Effect of low growth temperature on coupling between electron transport and proton flux in Vicia faba thylakoids

Coupling between electron transport and proton flux has been compared in chloroplasts from Vicia faba (cv. Windsor) plants grown at 20 and 5°C. Proton uptake by warm-grown thylakoids was sensitive to external pH and stimulated by micromolar adenine nucleotide above pH 7.0. Electron transport was modulated by pH, adenine nucleotide and energy transfer inhibitors (triphenyltin and Hg²⁺). By contrast, proton uptake by cold-grown thylakoids was generally lower and was insensitive to micromolar ATP. The rate of non-phosphorylating electron flow in cold-grown thylakoids was relatively insensitive to pH and Hg²⁺ and was not modulated by adenine nucleotides or triphenyltin. Stimulation of electron transport by phosphorylating conditions in cold-grown thylakoids was generally lower and insensitive to pH. It is concluded that the control of proton efflux through CF₀-CF₁ differs in thylakoids of V. faba grown at warm and cold temperatures.     

New results about structure, function and regulation of the chloroplast ATP synthase (CF0CF1)

The chloroplast ATP synthase utilises the energy of a transmembrane electrochemical proton gradient to drive the synthesis of ATP from ADP and phosphate. This multi-subunit thylakoid membrane-bound enzyme consists of a proton channel, CF₀, and an extrinsic catalytic sector, CF₁. Stimulated by the elucidation of a three-dimensional partial structure of the mitochondrial enzyme, substantial progress has been made to understand the catalytic mechanism and interesting hypotheses have been proposed about the molecular mechanism of energy coupling. The review discusses the present state of knowledge concerning the structure, molecular genetics, catalytic mechanism, energy coupling and regulation of this important enzyme involved in photophosphorylation.     

Correlated responses of chickens to selection for production of antibodies to sheep erythrocytes

A bi directional selection experiment was conducted to measure 5-day antibody titers to sheep erythrocytes in White Leghorn chickens. There was an immediate response to selection with significant differences between lines for the selected trait found in the S₁ and all subsequent generations. Comparisons of S₆, S₇ and S₈ generation females revealed differences between lines in disease resistance and in certain reproductive traits such as age at first egg, percentage hen-day egg production, percentage fertility and duration of fertility. The implications of these correlated responses are important to selection programs for general disease resistance.     

Lack of correlation between effects of tentoxin on chloroplast coupling factor and chloroplast ultrastructure

The mediation of tentoxin-induced chlorosis through inhibition of chloroplast coupling factor 1 (CF₁) ATPase activity was investigated through an examination of the effects of tentoxin on electrophoretically-separated CF₁ ATPases from sensitive and insensitive Nicotiana species. Sensitive species exhibited three major ATPases, only one of which was inhibited at some concentrations of tentoxin. Insensitive Nicotiana species showed the same three "isozymes"upon electrophoresis but none of the isozymes were tentoxin sensitive. CF₁ isolated from Zea mays L. cv. Pioneer 3541, which is insensitive to tentoxin in vivo based on lack of chlorosis, exhibited two ATPases, one of which was sensitive to tentoxin. The concentration/activity relationships between tentoxin and ATPase inhibition of the sensitive isozyme did not correlate well with the chlorosis induced at similar levels of tentoxin in vivo. Both Oenothera hookeri Torr. & Gray and the CF₁-deficient I iota mutant derived from it are sensitive to tentoxin as determined by loss of chlorophyll and ultrastructural changes typical of the tentoxin syndrome. These results support a mechanism of action different from inhibition of CF¹ for tentoxin-induced chlorosis.     

The role of the chloroplast coupling factor in the inactivation of thylakoid membranes at low temperatures

Thylakoids isolated from spinach leaves ( Spinacia oleracea L. cv. Monatol) were exposed to variable low temperatures under non-freezing conditions. After incubation, changes in the activities of several photochemical reactions and physical properties of the membranes were measured at room temperature.
Cyclic photophosphorylation was strictly dependent on the temperature and the electrolyte concentration: decrease in temperature and increase in NaCl concentration enhanced membrane damage. Inactivation of photophosphorylation was accompanied by stimulation of non-cyclic electron transport, increase in proton permeability and decrease in δpH. When dicyclohexylcarbodiimide was added, the proton gradient became completely restored. The temperature- and salt-dependent breakdown of photophosporylation was closely related to the release of the chloroplast coupling factor (CF₁) from the membranes. The addition of Mg²⁺, very low concentrations of ATP or ADP, or higher concentrations of low-molecular-weight polyols prior to temperature treatment prevented thylakoid damage.
The data indicate that inactivation of photophosphorylation of thylakoids at low temperatures is determined to a considerable extent by the cold lability of the CF₁. As a consequence, it must be concluded that damage of biomembranes caused by freezing is not due solely to changes resulting from the ice formation but additionally by temperature-dependent alterations of cold-labile proteins. Moreover, the data explain the mechanism of non-colligative cryoprotection of isolated thylakoid membranes.     

MICROHETEROGENEITY OF BRAIN CYTOPLASMIC AND SYNAPTOPLASMIC ACTINS

Abstract— Actin present in whole rat brain cytoplasm and in synaptosomes was purified by DNase I affinity chromatography. By use of two-dimensional gels and one-dimensional isoelectric focusing gels, brain actin was shown to be composed of two isomeric forms. By comparison with muscle actins, brain actins were identified as the β and γ isomers. Muscle type α actin is not present in brain. Synaptosomal protein with high affinity for DNase I is primarily composed of β and γ actin, however, two minor synaptosomal proteins, S₁ and S₂, with similar DNase I affinity were also isolated. S₁1 and S₂ have the same apparent molecular weight as whole brain actin, are more acidic than the major actin forms and are distinct from a actin. Relative to β and γ actin, the content of S₁ and S₂ is 3-fOld greater in synaptosomes when compared to similar non-synaptosomal species. The results demonstrate heterogeneity of brain actins and compartmentalization of brain proteins with high affinity for DNase I at the synapse. It was also shown that tubulin has selective affinity for the DNase I-actin complex.     

Depletion of stromal Pi induces high 'energy-dependent' antenna exciton quenching (qE) by decreasing proton conductivity at CFO-CF1 ATP synthase

This work tests two models to account for the effects of depletion of stromal inorganic phosphate (P_i), which results in down-regulation of light capture via the exciton quenching (q_E) mechanism and has been proposed to act in feedback regulation of the light reactions. In both models, antenna down-regulation is activated by acidification of the lumen, despite the fact that linear electron flow (LEF) (and associated proton flux) is decreased upon P_i depletion. In one model, an imbalance of ATP or NADPH activates cyclic electron transfer around photosystem I (CEF1), increasing proton influx to the lumen. In the second, the effective conductivity of the CF_O-CF₁ ATP synthase to protons ( g _H⁺) is decreased, retarding proton efflux from the lumen. Sequestering of P_i by mannose infiltration increased sensitivities of q_E and pmf to LEF. The effects were attributable to decreases in g _H⁺, but not to CEF1 and were largely reversed by subsequent P_i feeding. Rapid recovery of g _H⁺ in the dark suggested that dark-labile metabolic pools are responsible for regulation of the ATP synthase. Overall, these results support models where accumulation of Benson–Calvin cycle intermediates or lowering of stromal P_i below its K _Mat the ATP synthase, retards proton efflux from the lumen, leading to build-up of pmf and subsequent down-regulation of photosynthetic light capture.     

Dose responses of cytokinins

Analysis of published data for the most widely used bioassays for cytokinins reveals that the dose responses are usually subsensitive, i.e. that S₉₀/S₁₀> 81. The data are. in general, insufficiently detailed and/or precise for the kinetics (cooperative, multiphasic or more complex) of the subsensitive responses to be determined. Genus-and species-specific differences in response type were found for callus growth, where the responses are ultrasensitive (S₉₀/S₁₀<81) for tabacco and Phaseolus Iunatus but mostly subsensitive for soybean and P. vulgaris.     

Effect of light quality (blue, red) and fluence rate on the synthesis of pigments and pigment-proteins in maize and black pine mesophyll chloroplasts

Maize ( Zea mays L. hybrid ZP-704) and black pine ( Pinus nigra Arn.) were grown for five days at low fluence rate (0.4–4.0, μmol m^–2 s⁻¹) in blue or red light. Compared to red light of the same fluence rate, blue light effects in maize were repressive for the accumulation of Chita, b , carotenoids and light-harvesting complex-2 (LHC-2) proteins. The maximal reduction of proteins bound to the light-harvesting complex of photosystem 2 and pigments was attained at different fluence rate levels. In black pine, blue light compared to the red of the same fluence rate level either activated or reduced accumulation of pigments and LHC proteins, the effect being dependent on its fluence rate level. At fluence less than 3.0 μmol m⁻² s⁻¹ blue light was more efficient for the synthesis of Chi a, b and carotenoids, hut for LHC-2 complexes, fluence rates between 0.4 and 1.5 [μmol m⁻² s⁻¹ were more effective. In pine the effects of the two lights on the accumulation of pigments and LHC proteins were demonstrated separately and were dependent on fluence rate level. This suggests irradianoe-controlled activation/deactivation of the photoreceptor at the level of the cell.     

Ecological consequences of photomovement and photobleaching in the marine flagellate Cryptomonas maculata

Abstract The marine flagellate Cryptomonas maculata is bleached and eventually killed by exposure to even moderate white-light fluence rates. Bleaching affects all of its photosynthetic pigments and the kinetics depend on the fluence rate of the radiation the organisms are exposed to. Nitrogen-deficient cells which show a reduced pigment concentration and impaired photosynthetic efficiency tolerate bleaching white-light exposure far better than the normally colored cells. In their natural environment the organisms escape this situation by a pronounced negative phototaxis at fluence rates above 3.6 klx (= 15 W.m⁻²), while they show positive phototaxis at lower fluence rates. In nitrogen-deficient cells, however, though being less prone to photobleaching, negative phototaxis commences even at a fluence rate of about 830 lx (= 3.5 W.m⁻²). The ecological consequences of the remarkable light sensitivity and the phototactic orientation are being discussed.     

Dose responses of gibberellins

To determine the response type, published data for the most widely used bioassays for gibberellins have been analyzed by means of a computer program for estimating sensitivity parameters, or by interpolation. The dose response data are almost uniformly subsensitive, i. e. more than an 81-fold increase in external gibberellin concentration is required for a change from 10 to 90% of maximal response (S₉₀/S₁₀). Data for the interaction of gibberellins with artificial membranes are, in contrast, markedly ultrasensitive (S₉₀/S₁₀± 10). This difference further strengthens the view that lipid structures do not function as receptors for gibberellins. Most of the subsensitive dose responses for gibberellins can be quite precisely represented by cooperative isotherms. However, available data are insufficiently detailed for an unequivocal choice among cooperative, multiphasic or more complex kinetics.     

Importance of oxidative electron transport over oxidative phosphorylation in optimizing photosynthesis in mesophyll protoplasts of pea (Pisum sativum L.)

The role of mitochondrial respiration in optimizing photosynthesis was assessed in mesophyll protoplasts of pea ( Pisum sativum L., cv. Arkel) by using low concentrations of oligomycin (an inhibitor of oxidative phosphorylation), antimycin A (inhibits cytochrome pathway of electron transport) and salicylhydroxamic acid (SHAM, an inhibitor of alternative oxidase). All three compounds decreased the rate of photosynthetic O₂ evolution in mesophyll protoplasts, but did not affect chloroplast photosynthesis. The inhibition of photosynthesis by these mitochondrial inhibitors was stronger at optimal CO₂ (1.0 m M NaHCO₃) than that at limiting CO₂ (0.1 m M NaHCO₃). We conclude that mitochondrial metabolism through both cytochrome and alternative pathways is essential for optimizing photosynthesis at limiting as well as at optimal CO₂. The ratios of ATP to ADP in whole protoplast extracts were hardly affected, despite the marked decrease in their photosynthetic rates by SHAM. Similarly, the decrease in the ATP/ADP ratio by oligomycin or antimycin A was more pronounced at limiting CO₂ than at optimal CO₂. The mitochondrial oxidative electron transport, through both cytochrome and alternative pathways, therefore akppears to be more important than oxidative phosphorylation in optimizing photosynthesis, particularly at limiting CO₂ (when ATP demand is expected to be low). Our results also confirm that the alternative pathway has a significant role in contributing to the cellular ATP, when the cytochrome pathway is limited.     

Relationship between the bovine major histocompatibility complex (BoLA), erythrocyte markers and susceptibility to mastitis in Icelandic cattle

Summary. Milk and blood samples were obtained from three Icelandic dairy herds. The herds were monitored regularly for mastitis incidence. Cell counts, adenosine triphosphate (ATP) and antitrypsin levels of the milk samples were recorded. In addition, red cell and BoLA typing were performed on the blood. Although cell counts and ATP levels showed significant associations with mastitis, antitrypsin levels did not. Red blood cell antigens N'₂ and S₁ and the lymphocyte antigen detected by the monoclonal antibody M7 were associated with low cell counts, whilst BoLA w6 and w6.1 were associated with high cell counts. BoLA w6.2 and w11 showed significant association with high antitrypsin levels. Only ED116 showed a significant association with mastitis.     

Diurnal changes in phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase properties in the natural environment: interplay of light and temperature in a C4 thermophile

Activities of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) were measured in leaf extracts of field grown Amaranthus paniculatus L. (C₄) during a natural diurnal irradiance and temperature pattern. Enzyme assays were run at both fixed (30°C) and the corresponding leaf temperature at the time of harvest. Light activation of PEP carboxylase (PEPCase) at fixed assay temperatures was expressed as a decrease in S_0–5 (PEP) after a threshold (> 330 μmol m^–2 s^–1) photon fluence rate was surpassed at noon. Earlier in the morning, increase in apparent enzyme affinity for PEP was observed when the assay was run at leaf temperature, indicating a physiologically meaningfull effect of temperature on S^0.5 (PEP). The 3.3-fold increase in PEPCase activity at low PEP and fixed assay temperature between the minimal and maximal irradiance and temperature hours of the day, became 12.8-, 11.5- and 7.4-fold when assays were run at the corresponding leaf temperature during three diurnal cycles with respective temperature differences (max minus min) of 9.0, 8.3 and 7.4°C. The extent of malate inhibition was the same for both day and night forms of PEPCase assayed at 35°C, but increased considerably with night enzyme at 25°C. The results indicate that light increases the apparent affinity of PEPCase for PEP and that at lower temperatures malate becomes more inhibitory. Pyruvate orthophosphate dikinase activity started to increase immediately after sunrise and the 10-fold increase at fixed temperature became 14.8-, 14.2- and 13.1-fold when assays were run at the above leaf temperatures. This indicates that the light effect predominates with pyruvate, orthophosphate dikinase, while with phosphoenolpyravate carboxylase, light and temperature co-operate to increase the day enzyme activities.     

Stimulation of respiration by Pb2+ in detached leaves and mitochondria of C3 and C4 plants

The exposure of detached leaves of C₃ plants (pea, barley) and C₄ plant (maize) to 5 m M Pb (NO₃)₂ for 24 h caused a reduction of their photosynthetic activity by 40–60%, whereas the respiratory rate was stimulated by 20–50%. Mitochondria isolated from Pb²⁺-treated pea leaves oxidized substrates (glycine, succinate, malate) at higher rates than mitochondria from control leaves. The respiratory control (RCR) and the ADP/O ratio were not affected. Pb²⁺ caused an increase in ATP content and the ATP/ADP ratio in pea and maize leaves. Rapid fractionation of barley protoplasts incubated at low and high CO₂ conditions, indicated that the increased ATP/ADP ratio in Pb²⁺-treated leaves resulted mainly from the production of mitochondrial ATP. The measurements of membrane potential of mitochondria with a TPP⁺-sensitive electrode further showed that mitochondria isolated from Pb²⁺-treated leaves had at least as high membrane potential as mitochondria from control leaves. The activity of NAD-malate dehydrogenase in the protoplasts from barley leaves treated with Pb²⁺ was 3-fold higher than in protoplasts from control leaves. The activities of photorespiratory enzymes NADH-hydroxypyruvate reductase and glycolate oxidase as well as of NAD-malic enzyme were not affected. The presented data indicate that stimulation of respiration in leaves treated by lead is in a close relationship with activation of malate dehydrogenase and stimulation of the mitochondrial ATP production. Thus, respiration might fulfil a protective role during heavy metal exposure.     

Nitrogen fixation by Baltic cyanobacteria is adapted to the prevailing photon flux density

N₂ fixation, measured as acetylene reduction, was studied in laboratory cultures and in natural assemblages (both as a mixed population and as individually picked colonies) of the heterocystous cyanobacteria Aphanizomenon sp. and Nodularia spp. from the Baltic Sea. During a diurnal cycle of alternating light and darkness, these organisms reduced acetylene predominantly during the period of illumination, although considerable activity was also observed during the dark period. In both laboratory cultures and natural populations N₂ fixation was saturated below a photon flux density of 600 μm⁻² s⁻¹. In cyanobacterial blooms in the Baltic Sea, nitrogenase activity was mostly confined to the surface layers. Samples collected from greater depths did not possess the same capacity for acetylene reduction as samples from the surface itself, even when incubated at the photon flux density prevailing in surface waters. This suggests that, with respect to N₂ fixation, Baltic cyanobacteria are adapted to the intensity of illumination that they are currently experiencing.     

Light dependent activation of CO2 assimilation and the ratio of Rubisco activase to Rubisco during leaf aging of rice (Oryza sativa)

The effects of the ratio of Rubisco activase to Rubisco (activase/Rubisco ratio) on light dependent activation of CO₂ assimilation were investigated during leaf aging of rice. Changes of photosynthetic CO₂ gas exchange rates in relation to step increases of light intensity from two photon flux densities of 60 µmol m⁻² s⁻¹ (low initial PFD) and 500 µmol m⁻² s⁻¹ (high initial PFD) to saturated PFD of 1 800 µmol m⁻² s⁻¹ were measured. These photosynthetic activation processes were considered to be limited by the Rubisco activation rate when analyzed by the relaxation method. The relaxation time of low initial PFD gradually declined from 3 to 33 days after leaf emergence and showed high and negative correlation to the activase/Rubisco ratio. The initial rate of Rubisco activation under low initial PFD linearly correlated to the amounts of Rubisco activase, whereas these were almost constant from 3 to 23 days after leaf emergence. But these correlations could not be recognized in the case of high initial PFD. Moreover, the relaxation times were more sensitive to intercellular CO₂ concentration (C_i) under high initial PFD than under low initial PFD, especially, at C_i below 300 µl l⁻¹. These results suggest the involvement of the activase/Rubisco ratio in the photosynthetic activation under relatively low initial PFD, and the limitation of photosynthetic activation under relatively high initial PFD by Rubisco carbamylation during leaf aging of rice.     

ASYMMETRIC, BIMODAL TRADE-OFFS DURING ADAPTATION OF METHYLOBACTERIUM TO DISTINCT GROWTH SUBSTRATES

Trade-offs between selected and nonselected environments are often assumed to exist during adaptation. This phenomenon is prevalent in microbial metabolism, where many organisms have come to specialize on a narrow breadth of substrates. One well-studied example is methylotrophic bacteria that can use single-carbon (C₁) compounds as their sole source of carbon and energy, but generally use few, if any, multi-C compounds. Here, we use adaptation of experimental populations of the model methylotroph, Methylobacterium extorquens AM1, to C₁ (methanol) or multi-C (succinate) compounds to investigate specialization and trade-offs between these two metabolic lifestyles. We found a general trend toward trade-offs during adaptation to succinate, but this was neither universal nor showed a quantitative relationship with the extent of adaptation. After 1500 generations, succinate-evolved strains had a remarkably bimodal distribution of fitness values on methanol: either an improvement comparable to the strains adapted on methanol or the complete loss of the ability to grow on C₁ compounds. In contrast, adaptation to methanol resulted in no such trade-offs. Based on the substantial, asymmetric loss of C₁ growth during growth on succinate, we suggest that the long-term maintenance of C₁ metabolism across the genus Methylobacterium requires relatively frequent use of C₁ compounds to prevent rapid loss.     

Insulin-stimulated ribosomal protein synthesis in maize embryonic axes during germination

Addition of insulin to maize seed ( Zea mays L. cv. Chalqueño) was found to accelerate germination and seedling growth. Insulin-stimulated maize axes showed enhancement of 35 S-methionine incorporation into ribosomal proteins (rp) and mobilization of S₆ rp mRNA into polysomes. Increase in S₆ rp phosphorylation of the small ribosomal subunit (40S) was observed in 32 P-orthophosphate pulse-labeled experiments when maize axes were stimulated by insulin. Application of either wortmannin or rapamycin, inhibitors of protein kinases of the insulin transduction pathway, abolished the insulin stimulatory effect on S₆ rp phosphorylation and on ribosomal protein synthesis. The above data are interpreted as an indication of the existence of an insulin-stimulated signal transduction pathway in maize tissues that is involved in the regulation of translation.