrast, aluminium stimulated the influx of K⁺(⁸⁶Rb⁺) in cvs Primahill, Monohill and Regina in the absence of DNP. Thus, the Al effects on active and passive K⁺(⁸⁶Rb⁺) influx are different. The total influx of K⁺(⁸⁶Rb⁺) increased in the presence of Al and might be connected to an active exclusion of Al. Regina is the least Al-sensitive cultivar, probably because Al interferes less with the Ca²⁺ fluxes and because this cultivar actively excludes phosphate in the presence of Al. Thus Al-phosphate precipitation within the plant could be avoided.     

Influence of temperature on root development and phosphate influx in winter wheat grown at different P levels

Root development was studied in winter wheat ( Triticum aestivum L. cv Starke II) grown at 5,10, 15 and 20°C in nutrient solutions with phosphate concentrations of 10, 100 or 1000 μM . The plants were grown for 38 days (5 and 10°C), 19 days (15°C) or 14 days (20°C). At the end of the cultivation period the phosphate influx in the roots was determined with ³²P-phosphate. Root development (lateral and seminal roof length and number) was monitored throughout the cultivation period on the same individuals by repeated (approximately every second day) photocopying of the roots for measurements with digitizer and appropriate software. The 5°C treatment yielded no laterals, and the seminals were only slightly affected by the different phosphate treatments. The 10 μM phosphate treatment gave high root:shoot dry weight ratio, high average lateral root length and high specific root length [m root (g root fresh weight)^-1]. The 1000 μM phosphate treatment yielded the highest number of laterals per m seminal root, and usually also the highest absolute numbers. Phosphate influx decreased with increased P status of the roots. It is argued that phosphate influx is dependent on factors such as P status, root geometry and relative root extension rate.     

Phosphorus nutrition of jarrah (Eucalyptus marginata) seedlings

Summary Effects of calcium phosphate supply on plant dry matter and phosphorus concentrations of parts of jarrah (Eucalyptus marginata) seedlings grown in a lateritic topsoil from the jarrah forest were examined in two glasshouse trials. Phosphorus deficiency depressed root and shoot dry weights and severely deficient leaves were smal and purple with prominent red major veins. Phosphorus deficiency severely reduced stem phosphorus levels (0.5% to 0.02%, experiment 1). Phosphorus concentrations were higher in bark than wood and the amount of phosphorus in the bark was sensitive to stem age and phosphate supply. Phosphorus adequate plants had bark phosphorus concentrations in the range 0.2–0.9% compared to <0.1% in deficient plants (experiment 2). Jarrah leaves accumulated dry matter up to 80 days after expansion and some leaves exported phosphorus during this period. Bark analysis may therefore be preferable to leaf analysis for detecting phosphorus deficiency in this species.     

Effects of P-fertilization and inoculation by two vesicular-arbuscular mycorrhizal fungi on growth and nodulation ofCalopogonium caeruleum

The growth response ofCalopogonium caeruleum, a leguminous covercrop in plantation agriculture, to inoculation with two vesicular-arbuscular mycorrhizal (VAM) fungi was investigated in five phosphorus (P)-deficient soils supplied with various levels of rock phosphate. Significant shoot yield increases over the uninoculated controls were obtained in most sterilised or unsterilised soils at all applied P levels, although the inoculant VAM fungi differed in their effectiveness in the soils used. Responses in mycorrhizal root infections, P and nitrogen (N) concentrations in tops and plant nodulation varied. The results are discussed in relation to the edaphic environment of the mycorrhizal association.     

Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Carboxylate composition of root exudates does not relate consistently to a crop species' ability to use phosphorus from aluminium, iron or calcium phosphate sources

* The relationship between carboxylate release from roots and the ability of the species to utilize phosphorus from sparingly soluble forms was studied by comparing Triticum aestivum, Brassica napus, Cicer arietinum, Pisum sativum, Lupinus albus, Lupinus angustifolius and Lupinus cosentinii. * Plants were grown in sand and supplied with 40 mg P kg(-1) in the sparingly soluble forms AlPO(4), FePO(4) or Ca(5)OH(PO(4))(3), or as soluble KH(2)PO(4); control plants received no P. * The ability to utilize sparingly soluble forms of P differed between forms of P supplied and species. Pisum sativum and C. arietinum did not access AlPO(4) or FePO(4) despite releasing carboxylates into the rhizosphere. * Species accessed different forms of sparingly soluble P, but no species was superior in accessing all forms. We conclude that a single trait cannot explain access to different forms of sparingly soluble P, and hypothesize that in addition to carboxylates, rhizosphere pH and root morphology are key factors.