Redox State Of Electron Carriers Research Articles

Variable chlorophyll fluorescence and its use for assessing physiological condition of plant photosynthetic apparatus

Analysis of plant behavior under diverse environmental conditions would be impossible without the methods for adequate assessment of the processes occurring in plants. The photosynthetic apparatus and its reaction to stress factors provide a reliable source of information on plant condition. One of the most informative methods based on monitoring the plant biophysical characteristics consists in detection and analysis of chlorophyll a fluorescence. Fluorescence is mainly emitted by chlorophyll a from the antenna complexes of photosystem II (PSII). However, fluorescence depends not only on the processes in the pigment matrix or PSII reaction centers but also on the redox reactions at the PSII donor and acceptor sides and even in the entire electron transport chain. Presently, a large variety of fluorometers from various manufacturers are available. Although application of such fluorometers does not require specialized training, the correct interpretation of the results would need sufficient knowledge for converting the instrumental data into the information on the condition of analyzed plants. This review is intended for a wide range of specialists employing fluorescence techniques for monitoring the physiological plant condition. It describes in a comprehensible way the theoretical basis of light emission by chlorophyll molecules, the origin of variable fluorescence, as well as relations between the fluorescence parameters, the redox state of electron carriers, and the light reactions of photosynthesis. Approaches to processing and analyzing the fluorescence induction curves are considered in detail on the basis of energy flux theory in the photosynthetic apparatus developed by Prof. Reto J. Strasser and known as a “JIP-test.” The physical meaning and relation of each calculated parameter to certain photosynthetic characteristics are presented, and examples of using these parameters for the assessment of plant physiological condition are outlined.

Reactive oxygen species and nitric oxide in plant mitochondria: origin and redundant regulatory systems

Plant mitochondria differ from their mammalian counterparts in many respects, which are due to the unique and variable surroundings of plant mitochondria. In green leaves, plant mitochondria are surrounded by ample respiratory substrates and abundant molecular oxygen, both resulting from active photosynthesis, while in roots and bulky rhizomes and fruit carbohydrates may be plenty, whereas oxygen levels are falling. Several enzymatic complexes in mitochondrial electron transport chain (ETC) are capable of reactive oxygen species (ROS) formation under physiological and pathological conditions. Inherently connected parameters such as the redox state of electron carriers in the ETC, ATP synthase activity and inner mitochondrial membrane potential, when affected by external stimuli, can give rise to ROS formation via complexes I and III, and by reverse electron transport (RET) from complex II. Superoxide radicals produced are quickly scavenged by superoxide dismutase (MnSOD), and the resulting H(2)O(2) is detoxified by peroxiredoxin-thioredoxin system or by the enzymes of ascorbate-glutathione cycle, found in the mitochondrial matrix. Arginine-dependent nitric oxide (NO)-releasing activity of enzymatic origin has been detected in plant mitochondria. The molecular identity of the enzyme is not clear but the involvement of mitochondria-localized enzymes responsible for arginine catabolism, arginase and ornithine aminotransferase has been shown in the regulation of NO efflux. Besides direct control by antioxidants, mitochondrial ROS production is tightly controlled by multiple redundant systems affecting inner membrane potential: NAD(P)H-dependent dehydrogenases, alternative oxidase (AOX), uncoupling proteins, ATP-sensitive K(+) channel and a number of matrix and intermembrane enzymes capable of direct electron donation to ETC. NO removal, on the other hand, takes place either by reactions with molecular oxygen or superoxide resulting in peroxynitrite, nitrite or nitrate ions or through interaction with non-symbiotic hemoglobins or glutathione. Mitochondrial ROS and NO production is tightly controlled by multiple redundant systems providing the regulatory mechanism for redox homeostasis and specific ROS/NO signaling.

Theoretical analysis of inductive effects in higher plant photosynthesis

The proposed mathematical model of the light stages of photosynthesis in higher plants takes into account the regulatory process of excitation energy redistribution between photosystems depending on the redox state of electron carriers. Nonmonotonic changes in the intensity of pigment fluorescence upon switching on the excitation light (delayed fluorescence induction) are described. The time required for fluorescence to reach its peak intensity and the degree of fluorescence quenching depending on light intensity are analyzed. Th kinetics of light-induced changes in EPR I signal upon switching from the far-red to white light is simulated.

Redox control of psbA gene expression in the cyanobacterium Synechocystis PCC 6803. Involvement of the cytochrome b(6)/f complex.

We investigated the role of the redox state of the photosynthetic and respiratory electron transport chains on the regulation of psbA expression in Synechocystis PCC 6803. Different means to modify the redox state of the electron carriers were used: (a) dark to oxidize the whole electron transport chain; (b) a shift from dark to light to induce its reduction; (c) the chemical interruption of the electron flow at different points to change the redox state of specific electron carriers; and (d) the presence of glucose to maintain a high reducing power in darkness. We show that changes in the redox state of the intersystem electron transport chain induce modifications of psbA transcript production and psbA mRNA stability. Reduction of the intersystem electron carriers activates psbA transcription and destabilizes the mRNA, while their oxidation induces a decrease in transcription and a stabilization of the transcript. Furthermore, our data suggest that the redox state of one of the electron carriers between the plastoquinone pool and photosystem I influences not only the expression of the psbA gene, but also that of other two photosynthetic genes, psaE and cpcBA. As a working hypothesis, we propose that the occupancy of the Q(0) site in the cytochrome b(6)/f complex may be involved in this regulation.

Free-radical-induced mutation vs redox regulation: Costs and benefits of genes in organelles

The prokaryotic endosymbionts that became plastids and mitochondria contained genes destined for one of three fates. Genes required for free-living existence were lost. Most genes useful to the symbiosis were transferred to the nucleus of the host. Some genes, a small minority, were retained within the organelle. Here we suggest that a selective advantage of movement of genes to the nucleus is decreased mutation: plastids and mitochondria have high volume-specific rates of redox reactions, producing oxygen free radicals that chemically modify DNA. These mutations lead to synthesis of modified electron carriers that in turn generate more mutagenic free radicals—the “vicious circle” theory of aging. Transfer of genes to the nucleus is also advantageous in facilitating sexual recombination and DNA repair. For genes encoding certain key components of photosynthesis and respiration, direct control of gene expression by redox state of electron carriers may be required to minimize free radical production, providing a selective advantage of organelle location which outweighs that of location in the nucleus. A previous proposal for transfer of genes to the nucleus is an economy of resources in having a single genome and a single apparatus for gene expression, but this argument fails if any organellar gene is retained. A previous proposal for the retention of genes within organelles is that certain proteins are organelle-encoded because they cannot be imported, but there is now evidence against this view. Decreased free radical mutagenesis and increased sexual recombination upon transfer to the nucleus together with redox control of gene expression in organelles may now account for the slightly different gene distributions among nuclei, plastids, and mitochondria found in major eukaryote taxa. This analysis suggests a novel reason for uniparental inheritance of organelles and the evolution of anisogametic sex, and may also account for the occurrence of nitrogen fixation in symbionts rather than in nitrogen-fixing organelles.

Sites of inhibition of mitochondrial electron transport by cadmium

Cadmium is an extremely toxic environmental contaminant having a long half-life in humans. The greatest accumulation occurs in the liver and kidneys. Since mitochondria are the most sensitive targets, the effect of cadmium on the oxygen consumption and on the redox state of electron carriers of rat liver mitochondria has been evaluated. Cadmium markedly inhibits uncoupler-stimulated oxidation on various NADH-linked substrates as well as that of succinate. Experiments on specific segments of the respiratory chain showed that cadmium does not inhibit electron flow through cytochrome oxidase, whereas the inhibition of duroquinol oxidation clearly demonstrates an impairment of electron flow through site 2, the ubiquinone-b-cytochrome c 1 complex. On the basis of the ability of N,N,N′,N′ tetramethyl- p-phenylendiamine and 2,6-dichlorophenolinindophenol bypasses to relieve the cadmium inhibition of succinate oxidation and on the spectroscopic behaviour of the cytochrome b, the inhibition was found to take place before cytochrome b and, more precisely, between ubisemiquinone and cytochrome b T. Furthermore, the finding that cadmium induces a more oxidized state of cytochrome b in state 1 demonstrates the existence of a second point in which it inhibits electron transfer. Spectroscopic evidence demonstrates that cadmium induces an oxidation of NAD(P)H in mitochondria in states 1 and 4 and prevents the reduction of mitochondrial NAD(P) + by substrates, thus indicating that the site must be localized between NAD-linked substrates and respiratory chain.

State 1-State 2 transitions in the cyanobacterium Synechococcus 6301 are controlled by the redox state of electron carriers between Photosystems I and II

The mechanism by which state 1-state 2 transitions in the cyanobacterium Synechococcus 6301 are controlled was investigated by examining the effects of a variety of chemical and illumination treatments which modify the redox state of the plastoquinone pool. The extent to which these treatments modify excitation energy distribution was determined by 77K fluorescence emission spectroscopy. It was found that treatment which lead to the oxidation of the plastoquinone pool induce a shift towards state 1 whereas treatments which lead to the reduction of the plastoquinone pool induce a shift towards state 2. We therefore propose that state transitions in cyanobacteria are triggered by changes in the redox state of plastoquinone or a closely associated electron carrier. Alternative proposals have included control by the extent of cyclic electron transport around PS I and control by localised electrochemical gradients around PS I and PS II. Neither of these proposals is consistent with the results reported here.

Sites of inhibition of mitochondrial electron transport by rhein

The effect of rhein on the oxygen consumption, oxidative phosphorylation, ATPase activity and redox state of electron carriers of rat liver mitochondria has been studied. Rhein inhibits ADP- and uncoupler-stimulated respiration on various NAD-linked substrates and succinate, but stimulates state 4 respiration of mitochondria respiring on succinate. Experiments on specific segments of the respiratory chain showed that rhein does not inhibit electron flow through cytochrome oxidase. Electron flow through site 2, the ubiquinone-cytochrome b-cytochrome c 1 complex, was also unaffected by rhein, which failed to inhibit the oxidation of duroquinol. Rhein affects oxidative phosphorylation by inhibiting both electron transfer and ADP-driven H + uptake. The inhibition of succinate oxidation by rhein was found to take place at a point between succinate and ubiquinone, perhaps at the level of succinic dehydrogenase. Spectroscopic evidence demonstrated that rhein induces a NAD(P)H oxidation in mitochondria respiring either on endogenous substrates or on glutamate + malate, and an inhibition of the cytochrome b reduction by succinate. These observations, together with other evidence, suggest that rhein inhibits electron transport in rat liver mitochondria at the dehydrogenase-coenzyme level, particularly when the electron carriers are in a relatively oxidized state and/or when the inner membrane-matrix compartment is in the condensed state.

Regulation of photosynthesis in isolated spinach chloroplasts during orthophosphate limitation

The stromal concentration of orthophosphate in intact spinach chloroplasts (prepared in the absence of orthophosphate or pyrophosphate but supplied with both in the reaction medium) fell from a value of approx. 20 mM in the dark to a steady-state concentration of approx. 8 mM in the light. Chloroplasts illuminated in the absence of orthophosphate or pyrophosphate showed a similar trend. However, in this situation the stromal inorganic phosphate (P i) concentration rapidly decreased from approx. 10 mM in the dark to a constant steady-state concentration of between 1.5 and 2.5 mM in the light. This P i concentration was not further diminished (even though CO 2-dependent O 2 evolution had ceased) and was therefore considered to be stromal orthophosphate not freely available to metabolism. In the P i-deficient chloroplasts the rate of photosynthesis declined rapidly after 1–2 min in the light such that CO 2-dependent O 2 evolution ceased with 5 min of the onset of illumination. The decline in O 2 evolution was accompanied by an increase in the transthylakoid ΔpH (as measured by 9-aminoacridine fluorescence quenching) and in the high-energy state, non-photochemical component of chlorophyll fluorescence quenching (q E). Measurements of stromal metabolite concentrations showed that the ATP ADP ratio was decreased in the P i-deficient chloroplasts relative to chloroplasts illuminated in the presence of P i. The stromal concentration of glycerate 3-phosphate was comparable in the P i-deficient chloroplasts and those to which P i had been supplied. Chloroplasts which were illuminated in P i-free media showed a large accumulation of ribulose-1,5-bisphosphate relative to those supplied with P i, suggesting inhibition of ribulose-1,5-bisphosphate carboxylase under these conditions. When P i was added to chloroplasts illuminated in the absence of P i, both non-photochemical quenching (q E), photochemical quenching (q Q) and ΔpH increased. This suggests that electron transport was not limited by inability to discharge transthylakoid ΔpH. These observation are consistent with the hypothesis that P i limitation results in decreased ATP production by the thylakoid ATP synthase. The data presented here show that there are multiple sites of flux control exerted by low stromal P i in the chloroplast. At least three factors contribute to the inhibition of photosynthesis under phosphate limitation: (1) there appears to be a direct effect of P i on the energy-transducing system; (2) there is direct inhibition of the Calvin cycle decreasing the ability of the pathway to act as a sink for ATP and NADPH; and (3) feedback inhibition of primary processes occurs either via ΔpH or the redox state of electron carriers. However, ΔpH does not appear to be a limiting factor, but rather an inability to regenerate NADP as electron acceptor is suggested. The addition of DCMU to chloroplasts during illumination in the absence of P i for periods of up to 10 min showed that there was very little loss of variable fluorescence despite a 60% reduction in the capacity for O 2 evolution. This would suggest that photoinhibitory damage to Photosystem II was not the major cause of the inhibition of photosynthesis observed with low P i.

Versatile spectrophotometer for photosynthesis (light-induced changes in absorbance and fluorescence yield, circular and linear dichroism) and other biophysical measurements

The construction of a versatile, single-beam spectrophotometer expanded from a previously reported circular dichrometer utilizing an elasto-optic modulator (EOM) for producing circular polarization, is described. The new spectrophotometer is capable of measuring circular dichroism as well as linear dichroism and light-induced changes in absorbance and fluorescence yield, the latter being two of the most commonly measured properties in photosynthesis research. The use of an EOM, along with a phase-sensitive detector, affords high sensitivity and at the same time, in conjuction with a mechanical phosphoroscopic device, a high degree of immunity from artifacts in studies employing actinic light. A description is given of the generation and use of timing signals essential for control and signal processing. Its performance is illustrated by examples drawn from light-induced absorbance and fluorescence-yield changes associated with photosynthetic electron transport and changes in redox state of electron carriers. This photometer, with very little modification, may also be used to perform a host of other types of measurements.

Energetics of photosynthesis in Zea mays. II. Studies of the flash-induced electrochromic shift and fluorescence induction in mesophyll chloroplasts

Patterns of electron transfer in isolated mesophyll chloroplasts of maize ( Zea mays L.) were studied in the presence of the physiological substrates, oxaloacetate, 3-phosphoglycerate and pyruvate. Flash-induced absorbance changes due to the electrochromic pigment band-shift (P-518) were used to estimate relative electron flow rates through the cyclic and non-cyclic pathways of electron transport. Further information on the redox state of electron carriers and the activity of coupled electron flow was obtained from measurements of fluorescence induction and of actinic-light-induced fluorescence changes. The results demonstrate the importance of correct redox poising for optimal rates of photosynthesis and are discussed in relation to the operation and regulation of photosynthesis in the C 4 system.

Light-dependent inhibitory action of p-nitrothiophenol on Photosystem II in relation to the redox state of electron carriers

The photosystem-II activity of chloroplasts was inhibited by the treatment with p-nitrothiophenol (NphSH) in the light, and the inhibition was accompanied by a change of the fluorescence spectrum. Aromatic mercaptans examined were active in causing this inhibition and fluorescence change. These effects of p-nitrothiophenol were highly accelerated by blocking the electron transport on the oxidation side of photosystem II by carbonyl cyanide- m-chlorophenylhydrazone (CCCP) or Tris · HCl or heat pre-treatment, whereas these were suppressed by blocking the transport on the reduction side by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). It was deduced that the site of NphSH action in the electron transport chain is closer to the reaction center of photosystem II that the blocking site of CCCP or Tris · HCl or heat, and that such a site in photosystem II is exposed to be modified with NphSH when electron carriers on the oxidation side of photosystem II are oxidized by illumination.