Control of CO2 fixation. Regulation of spinach ribulose-5-phosphate kinase by stromal metabolite levels

The effect of stromal metabolites on the light-activated form of ribulose-5-phosphate kinase was studied with the enzyme rapidly extracted from illuminated spinach chlorplasts. In some instances, the effect of metabolites on the dark-inactivated enzyme extracted from darkened chloroplasts was also investigated. (1) The light-activated form of the enzyme is competitively inhibited with respect to ribulose 5-phosphate by 6-phosphogluconate, ribulose 1,5-bisphosphate, 3-phosphoglycerate and phosphate. Also, fructose 1,6-bisphosphate is inhibitory. All these compounds, except ribulose 1,5-bisphosphate, show an increasing inhibitory effect at lower pH values. Therefore, in the presence of these inhibitors, ribulose-5-phosphate kinase becomes strongly pH dependent. These compounds also exert an inhibitory effect on the dark-inactivated enzyme. (2) The assay of stromal levels of 6-phosphogluconate showed that this compound increased dramatically during a light-dark transient. (3) The dark-inactivated form of ribulose-5-phosphate kinase is strongly inhibited by ADP, the inhibition being competitive with respect to ATP. (4) A simulation of stromal metabolite levels in the enzyme activity assay indicates that in illuminated chloroplasts ribulose-5-phosphate kinase attains only about 4% of its maximal activity. When the fully light-activated enzyme is assayed under conditions occurring in the stroma in the dark, the activity is further decreased by a factor of 20. The same assay with the dark-inactivated enzyme yields an activity of virtually zero. (5) These results demonstrate that in the chloroplasts ribulose-5-phosphate kinase can not only be very efficiently switched off in the dark, but also be subjected to fine control during the illuminated state through the action of stromal metabolites.     

Photoactivation of the manganese catalyst of O2 evolution. I. Biochemical and kinetic aspects

Photosynthetic O₂ evolution requires a Mn complex which is activated by light. An analysis of this activation process yielded the following results:

1.

1. In any given illumination, the time course is first order, the rate being proportional to the number of inactive O₂-evolving System II trapping centers (the quantum yield being invariant).     

Pyridine nucleotides and H2 as electron donors to the respiratory and photosynthetic electron-transfer chains and to nitrogenase in Anabaena heterocysts

The pathways through which NADPH, NADH and H₂ provide electrons to nitrogenase were examined in anaerobically isolated heterocysts. Electron donation in freeze-thawed heterocysts and in heterocyst fractions was studied by measuring O₂ uptake, acetylene reduction and reduction of horse heart cytochrome c. In freeze-thawed heterocysts and membrane fractions, NADH and H₂ supported cyanide-sensitive, respiratory O₂ uptake and light-enhanced, cyanide-insensitive uptake of O₂ resulting from electron donation to O₂ at the reducing side of Photosystem I. Membrane fractions also catalyzed NADH-dependent reduction of cytochrome c. In freeze-thawed heterocysts and soluble fractions from heterocysts, NADPH donated electrons in dark reactions to O₂ or cytochrome c through a pathway involving ferredoxin:NADP reductase; these reactions were only slightly influenced by cyanide or illumination. In freeze-thawed heterocysts provided with an ATP-generating system, NADH or H₂ supported slow acetylene reduction in the dark through uncoupler-sensitive reverse electron flow. Upon illumination, enhanced rates of acetylene reduction requiring the participation of Photosystem I were observed with NADH and H₂ as electron donors. Rapid NADPH-dependent acetylene reduction occurred in the dark and this activity was not influenced by illumination or uncoupler. A scheme summarizing electron-transfer pathways between soluble and membrane components is presented.     

The effects of mono- and divalent salts on the O2-evolution activity and low temperature multiline EPR spectrum of Photosystem II preparations from spinach

The ability of salts to inhibit the O₂-evolution activity of PS II preparations is shown to parallel closely the Hofmeister series, suggesting that inhibition is related to the solubility of the 16, 24 and 33 kDa proteins in these salt solutions. An examination of the effect of salt inactivation on the low temperature multiline EPR signal indicates that the release of either the 16 and 24 kDa proteins, or additionally the 33 kDa protein blocks or greatly reduces the efficiency of the advancement of the water-splitting complex to the S₂-state; under some conditions, this inhibition is reversible.     

The role of chloride in O2 evolution by thylakoids from salt-tolerant higher plants

(1) Thylakoids isolated from leaves of two salt-tolerant higher plant species were found to require high (greater than 250 mM) concentrations of Cl⁻ for maximal rates of photosynthetic O₂ evolution and maximum variable chlorophyll a fluorescence yield. These activities were also tolerant to extremely high (2–3 M) salt concentrations. Their pH dependence was markedly different in the absence and presence of sufficient salt levels. (2) When Cl⁻ was provided as CaCl₂, as opposed to MgCl₂, KCl or NaCl, higher rates of O₂ evolution were obtained, suggesting that Ca²⁺ has an important role in Photosystem II reactions. (3) The site of Cl⁻ action was located on the electron donor side of Photosystem II. (4) O₂ evolution in the presence of optimal Cl⁻ concentrations showed a pH dependence closely matched by that of ³⁵Cl-NMR line broadening, which is indicative of Cl⁻ binding. This pH-dependent ³⁵Cl-NMR line-width broadening was not altered significantly by treatment of the thylakoids with EDTA; it was, however, abolished by heat treatment. (5) Only anions with similar ionic radii (Br⁻, NO⁻₃) were effective in replacing Cl⁻. Small anions such as F⁻ and OH⁻ were inhibitory; larger ions had no effect. The inhibition by F⁻ is due, at least in part, to displacement of Cl⁻. The selectivity is attributed to a combination of steric and ionic field effects. (6) It is proposed that Cl⁻ facilitates Photosystem II electron transport by reversible ionic binding to the O₂-evolving complex itself or to the thylakoid membrane in close proximity to it.     

Oscillation of delayed luminescence from PS II: recombination of S2Q−B and S3Q−B

Luminescence decaying in the seconds to minutes time scale was studied in spinach chloroplasts and the following results were obtained: (1) After a series of flashes a slow phase which decays in the tens of seconds to minutes time scale was observed to oscillate with a pattern characteristic of S₂Q^?_B and S₃Q^?_B recombination. This phase was lost upon Tris-treatment or upon the addition of DCMU. (2) After every flash a small faster phase of luminescence decaying in the seconds time scale was also present. This phase progressively increased with increasing numbers of flashes but when methyl viologen was present no such progressive increase of this phase occurred. In the presence of DCMU this seconds time scale luminescence was greatly increased. This phase of luminescence is attributed to S₂Q^?_A recombination. (3) Tris-treatment resulted in the appearance of an even faster phase of luminescence which may be due to Z⁺Q^?_B recombination. These results demonstrate a close correlation of the kinetics of luminescence decay with thermoluminescence emission temperature.     

Partial disintegration and reconstitution of the photosynthetic oxygen evolution system. Binding of 24 kilodalton and 18 kilodalton polypeptides

Treatment with 1 M NaCl almost totally removed two polypeptides of 24 and 18 kDa from the Photosystem II particles of spinach chloroplasts and reduced the oxygen-evolution activity by about half. Both polypeptides were able to rebind to the NaCl-treated particles in a low-salt medium. The rebinding of the 24 kDa polypeptide showed a saturation curve whose maximum level was close to that naturally occurring in the untreated particles. In parallel with the amount of rebound 24 kDa polypeptide, the oxygen-evolution activity was recovered. The 18 kDa polypeptide bound to the NaCl-treated particles without saturation. When the 18 kDa polypeptide was added to the particles previously treated with NaCl and then supplemented with a saturating amount of 24 kDa polypeptide, there appeared, in addition to the binding without saturation, another binding of the 18 kDa polypeptide with saturation to a maximum level close to that naturally occurring in the untreated particles. The 18 kDa polypeptide did not restore the oxygen-evolution activity. These findings suggest that there are specific binding sites; one for the 24 kDa polypeptide located on the Photosystem II particles, and the other for the 18 kDa polypeptide on the 24 kDa polypeptide.     

Photosynthetic nature of nitrate uptake and reduction in the cyanobacterium Anacystis nidulans

The photosynthetic nature of the initial stages of nitrate assimilation, namely, uptake and reduction of nitrate, has been investigated in cells of the cyanobacterium Anacystis nidulans treated with l-methionine dl-sulfoximine to prevent further assimilation of the ammonium resulting from nitrate reduction. The light-driven utilization of nitrate or nitrite by these cells results in ammonium release and is associated with concomitant oxygen evolution. Stoichiometry values of about 2 mol oxygen evolved per mol nitrate reduced to ammonium and 1.5 mol oxygen per mol nitrite have been determined in the presence of CO₂, as well as in its absence, with nitrate or nitrite as the only Hill reagent. This indicates that in A. nidulans water photolysis directly provides, without the need for carbon metabolites, the reducing power required for the in vivo reduction of nitrate and nitrite to ammonium, processes which are besides strongly inhibited when the operation of the photosynthetic noncyclic electron flow is blocked. Evidence indicating the participation of concentrative transport system(s) in the uptake of nitrate and nitrite by A. nidulans is also presented. The operation of these energy-requiring systems seems to account for the sensitivity to ATP-synthesis inhibitors exhibited by nitrate and nitrite utilization in l-methionine dl-sulfoximine-treated cells. The utilization of nitrate by A. nidulans cells, concomitant with oxygen evolution, can therefore be considered as a genuinely CO₂-independent photosynthetic process that makes direct use of photosynthetically generated assimilatory power.     

The influence of carotenoids on the conformation of chlorophyll-protein complexes isolated from the cyanobacterium Plectonema boryanum. Absorption and circular dichroism study

Thylakoid membranes of the cyanobacterium Plectonema boryanum solubilized with Triton X-100 can be resolved into three fractions of pigment-protein complexes (Hladík, J. and Sofrová, D. (1981) Photosynthetica 15, 490–503). Fraction I contained relatively the highest amount of carotenoids as well as monomeric forms of chlorophyll a, Fractions II and III contained chlorophyll-protein complexes with a characteristic exciton-split circular dichroism in the red region. It has been shown that fraction III is an oligomeric form of the chlorophyll-protein complex of fraction II. Circular dichroism spectra indicate that, different from fraction II, fraction III contains specifically oriented and space-fixed molecules of carotenoids. Thermal dissociation of fracion III to fraction II is accompanied by disappearance of the positive circular dichroism effect of carotenoids in the 500–550 nm region, thus causing deorganization of the carotenoids, proceeding in parallel to the geometrical rearrangement of chlorophyll molecules. Extraction of the carotenoids of fraction III with heptane is acompanied by dissociation of fraction III. We assume that the observed effects are due to binding of the two pigments to the protein component of the complex and that carotenoids can mediate a part of the interactions which stabilize the structure of pigment-protein complexes. Thus, besides the light-harvesting and protective functions, carotenoids can also play a structural role.     

Calcium ions can be substituted for the 24-kDa polypeptide in photosynthetic oxygen evolution

Photosystem II particles were prepared from spinach chloroplasts with Triton X-100, and treated with 1.0 M NaCl to remove polypeptides of 24 kDa and 18 kDa and to reduce the photosynthetic oxygen-evolution activity by about half. Oxygen-evolution activity was restored almost to the original level with 10 mM Ca²⁺, in a similar manner to the rebinding of 24-kDa polypeptide. Other cations such as magnesium, sodium and manganese ions could not restore any oxygen-evolution activity. These observations, together with a kinetic analysis, suggest that Ca²⁺ can be substituted for the 24-kDa polypeptide in photosynthetic oxygen evolution in Photosystem II particles.     

The interaction of the reaction center secondary quinone with the ubiquinone-cytochrome c2 oxidoreductase in Rhodopseudomonas sphaeroides chromatophores

(1) Two populations of reaction centers in the chromatophore membrane can be distinguished under some conditions of initial redox poise (300 mV < E_h < 400 mV): those which transfer a reducing equivalent after the first flash from the secondary quinone (Q_II) of the reaction center to cytochrome b of the ubiquinone-cytochrome c₂ oxidoreductase; and those which retain the reducing equivalent on Q^?_II until a second flash is given. These two populations do not exchange on a time scale of tens of seconds. (2) At redox potentials higher than 400 mV, Q^?_II generated after the first flash is no longer able to reduce cytochrome b-560 even in those reaction centers associated with an oxidoreductase. Under these conditions, doubly reduced Q_II generated by a second flash is required for cytochrome b reduction, so that the Q_II effectively functions as a two-electron gate into the oxidoreductase at these high potentials. (3) At redox potentials below 300 mV, although the two populations of Q_II are no longer distinguishable, cytochrome b reduction is still dependent on only part of the reaction center population. (4) Proton binding does not oscillate under any condition tested.     

luxS基因缺失对禽致病性大肠杆菌O1、O2和O78三种血清型分离株的生物学特性影响

【目的】LuxS/AI-2型密度群体感应系统产生的自诱导信号分子AI-2(AI-2的产生需要luxS基因编码的Lux S蛋白参与)参与对细菌众多生理功能的调控。探讨luxS对不同血清型禽致病性大肠杆菌(Avian Pathogenicity Escherichia coli,APEC)生物学特性的影响。【方法】本研究以APEC优势血清型APECO_1(O_1血清型)、DE17(O_2血清型)、E940(O_(78)血清型)及其相应luxS缺失株为研究对象,对野生株和缺失株的生长特性、生物被膜形成、rdar(red,dry and rough)形态、运动性和耐药性等特性进行分析。【结果】luxS基因的缺失不影响APEC生长特性,但导致APEC不能产生AI-2;此外,luxS基因的缺失显著降低APECO_1和E940的生物被膜形成(P0.05),而DE17的生物被膜形成无显著变化。对各菌株的rdar形态和运动性检测结果表明,luxS基因的缺失改变了APECO_1的rdar形态,对DE17和E940并无影响;显著降低了APECO_1和DE17运动能力,对E940并无影响。荧光定量PCR检测结果表明,luxS基因的缺失显著降低APECO_1、DE17和E940与细菌运动性相关的鞭毛基因fli G和fli I的转录水平(P0.05)。此外,对各菌株的耐药性检测结果表明,luxS基因缺失导致APECO_1对头孢吡肟和丁胺卡那由耐药变为高敏,同时对氯霉素与E940相同由高敏变为耐药,但对DE17的耐药性无显著改变。【结论】luxS对APEC的生物学特性具有重要的调控作用,且这种调控具有菌株特异性。     

Biochemical characterization of a highly active O2-evolving Photosystem II preparation from maize

An O₂-evolving Photosystem (PS) II preparation was isolated from maize by a Triton X-100 procedure (Kuwabara, T. and Murata, N. (1982) Plant Cell Physiol. 23, 533–539). A highly active O₂-evolving preparation was obtained which evolved O₂ at 76% the rate of fresh chloroplasts (H₂O → 2,6-dichloro-p-benzoquinone) and was very sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. There was no detectable PS I activity in the preparation (2,3,5,6-tetramethyl-p-phenylenediamine → methyl viologen). When analyzed by lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis the O₂-evolving preparation was shown to be highly depleted in CP I, CF₁, and devoid of cytochromes f and b-563 (the absence of which was confirmed by difference spectroscopy). The preparation was enriched in the PS II reaction center polypeptides I and II, the 34 kDa polypeptide (Metz, J., Wong, J. and Bishop, N.I. (1980) FEBS Lett. 114, 61–66), the Coomassie blue-stainable 32 kDa polypeptide (Kuwabara, T. and Murata, N. (1979) Biochim. Biophys. Acta 581, 228–236), LHCP-associated polypeptides and cytochrome b-559. Polypeptides of unknown function at 40.5, 25, 24, 22, 16.6 and 14 kDa were also present in the O₂-evolving preparation. Triton X-114 phase partitioning (Bricker, T.M. and Sherman, L.A. (1982) FEBS Lett. 149, 197–202) indicated that the majority of these polypeptides were intrinsic. Only the polypeptides at 32, 25, 24 and 14 kDa were extrinsic. When examined by the octylglucoside procedure of Camm and Green (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) the PS II O₂-evolving preparation was shown to contain the chlorophyll-proteins CP 27, CP 29, CP II1, D, and CP a-1 and CP a-2. Chlorophyll-proteins associated with PS I were highly depleted. The visible absorption spectra indicated an enrichment of chlorophyll b and carotenoids in the preparation. The 77 K fluorescence emission spectrum (excitation wavelength = 435 nm) exhibits a strong F-686 with little F-695 shoulder and a broad, low-intensity F-735 emission.     

Bacteriochlorophyll a cation radical in solution and in reaction centers of Rhodopseudomonas sphaeroides. Resonance Raman scattering

Resonance Raman spectra of the π-cation of bacterio-chlorophyll a in solution at 30 K are reported and discussed. Outer C

C bonds of the pyrroles and the methine bridges are weakened by the ionization, while C

N and Mg-N bonds remain essentially unaffected. Resonance Raman spectra of reaction centers suggest that the positive charge on P-870⁺ should be localized on a single bacteriochlorophyll molecule by the lifetime of the scattering process (≈ 10^?13 s).     

Picosecond fluorescence kinetics in spinach chloroplasts at room temperature. Effects of Mg2+

Single-photon timing with picosecond resolution is used to investigate the effect of Mg²⁺ on the room-temperature fluorescence decay kinetics in broken spinach chloroplasts. In agreement with an earlier paper (Haehnel, W., Nairn, J.A., Reisberg, P. and Sauer, K. (1982) Biochim. Biophys. Acta 680, 161–173), we find three components in the fluorescence decay both in the presence and in the absence of Mg²⁺. The behavior of these components is examined as a function of Mg²⁺ concentration at both the F₀ and the F_max fluorescence levels, and as a function of the excitation intensity for thylakoids from spinach chloroplasts isolated in the absence of added Mg²⁺. Analysis of the results indicates that the subsequent addition of Mg²⁺ has effects which occur at different levels of added cation. At low levels of Mg²⁺ (less than 0.75 mM), there appears to be a decrease in communication between Photosystem (PS) II and PS I, which amounts to a decrease in the spillover rate between PS II and PS I. At higher levels of Mg²⁺ (about 2 mM), there appears to be an increase in communication between PS II units and an increase in the effective absorption cross-section of PS II, probably both of these involving the chlorophyll $\text{a}\text{b}$ light-harvesting antenna.     

Light scattering,chlorophyll fluorescence and state of the adenylate system in illuminated spinach leaves

Scattering of green light and chlorophyll fluorescence by spinach leaves kept in a stream of air or nitrogen were compared with leaf adenylate levels during illumination with blue, red or far-red light. Energy charge and ATP-ADP ratios exhibited considerable variability in different leaves both in the dark and in the light. Variability is explained by different possible states of the reaction oxidizing triose phosphate or reducing 3-phosphoglycerate. Except when oxygen levels were low, there was an inverse relationship between light scattering and chlorophyll fluorescence during illumination with blue or red light. When CO₂ was added to a stream of CO₂-free air, chlorophyll fluorescence increased, sometimes after a transient decrease, and both light scattering and leaf $\text{ATP}\text{ADP}$ ratios decreased. Similar observations were made when air was replaced by nitrogen under blue or high-intensity red light. Under these conditions, over-reduction caused inhibition of electron transport and phosphorylation in chloroplasts. However, when air was replaced by nitrogen during illumination with low-intensity red light or far-red light, light scattering increased instead of decreasing. Under these light conditions, $\text{ATP}\text{ADP}$ ratios were maintained in the light. They decreased drastically only after darkening. Although $\text{ATP}\text{ADP}$ ratios responded faster than light scattering or the slow secondary decline of chlorophyll fluorescence due to illumination, it appeared that in the steady state, light scattering and chlorophyll fluorescence are useful indicators of the phosphorylation state of the leaf adenylate system at least under aerobic conditions, when chloroplast and extrachloroplast adenylate systems can effectively communicate.     

Effect of exogenous gibberellin A4/7 on tracheid production, longitudinal growth and the levels of indole‐3‐acetic acid and gibberellins A4, A7 and A9 in the terminal shoot of Pinus sylvestris seedlings

Synchronously dividing cultures of the unicellular green alga Scenedesmus obtusiusculus were cultivated for 24 or 70 h in medium high (1000 μM) or low (60 μM) in phosphorus. Aliquots of AlCl₃ (0, 37, 74, 111, 148, 185, or 222 μmol) were added daily to 1 l cell suspension at the end of the cell division phase. Algae were also grown in media with different pH, adjusted with HCl, in the absence of AlCl₃.
Effects of Al on cell metabolism vary with the intracellular Al concentration and with the concentration of Al available per cell. When the concentration of phosphorus is low, internal concentrations of Al are high and the chlorophyll content and the net dry matter production per cell increase, whereas the photosynthesis and the cell division are increased. Presence of Al in a low P medium decreases the pH of the medium down to 4.5. There are only small effects of Al in the presence of P, due to precipitation of most of the Al with P in the medium.
Despite the Al-induced decrease of the pH of the culture medium, effects caused by Al cannot be explained as a pH effect. Instead, the Al effect may, at least to some extent, be related to a decrease in availability of P in the metabolism, due to formation of aluminium phosphate inside the cell.     

Time-resolved infrared study of reactive species produced by flash photolysis of the hydroformylation catalyst precursor Co2(CO)6(PMePh2)2

Flash photolysis with time-resolved infrared (TRIR) spectroscopy was used to elucidate the photochemical reactivity of the hydroformylation catalyst precursor Co₂(CO)₆(PMePh₂)₂. Depending on reaction conditions, the net products of photolysis varied significantly. A model is presented that accounts for the net reactivity with two initial photoproducts, the 17-electron species Co(CO)₃(PMePh₂) and the coordinatively unsaturated dimer Co₂(CO)₅(PMePh₂)₂. No evidence was found for photochemical formation of Co₂(CO)₆(PMePh₂). Time-resolved spectroscopic studies allowed for the direct observation of transient species and for kinetics studies of certain reactions; for example, the reactions of Co(CO)₃PMePh₂ with CO and with PMePh₂ gave the respective rate constants 1.5 × 10⁵ and 1.2 × 10⁷ M⁻¹ s⁻¹, while the analogous reactions with Co₂(CO)₅(PMePh₂)₂ gave the rate constants of 2.6 × 10⁶ M⁻¹ s⁻¹ and 3.9 × 10⁷ M⁻¹ s⁻¹.