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.     

Metabolic Stability of Hippocampal Slice Preparations During Prolonged Incubation

Abstract: Hippocampal slices were prepared under three conditions: (1) in medium containing glucose and oxygen at 4°C; (2) as in (1), but at 37°C; (3) in medium devoid of glucose and oxygen at 37°C. The rates of recovery to roughly steady-state levels and through 8 h of incubation were monitored for energy metabolite levels and related parameters. In vitro stable values are compared with in situ hippocampal levels. Regardless of the conditions under which slices were prepared, metabolite levels required up to 3 h to stabilize, and these levels were maintained or improved through 8 h of incubation. Further, the maximal concentrations of metabolites were independent of the conditions of slice preparation. Total adenylates and total creatine levels reached 55% of those in vivo. Lactate decreased from the decapitation-induced high levels, but stabilized at concentrations about twice those in rapidly frozen brain. Cyclic AMP and cyclic GMP exhibited peak levels at 30 min of incubation, and cyclic GMP remained elevated for 3 h. Although all three methods of slice preparation resulted in similar metabolite profiles on incubation, the initial decreases in high energy phosphates were delayed by chilling. Most striking, the slices prepared in the absence of glucose and oxygen exhibited much smaller orthodromic evoked potentials in the dentate gyrus. The presence of glucose and oxygen during preparation of the slices appears to be critical to the electrophysiological response of the tissue.     

DIEL CHANGES IN PHASED-DIVIDING CULTURES OF CERATIUM FURCA (DINOPHYCEAE): NUCLEOTIDE TRIPHOSPHATES,ADENYLATE ENERGY CHARGE,CELL CARBON,AND PATTERNS OF VERTICAL MIGRATION1

The diel pattern of cell division, cell carbon, adenine nucleotides and vertical migration was determined for laboratory cultures of the photosynthetic marine dinoflagellate, Ceratium furca (Ehr.) Clap. & Lachm., entrained on an alternating 12:12 LD schedule at 20 C. Cell division was initiated during the latter portion of the dark period with ca. 30% of the population undergoing division. Cell C increased during the light period and exhibited a linear decrease with a loss of 33% during the dark period. ATP · cell^?1 increased during the light period and decreased by ca. 40–50% during the dark period. The diel patterns of cell C and ATP tended to “buffer” the magnitude of the change in C:ATP ratios around an overall mean value of 89. There was no obvious trend in the concentration of [GTP + UTP] · cell^?1 over the cell cycle. The cellular adenylate energy charge was maintained at values between 0.8 to 0.9 throughout the 24 h LD cycle, despite a ca. 40% decrease in total adenylates (A_T= ATP + ADP + AMP) during the dark period on 12:12 LD, and over a 68% decrease in ATP during 42 h of continuous darkness. These data lend experimental support to the theory of cellular metabolic control by the adenine nucleotides. With lateral illumination on 12:12 LD cycles, the cells began to concentrate at the surface of the experimental tubes shortly before the lights were turned on, and at the bottom of the tubes shortly before the lights were extinguished. This pattern continued for 6 days in continuous darkness, suggesting that the vertical migration pattern is independent of a phototactic response and may be under the control of an endogenous rhythm.     

Synthesis of amino acyl adenylates using thetert-butoxycarbonyl protecting group

Summary Alanyl, [¹⁴C]-alanyl, phenylalanyl, and methionyl adenylates have been synthesized in high yields and relatively good purities. Elemental analysis¹H nmr and ir spectra have been utilized for the characterization of these extremely labile compounds. The present synthesis, which uses readily available N-tert-butoxycarbonyl amino acids, is compared with previous methods.     

Effects of experimental anaemia on intra-erythrocytic phosphate levels in rainbow trout, Oncorhynchus mykiss

The concentrations of intra-erythrocytic adenylates (ATP, ADP and AMP) and guanylates (GTP, GDP and GMP) were determined in rainbow trout subjected to 10% blood removal every 12 h for 96 h. Haemoglobin concentration, [Hb], decreased from 6·043±0·617 to 0·957 ± 0·195 g dl⁻¹. This decrease in [Hb] was followed by a continuous increase in total organic phosphates, e.g. adenylates plus guanylates. Intra-erythrocytic NTP (ATP plus GTP) levels increased significantly after 48 h when haemoglobin concentration was 2·427 ± 0·256 g dl⁻¹. Although a significant increase in GDP levels in animals with [Hb] less than 1·677 ± 0·235 g dl⁻¹ was observed, the general increase in guanylate level was mainly due to the GMP which increased about 85-fold during the experimental period. It is suggested that the erythrocytes of anaemic rainbow trout have the capacity to increase NTP/Hb₄ ratios which may represent an advantage for anaemic fish.     

Light induction of alternative pathway capacity in leaf slices of Belgium endive

The effect of light on the development of the capacity for alternative pathway respiration was investigated in leaf slices of Belgium endive (Cichorum intybus L. cv. deliva). Dark-grown plants possessed little capacity for the cyanide-insensitive alternative pathway. In contrast, plants grown in continuous light had significant alternative pathway capacity. Light-grown plants also had substantially higher concentrations of ethanol-soluble carbohydrates in their leaves than plants grown in complete darkness. Despite these differences in leaf carbohydrate status and alternative pathway capacity of light- and dark-grown leaf tissue, no differences were found in the activity of the alternative pathway, which was negligible in both treatments. Dark-grown plants were adenylate restricted, as indicated by the increase in cytochrome pathway activity following uncoupling. Adenylates did not limit respiration in light-grown leaf tissue. Plants that had been grown for 8d in complete darkness were also transferred to continuous light. Respiration of dark controls steadily declined over 11d following the transfer of plants to the light, due primarily to a decrease in cytochrome pathway activity. No such decline was observed in the plants transferred to continuous light. Transfer to continuous light led to significant increases in alternative pathway capacity relative to the dark controls. Alternative pathway activity remained negligible in both the dark controls and in plants transferred to continuous light. The results of this study suggest then that light per se may be responsible for the induction of alternative pathway capacity in Belgium endive.     

Effects of the herbicide atrazine on adenine nucleotide levels in Zostera marina L. (eelgrass)

Response of adenine nucleotides (ATP, ADP, AMP) and adenylate energy charge (EC) to atrazine, a triazine herbicide, was evaluated as an indicator of metabolic state in Zostera marina L. (eelgrass), a submerged marine angiosperm. Short-term (6 h) atrazine stress reduced ATP and total adenylates (AT) at both 10 and 100 ppb, but EC remained constant. Net productivity decreased at 100, but not at 10 ppb atrazine over 6 h. Long-term (21 day) atrazine stress was evidenced by growth inhibition and 50% mortality near 100 ppb. EC was reduced at 0.1, 1.0 and 10 ppb atrazine, but ATP and EC increased with physiological response to severe stress (100 ppb) after 21 days. Apparently, ATP and AT decrease over the short-term but rebound over the long-term with severe atrazine stress, increasing beyond control levels before plant death results. Supplementing adenine nucleotide and EC results with more conventional quantitative analyses should afford greater knowledge of physiological response to environmental variation.     

Diurnal changes in adenylates and nicotinamide nucleotides in sugar beet leaves

Sugar beets (Beta vulgaris L. cv. F58-554H1) were cultured hydroponically in growth chambers at 25°C, with a photon flux density of 500 mol m^-2s^-1. Measurements were made of net CO₂ exchange, leaf adenylates (ATP, ADP and AMP), and leaf nicotinamide nucleotides (NAD⁺, NADP⁺, NADH, NADPH), over the diurnal period (16h light/8 h dark) and during photosynthetic induction. All the measurements were carried out on recently expanded leaves from 5-week-old plants. When the lights were switched on in the growth chamber, the rate of photosynthetic CO₂ uptake, and the levels of leaf ATP and NADPH increased to a maximum in 30 min and remained there throughout the light period. The increase in ATP over the first few minutes of illumination was associated with the phosphorylation of ADP to ATP and the increase in NADPH with the reduction of NADP⁺; subsequently, the increase in ATP was associated with an increase in total adenylates while the increase in NADPH was associated with an accumulation of NADP⁺ and NADPH due to the light-driven phosphorylation of NAD⁺ to NADP⁺. On return to darkness, ATP and NADPH values decreased much more slowly, requiring 2 to 4 hours to reach minimum values. From these results we suggest that (i) the total adenylate and NADPH and NADP⁺ (but not NAD⁺ and NADH) pools increase following exposure to light; (ii) the increase in pool size is not accompanied by any large change in the energy or redox states of the system; and (iii) the measured ratios of ATP/ADP and NADPH/NADP⁺ for intact leaves are low and constant during steady-state illumination.Abbreviations AEC adenylate energy charge - DHAP dihydroxyacetone phosphate - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide - PES phenazine ethosulfate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - PFD photon flux density - Ru5P ribulose-5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

An AMP nucleosidase gene knockout in Escherichia coli elevates intracellular ATP levels and increases cold tolerance

Disparate psychrophiles (e.g. glacier ice worms, bacteria, algae and fungi) elevate steady-state intracellular ATP levels as temperatures decline, which has been interpreted as a compensatory mechanism to offset reductions in molecular motion and Gibb's free energy of ATP hydrolysis. In this study, we sought to manipulate steady-state ATP levels in the mesophilic bacterium, Escherichia coli, to investigate the relationship between cold temperature survivability and elevated intracellular ATP. Based on known energetic pathways and feedback loops, we targeted the AMP nucleotidase (amn) gene, which is thought to encode the primary AMP degradative enzyme in prokaryotes. By knocking out amn in wild-type E. coli DY330 cells using recombineering methodology, we generated a mutant (AMNk) that elevated intracellular ATP levels by more than 30% across its viable temperature range. As temperature was lowered, the relative ATP disparity between AMNk and DY330 cells increased to approximately 66% at 10 degrees C, and was approximately 100% after storage at 0 degrees C for 5-7 days. AMNk cells stored at 0 degrees C for 7 days displayed approximately fivefold higher cell viability than wild-type DY330 cells treated in the same manner.     

Effects of adenylates on the circadian interaction of KaiB with the KaiC complex in the reconstituted cyanobacterial Kai protein oscillator

Cyanobacteria are photosynthetic prokaryotes that possess circadian oscillators. Clock proteins, KaiA, KaiB, KaiC compose the central circadian oscillator, which can be reconstituted in vitro in the presence of ATP. KaiC has ATPase, autokinase, and autophosphatase enzymatic activities. These activities are modulated by protein–protein interactions among the Kai proteins. The interaction of KaiB with the KaiC complex shows a circadian rhythm in the reconstituted system. We previously developed a quantitative, real-time monitoring system for the dynamic behavior of the complex using fluorescence correlation spectroscopy. Here, we examined the effects of ATP and ADP on the rhythmic interaction of KaiB. We show that increased concentration of ATP or ADP shortened period length. Adding ADP to the Kai protein oscillation shifted its phase in a phase-dependent manner. These results provide insight into how circadian oscillation entrainment mechanism is linked to cellular metabolism.