RuBP Limitation of Photosynthetic Carbon Fixation during NH(3) Assimilation : Interactions between Photosynthesis, Respiration, and Ammonium Assimilation in N-Limited Green Algae

The effects of ammonium assimilation on photosynthetic carbon fixation and O₂ exchange were examined in two species of N-limited green algae, Chlorella pyrenoidosa and Selenastrum minutum. Under light-saturating conditions, ammonium assimilation resulted in a suppression of photosynthetic carbon fixation by S. minutum but not by C. pyrenoidosa. These different responses are due to different relationships between cellular ribulose bisphosphate (RuBP) concentration and the RuBP binding site density of ribulose bisphosphate carboxylase/oxygenase (Rubisco). In both species, ammonium assimilation resulted in a decrease in RuBP concentration. In S. minutum the concentration fell below the RuBP binding site density of Rubisco, indicating RuBP limitation of carboxylation. In contrast, RuBP concentration remained above the binding site density in C. pyrenoidosa. Compromising RuBP regeneration in C. pyrenoidosa with low light resulted in an ammonium-induced decrease in RuBP concentration below the RuBP binding site density of Rubisco. This resulted in a decrease in photosynthetic carbon fixation. In both species, ammonium assimilation resulted in a larger decrease in net O₂ evolution than in carbon fixation. Mass spectrometric analysis shows this to be a result of an increase in the rate of mitochondrial respiration in the light.     

Ammonium Assimilation Requires Mitochondrial Respiration in the Light : A Study with the Green Alga Selenastrum minutum

Mass spectrometric analysis of O₂ and CO₂ exchange in the green alga Selenastrum minutum (Naeg. Collins) provides evidence for the occurrence of mitochondrial respiration in light. Stimulation of amino acid synthesis by the addition of NH₄Cl resulted in nearly a 250% increase in the rate of TCA cycle CO₂ efflux in both light and dark. Ammonium addition caused a similar increase in cyanide sensitive O₂ consumption in both light and dark. Anaerobiosis inhibited the CO₂ release caused by NH₄Cl. These results indicated that the cytochrome pathway of the mitochondrial electron transport chain was operative and responsible for the oxidation of a large portion of the NADH generated during the ammonium induced increase in TCA cycle activity. In the presence of DCMU, ammonium addition also stimulated net O₂ consumption in the light. This implied that the Mehler reaction did not play a significant role in O₂ consumption under our conditions. These results show that both the TCA cycle and the mitochondrial electron transport chain are capable of operation in the light and that an important role of mitochondrial respiration in photosynthesizing cells is the provision of carbon skeletons for biosynthetic reactions.     

Interactions between Photosynthesis and Respiration in the Green Alga Chlamydomonas reinhardtii (Characterization of Light-Enhanced Dark Respiration)

The rate of respiratory O2 consumption by Chlamydomonas reinhardtii cell suspensions was greater after a period of photosynthesis than in the preceding dark period. This "light-enhanced dark respiration" (LEDR) was a function of both the duration of illumination and the photon fluence rate. Mass spectrometric measurements of gas exchange indicated that the rate of gross respiratory O2 consumption increased during photosynthesis, whereas gross respiratory CO2 production decreased in a photon fluence rate-dependent manner. The rate of postillumination O2 consumption provided a good measure of the O2 consumption rate in the light. LEDR was substantially decreased by the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or glycolaldehyde, suggesting that LEDR was photosynthesis-dependent. The onset of photosynthesis resulted in an increase in the cellular levels of phosphoglycerate, malate, and phosphoenolpyruvate, and a decrease in whole-cell ATP and citrate levels; all of these changes were rapidly reversed upon darkening. These results are consistent with a decrease in the rate of respiratory carbon flow during photosynthesis, whereas the increase in respiratory O2 consumption during photosynthesis may be mediated by the export of photogenerated reductant from the chloroplast. We suggest that photosynthesis interacts with respiration at more than one level, simultaneously decreasing the rate of respiratory carbon flow while increasing the rate of respiratory O2 consumption.     

In Vivo and in Vitro Studies of Glucose-6-Phosphate Dehydrogenase from Barley Root Plastids in Relation to Reductant Supply for NO2- Assimilation

Pyridine nucleotide pools were measured in intact plastids from roots of barley (Hordeum vulgare L.) during the onset of NO2- assimilation and compared with the in vitro effect of the NADPH/NADP ratio on the activity of plastidic glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) from N-sufficient or N-starved roots. The NADPH/NADP ratio increased from 0.9 to 2.0 when 10 mM glucose-6-phosphate was supplied to intact plastids. The subsequent addition of 1 mM NaNO2 caused a rapid decline in this ratio to 1.5. In vitro, a ratio of 1.5 inactivated barley root plastid G6PDH by approximately 50%, suggesting that G6PDH could remain active during NO2- assimilation even at the high NADPH/NADP ratios that would favor a reduction of ferredoxin, the electron donor of NO2- reductase. Root plastid G6PDH was sensitive to reductive inhibition by dithiothreitol (DTT), but even at 50 mM DTT the enzyme remained more than 35% active. In root plastids from barley starved of N for 3 d, G6PDH had a substantially reduced specific activity, had a lower Km for NADP, and was less inhibited by DTT than the enzyme from N-sufficient root plastids, indicating that there was some effect of N starvation on the G6PDH activity in barley root plastids.     

The Z-conformation of poly(dA-dT).poly(dA-dT) in solution as studied by ultraviolet resonance Raman spectroscopy

Poly(dA-dT).poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni2+ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl2 in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm-1 coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm-1 region characterizing local chemical interactions of the Ni2+ ions with the adenine N7 position, iii) lines at about 1483- and 1582 cm-1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680- and 1733 cm-1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT).poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.     

Kinetics of exchangeable protons in Z DNA: a UV resonance Raman study.

We have studied the hydrogen-deuterium exchange kinetics of the exchangeable protons of the poly(dG-dC).poly(dG-dC) in the Z form of the polymer, using resonance Raman spectroscopy with 257 nm and 284 nm excitation wavelengths. In our experimental conditions (4.5 M NaCl, phosphate buffer pH7, 2 degrees C) the two amino protons and the imino proton of guanine are exchanged with the same exchange half-time of 13 min, whereas the two amino protons of cytosine are exchanged with the same exchange half-time of 51 min.     

Mitochondrial Respiration Can Support NO(3) and NO(2) Reduction during Photosynthesis : Interactions between Photosynthesis, Respiration, and N Assimilation in the N-Limited Green Alga Selenastrum minutum

Mass spectrometric analysis shows that assimilation of inorganic nitrogen (NH₄⁺, NO₂⁻, NO₃⁻) by N-limited cells of Selenastrum minutum (Naeg.) Collins results in a stimulation of tricarboxylic acid cycle (TCA cycle) CO₂ release in both the light and dark. In a previous study we have shown that TCA cycle reductant generated during NH₄⁺ assimilation is oxidized via the cytochrome electron transport chain, resulting in an increase in respiratory O₂ consumption during photosynthesis (HG Weger, DG Birch, IR Elrifi, DH Turpin [1988] Plant Physiol 86: 688-692). NO₃⁻ and NO₂⁻ assimilation resulted in a larger stimulation of TCA cycle CO₂ release than did NH₄⁺, but a much smaller stimulation of mitochondrial O₂ consumption. NH₄⁺ assimilation was the same in the light and dark and insensitive to DCMU, but was 82% inhibited by anaerobiosis in both the light and dark. NO₃⁻ and NO₂⁻ assimilation rates were maximal in the light, but assimilation could proceed at substantial rates in the light in the presence of DCMU and in the dark. Unlike NH₄⁺, NO₃⁻ and NO₂⁻ assimilation were relatively insensitive to anaerobiosis. These results indicated that operation of the mitochondrial electron transport chain was not required to maintain TCA cycle activity during NO₃⁻ and NO₂⁻ assimilation, suggesting an alternative sink for TCA cycle generated reductant. Evaluation of changes in gross O₂ consumption during NO₃⁻ and NO₂⁻ assimilation suggest that TCA cycle reductant was exported to the chloroplast during photosynthesis and used to support NO₃⁻ and NO₂⁻ reduction.     

Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum

Nitrate-limited chemostat cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) were used to determine the effects of nitrogen addition on photosynthesis, dark respiration, and dark carbon fixation. Addition of NO₃⁻ or NH₄⁺ induced a transient suppression of photosynthetic carbon fixation (70 and 40% respectively). Intracellular ribulose bisphosphate levels decreased during suppression and recovered in parallel with photosynthesis. Photosynthetic oxygen evolution was decreased by N-pulsing under saturating light (650 microeinsteins per square meter per second). Under subsaturating light intensities (<165 microeinsteins per square meter per second) NH₄⁺ addition resulted in O₂ consumption in the light which was alleviated by the presence of the tricarboxylic acid cycle inhibitor fluoroacetate. Addition of NO₃⁻ or NH₄⁺ resulted in a large stimulation of dark respiration (67 and 129%, respectively) and dark carbon fixation (360 and 2080%, respectively). The duration of N-induced perturbations was dependent on the concentration of added N. Inhibition of glutamine 2-oxoglutarate aminotransferase by azaserine alleviated all these effects. It is proposed that suppression of photosynthetic carbon fixation in response to N pulsing was the result of a competition for metabolites between the Calvin cycle and nitrogen assimilation. Carbon skeletons required for nitrogen assimilation would be derived from tricarboxylic acid cycle intermediates. To maintain tricarboxylic acid cycle activity triose phosphates would be exported from the chloroplast. This would decrease the rate of ribulose bisphosphate regeneration and consequently decrease net photosynthetic carbon accumulation. Stoichiometric calculations indicate that the Calvin cycle is one source of triose phosphates for N assimilation; however, during transient N resupply the major demand for triose phosphates must be met by starch or sucrose breakdown. The effects of N-pulsing on O₂ evolution, dark respiration, and dark C-fixation are shown to be consistent with this model.     

Ultraviolet Raman spectroscopy of polyribouridylic acid: Excitation profile of the hypochromism induced by order–disorder transition

Raman spectra of polyribouridylic acid excited in the uv region, from 363 to 290 nm, are reported. The conformational changes of the polymer from random coil to ordered structure with stacked bases at high and low temperature, respectively, are reflected by important changes in the Raman line intensities; this Raman hypochromism is itself a function of the excitation wavelength—its profile has been determined and shows negative values in the region of 290 nm (near resonance), i.e., hypochromism becomes hyperchromism. Thus the knowledge of the hypochromism excitation profile is important in following order–disorder transition of a polymer using resonance Raman spectroscopy. Theoretical attempts are proposed for explanation, involving not only the relative variations of the molar extinction coefficient on the order-disorder transition of the polymer, but also the damping factors of the vibronic levels. The theoretical curve is found to fit adequately the experimental data over the excitation range, using only the frequency of the O-O transition of uracil and a vibronic linewidth of 2200 cm^?1.