Publications by authors named "Szilvia Z Tóth"

33 Publications

Thin cell layer cultures of Chlamydomonas reinhardtii L159I-N230Y, pgrl1 and pgr5 mutants perform enhanced hydrogen production at sunlight intensity.

Bioresour Technol 2021 Aug 27;333:125217. Epub 2021 Apr 27.

Institute of Plant Biology, Biological Research Centre, Szeged, Temesvári krt. 62, H-6726 Szeged, Hungary. Electronic address:

Photobiological hydrogen (H) production is a promising renewable energy source. HydA hydrogenases of green algae are efficient but O-sensitive and compete for electrons with CO-fixation. Recently, we established a photoautotrophic H production system based on anaerobic induction, where the Calvin-Benson cycle is inactive and O scavenged by an absorbent. Here, we employed thin layer cultures, resulting in a three-fold increase in H production relative to bulk CC-124 cultures (50 µg chlorophyll/ml, 350 µmol photons m s). Productivity was maintained when increasing the light intensity to 1000 µmol photons ms and the cell density to 150 µg chlorophyll/ml. Remarkably, the L159I-N230Y photosystem II mutant and the pgrl1 photosystem I cyclic electron transport mutant produced 50% more H than CC-124, while the pgr5 mutant generated 250% more (1.2 ml H/ml culture in six days). The photosynthetic apparatus of the pgr5 mutant and its in vitro HydA activity remained remarkably stable.
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http://dx.doi.org/10.1016/j.biortech.2021.125217DOI Listing
August 2021

Ascorbate inactivates the oxygen-evolving complex in prolonged darkness.

Physiol Plant 2021 Feb 6;171(2):232-245. Epub 2020 Dec 6.

Institute of Plant Biology, Biological Research Centre, Szeged, Hungary.

Ascorbate (Asc, vitamin C) is an essential metabolite participating in multiple physiological processes of plants, including environmental stress management and development. In this study, we acquired knowledge on the role of Asc in dark-induced leaf senescence using Arabidopsis thaliana as a model organism. One of the earliest effects of prolonged darkness is the inactivation of oxygen-evolving complexes (OEC) as demonstrated here by fast chlorophyll a fluorescence and thermoluminescence measurements. We found that inactivation of OEC due to prolonged darkness was attenuated in the Asc-deficient vtc2-4 mutant. On the other hand, the severe photosynthetic phenotype of a psbo1 knockout mutant, lacking the major extrinsic OEC subunit PSBO1, was further aggravated upon a 24-h dark treatment. The psbr mutant, devoid of the PSBR subunit of OEC, performed only slightly disturbed photosynthetic activity under normal growth conditions, whereas it showed a strongly diminished B thermoluminescence band upon dark treatment. We have also generated a double psbo1 vtc2 mutant, and it showed a slightly milder photosynthetic phenotype than the single psbo1 mutant. Our results, therefore, suggest that Asc leads to the inactivation of OEC in prolonged darkness by over-reducing the Mn-complex that is probably enabled by a dark-induced dissociation of the extrinsic OEC subunits. Our study is an example that Asc may negatively affect certain cellular processes and thus its concentration and localization need to be highly controlled.
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http://dx.doi.org/10.1111/ppl.13278DOI Listing
February 2021

H Transport by K EXCHANGE ANTIPORTER3 Promotes Photosynthesis and Growth in Chloroplast ATP Synthase Mutants.

Plant Physiol 2020 04 10;182(4):2126-2142. Epub 2020 Feb 10.

Max Planck Institute of Molecular Plant Physiology, Wissenschaftspark Golm, Am Mühlenberg 1, 14476 Potsdam, Germany

The composition of the thylakoid proton motive force (pmf) is regulated by thylakoid ion transport. Passive ion channels in the thylakoid membrane dissipate the membrane potential (Δψ) component to allow for a higher fraction of pmf stored as a proton concentration gradient (ΔpH). K/H antiport across the thylakoid membrane via K+ EXCHANGE ANTIPORTER3 (KEA3) instead reduces the ΔpH fraction of the pmf. Thereby, KEA3 decreases nonphotochemical quenching (NPQ), thus allowing for higher light use efficiency, which is particularly important during transitions from high to low light. Here, we show that in the background of the Arabidopsis () chloroplast (cp)ATP synthase assembly mutant , with decreased cpATP synthase activity and increased pmf amplitude, KEA3 plays an important role for photosynthesis and plant growth under steady-state conditions. By comparing single with double mutants, we demonstrate that in the background loss of KEA3 causes a strong growth penalty. This is due to a reduced photosynthetic capacity of mutants, as these plants have a lower lumenal pH than mutants, and thus show substantially increased pH-dependent NPQ and decreased electron transport through the cytochrome complex. Overexpression of in the background reduces pH-dependent NPQ and increases photosystem II efficiency. Taken together, our data provide evidence that under conditions where cpATP synthase activity is low, a KEA3-dependent reduction of ΔpH benefits photosynthesis and growth.
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http://dx.doi.org/10.1104/pp.19.01561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140953PMC
April 2020

Light Control of Salt-Induced Proline Accumulation Is Mediated by ELONGATED HYPOCOTYL 5 in .

Front Plant Sci 2019 10;10:1584. Epub 2019 Dec 10.

Institute of Plant Biology, Biological Research Centre, Szeged, Hungary.

Plants have to adapt their metabolism to constantly changing environmental conditions, among which the availability of light and water is crucial in determining growth and development. Proline accumulation is one of the sensitive metabolic responses to extreme conditions; it is triggered by salinity or drought and is regulated by light. Here we show that red and blue but not far-red light is essential for salt-induced proline accumulation, upregulation of () and downregulation of () genes, which control proline biosynthetic and catabolic pathways, respectively. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the transcription factor ELONGATED HYPOCOTYL 5 (HY5) binds to G-box and C-box elements of and a C-box motif of . Salt-induced proline accumulation and expression were reduced in the double mutant, suggesting that HY5 promotes proline biosynthesis through connecting light and stress signals. Our results improve our understanding on interactions between stress and light signals, confirming HY5 as a key regulator in proline metabolism.
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http://dx.doi.org/10.3389/fpls.2019.01584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6914869PMC
December 2019

Elimination of the flavodiiron electron sink facilitates long-term H photoproduction in green algae.

Biotechnol Biofuels 2019 5;12:280. Epub 2019 Dec 5.

1Molecular Plant Biology, Department of Biochemistry, University of Turku, 20014 Turku, Finland.

Background: The development of renewable and sustainable biofuels to cover the future energy demand is one of the most challenging issues of our time. Biohydrogen, produced by photosynthetic microorganisms, has the potential to become a green biofuel and energy carrier for the future sustainable world, since it provides energy without CO emission. The recent development of two alternative protocols to induce hydrogen photoproduction in green algae enables the function of the O-sensitive [FeFe]-hydrogenases, located at the acceptor side of photosystem I, to produce H for several days. These protocols prevent carbon fixation and redirect electrons toward H production. In the present work, we employed these protocols to a knockout mutant lacking flavodiiron proteins (FDPs), thus removing another possible electron competitor with H production.

Results: The deletion of the FDP electron sink resulted in the enhancement of H photoproduction relative to wild-type . Additionally, the lack of FDPs leads to a more effective obstruction of carbon fixation even under elongated light pulses.

Conclusions: We demonstrated that the rather simple adjustment of cultivation conditions together with genetic manipulation of alternative electron pathways of photosynthesis results in efficient re-routing of electrons toward H photoproduction. Furthermore, the introduction of a short recovery phase by regular switching from H photoproduction to biomass accumulation phase allows to maintain cell fitness and use photosynthetic cells as long-term H-producing biocatalysts.
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http://dx.doi.org/10.1186/s13068-019-1618-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894204PMC
December 2019

Ascorbate Deficiency Does Not Limit Nonphotochemical Quenching in .

Plant Physiol 2020 01 29;182(1):597-611. Epub 2019 Oct 29.

Institute of Plant Biology, Biological Research Centre, Szeged, Hungary

Ascorbate (Asc; vitamin C) plays essential roles in development, signaling, hormone biosynthesis, regulation of gene expression, stress resistance, and photoprotection. In vascular plants, violaxanthin de-epoxidase requires Asc as a reductant; thereby, Asc is required for the energy-dependent component of nonphotochemical quenching (NPQ). To assess the role of Asc in NPQ in green algae, which are known to contain low amounts of Asc, we searched for an insertional mutant affected in the gene encoding GDP-l-Gal phosphorylase, which catalyzes the first committed step in the biosynthesis of Asc. The knockout mutant was viable and, depending on the growth conditions, contained 10% to 20% Asc relative to its wild type. When was grown photomixotrophically at moderate light, the zeaxanthin-dependent component of NPQ emerged upon strong red illumination both in the mutant and in its wild type. Deepoxidation was unaffected by Asc deficiency, demonstrating that the Chlorophycean violaxanthin de-epoxidase found in does not require Asc as a reductant. The rapidly induced, energy-dependent NPQ component characteristic of photoautotrophic cultures grown at high light was not limited by Asc deficiency either. On the other hand, a reactive oxygen species-induced photoinhibitory NPQ component was greatly enhanced upon Asc deficiency, both under photomixotrophic and photoautotrophic conditions. These results demonstrate that Asc has distinct roles in NPQ formation in as compared to vascular plants.
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http://dx.doi.org/10.1104/pp.19.00916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945847PMC
January 2020

Paradigm Shift in Algal H Production: Bypassing Competitive Processes.

Trends Biotechnol 2019 11 4;37(11):1159-1163. Epub 2019 Jun 4.

School of Plant Sciences and Food Security, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv, 69978, Israel. Electronic address:

Hydrogen is a promising energy carrier, but producing it sustainably remains a challenge. Green algae can produce hydrogen photosynthetically using their efficient but oxygen-sensitive hydrogenases. Recent strategies aiming to bypass competing processes provide a promising route for scaling up algal hydrogen production.
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http://dx.doi.org/10.1016/j.tibtech.2019.05.001DOI Listing
November 2019

Water-splitting-based, sustainable and efficient H production in green algae as achieved by substrate limitation of the Calvin-Benson-Bassham cycle.

Biotechnol Biofuels 2018 19;11:69. Epub 2018 Mar 19.

1Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Temesvári krt. 62, 6726 Szeged, Hungary.

Background: Photobiological H production has the potential of becoming a carbon-free renewable energy source, because upon the combustion of H, only water is produced. The [Fe-Fe]-type hydrogenases of green algae are highly active, although extremely O-sensitive. Sulphur deprivation is a common way to induce H production, which, however, relies substantially on organic substrates and imposes a severe stress effect resulting in the degradation of the photosynthetic apparatus.

Results: We report on the establishment of an alternative H production method by green algae that is based on a short anaerobic induction, keeping the Calvin-Benson-Bassham cycle inactive by substrate limitation and preserving hydrogenase activity by applying a simple catalyst to remove the evolved O. Cultures remain photosynthetically active for several days, with the electrons feeding the hydrogenases mostly derived from water. The amount of H produced is higher as compared to the sulphur-deprivation procedure and the process is photoautotrophic.

Conclusion: Our protocol demonstrates that it is possible to sustainably use algal cells as whole-cell catalysts for H production, which enables industrial application of algal biohydrogen production.
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http://dx.doi.org/10.1186/s13068-018-1069-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858145PMC
March 2018

The mechanism of photosystem-II inactivation during sulphur deprivation-induced H production in Chlamydomonas reinhardtii.

Plant J 2018 05 31;94(3):548-561. Epub 2018 Mar 31.

Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.

Sulphur limitation may restrain cell growth and viability. In the green alga Chlamydomonas reinhardtii, sulphur limitation may induce H production lasting for several days, which can be exploited as a renewable energy source. Sulphur limitation causes a large number of physiological changes, including the inactivation of photosystem II (PSII), leading to the establishment of hypoxia, essential for the increase in hydrogenase expression and activity. The inactivation of PSII has long been assumed to be caused by the sulphur-limited turnover of its reaction center protein PsbA. Here we reinvestigated this issue in detail and show that: (i) upon transferring Chlamydomonas cells to sulphur-free media, the cellular sulphur content decreases only by about 25%; (ii) as demonstrated by lincomycin treatments, PsbA has a significant turnover, and other photosynthetic subunits, namely RbcL and CP43, are degraded more rapidly than PsbA. On the other hand, sulphur limitation imposes oxidative stress early on, most probably involving the formation of singlet oxygen in PSII, which leads to an increase in the expression of GDP-L-galactose phosphorylase, playing an essential role in ascorbate biosynthesis. When accumulated to the millimolar concentration range, ascorbate may inactivate the oxygen-evolving complex and provide electrons to PSII, albeit at a low rate. In the absence of a functional donor side and sufficient electron transport, PSII reaction centers are inactivated and degraded. We therefore demonstrate that the inactivation of PSII is a complex and multistep process, which may serve to mitigate the damaging effects of sulphur limitation.
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http://dx.doi.org/10.1111/tpj.13878DOI Listing
May 2018

Concentration Does Matter: The Beneficial and Potentially Harmful Effects of Ascorbate in Humans and Plants.

Antioxid Redox Signal 2018 11 1;29(15):1516-1533. Epub 2017 Dec 1.

2 Laboratory of Biochemistry and Molecular Biology, Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics , Budapest, Hungary .

Significance: Ascorbate (Asc) is an essential compound both in animals and plants, mostly due to its reducing properties, thereby playing a role in scavenging reactive oxygen species (ROS) and acting as a cofactor in various enzymatic reactions. Recent Advances: Growing number of evidence shows that excessive Asc accumulation may have negative effects on cellular functions both in humans and plants; inter alia it may negatively affect signaling mechanisms, cellular redox status, and contribute to the production of ROS via the Fenton reaction.

Critical Issues: Both plants and humans tightly control cellular Asc levels, possibly via biosynthesis, transport, and degradation, to maintain them in an optimum concentration range, which, among other factors, is essential to minimize the potentially harmful effects of Asc. On the contrary, the Fenton reaction induced by a high-dose Asc treatment in humans enables a potential cancer-selective cell death pathway.

Future Directions: The elucidation of Asc induced cancer selective cell death mechanisms may give us a tool to apply Asc in cancer therapy. On the contrary, the regulatory mechanisms controlling cellular Asc levels are also to be considered, for example, when aiming at generating crops with elevated Asc levels.
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http://dx.doi.org/10.1089/ars.2017.7125DOI Listing
November 2018

On the pathways feeding the H production process in nutrient-replete, hypoxic conditions. Commentary on the article "Low oxygen levels contribute to improve photohydrogen production in mixotrophic non-stressed ", by Jurado-Oller et al., Biotechnology for Biofuels, published September 7, 2015; 8:149.

Biotechnol Biofuels 2017 4;10:116. Epub 2017 May 4.

Biological Research Centre Szeged, Hungarian Academy of Sciences, Temesvári krt. 62, Szeged, 6726 Hungary.

Background: Under low O concentration (hypoxia) and low light, cells can produce H gas in nutrient-replete conditions. This process is hindered by the presence of O, which inactivates the [FeFe]-hydrogenase enzyme responsible for H gas production shifting algal cultures back to normal growth. The main pathways accounting for H production in hypoxia are not entirely understood, as much as culture conditions setting the optimal redox state in the chloroplast supporting long-lasting H production. The reducing power for H production can be provided by photosystem II (PSII) and photofermentative processes during which proteins are degraded via yet unknown pathways. In hetero- or mixotrophic conditions, acetate respiration was proposed to indirectly contribute to H evolution, although this pathway has not been described in detail.

Main Body: Recently, Jurado-Oller et al. (Biotechnol Biofuels 8: 149, 7) proposed that acetate respiration may substantially support H production in nutrient-replete hypoxic conditions. Addition of low amounts of O enhanced acetate respiration rate, particularly in the light, resulting in improved H production. The authors surmised that acetate oxidation through the glyoxylate pathway generates intermediates such as succinate and malate, which would be in turn oxidized in the chloroplast generating FADH and NADH. The latter would enter a PSII-independent pathway at the level of the plastoquinone pool, consistent with the light dependence of H production. The authors concluded that the water-splitting activity of PSII has a minor role in H evolution in nutrient-replete, mixotrophic cultures under hypoxia. However, their results with the PSII inhibitor DCMU also reveal that O or acetate additions promoted acetate respiration over the usually dominant PSII-dependent pathway. The more oxidized state experienced by these cultures in combination with the relatively short experimental time prevented acclimation to hypoxia, thus precluding the PSII-dependent pathway from contributing to H production.

Conclusions: In , continuous H gas evolution is expected once low O partial pressure and optimal reducing conditions are set. Under nutrient-replete conditions, the electrogenic processes involved in H photoproduction may rely on various electron transport pathways. Understanding how physiological conditions select for specific metabolic routes is key to achieve economic viability of this renewable energy source.
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http://dx.doi.org/10.1186/s13068-017-0800-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418857PMC
May 2017

Identification and characterization of a stable intermediate in photosystem I assembly in tobacco.

Plant J 2017 May 27;90(3):478-490. Epub 2017 Mar 27.

Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476, Potsdam-Golm, Germany.

Photosystem I (PSI) is the most efficient bioenergetic nanomachine in nature and one of the largest membrane protein complexes known. It is composed of 18 protein subunits that bind more than 200 co-factors and prosthetic groups. While the structure and function of PSI have been studied in great detail, very little is known about the PSI assembly process. In this work, we have characterized a PSI assembly intermediate in tobacco plants, which we named PSI*. We found PSI* to contain only a specific subset of the core subunits of PSI. PSI* is particularly abundant in young leaves where active thylakoid biogenesis takes place. Moreover, PSI* was found to overaccumulate in PsaF-deficient mutant plants, and we show that re-initiation of PsaF synthesis promotes the maturation of PSI* into PSI. The attachment of antenna proteins to PSI also requires the transition from PSI* to mature PSI. Our data could provide a biochemical entry point into the challenging investigation of PSI biogenesis and allow us to improve the model for the assembly pathway of PSI in thylakoid membranes of vascular plants.
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http://dx.doi.org/10.1111/tpj.13505DOI Listing
May 2017

Regulation of ascorbate biosynthesis in green algae has evolved to enable rapid stress-induced response via the VTC2 gene encoding GDP-l-galactose phosphorylase.

New Phytol 2017 Apr 23;214(2):668-681. Epub 2017 Jan 23.

Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Temesvári krt. 62, H-6726, Szeged, Hungary.

Ascorbate (vitamin C) plays essential roles in stress resistance, development, signaling, hormone biosynthesis and regulation of gene expression; however, little is known about its biosynthesis in algae. In order to provide experimental proof for the operation of the Smirnoff-Wheeler pathway described for higher plants and to gain more information on the regulation of ascorbate biosynthesis in Chlamydomonas reinhardtii, we targeted the VTC2 gene encoding GDP-l-galactose phosphorylase using artificial microRNAs. Ascorbate concentrations in VTC2 amiRNA lines were reduced to 10% showing that GDP-l-galactose phosphorylase plays a pivotal role in ascorbate biosynthesis. The VTC2 amiRNA lines also grow more slowly, have lower chlorophyll content, and are more susceptible to stress than the control strains. We also demonstrate that: expression of the VTC2 gene is rapidly induced by H O and O resulting in a manifold increase in ascorbate content; in contrast to plants, there is no circadian regulation of ascorbate biosynthesis; photosynthesis is not required per se for ascorbate biosynthesis; and Chlamydomonas VTC2 lacks negative feedback regulation by ascorbate in the physiological concentration range. Our work demonstrates that ascorbate biosynthesis is also highly regulated in Chlamydomonas albeit via mechanisms distinct from those previously described in land plants.
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http://dx.doi.org/10.1111/nph.14425DOI Listing
April 2017

Ascorbate accumulation during sulphur deprivation and its effects on photosystem II activity and H2 production of the green alga Chlamydomonas reinhardtii.

Plant Cell Environ 2016 07 13;39(7):1460-72. Epub 2016 Apr 13.

Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Temesvári krt. 62, H-6726, Szeged, Hungary.

In nature, H2 production in Chlamydomonas reinhardtii serves as a safety valve during the induction of photosynthesis in anoxia, and it prevents the over-reduction of the photosynthetic electron transport chain. Sulphur deprivation of C. reinhardtii also triggers a complex metabolic response resulting in the induction of various stress-related genes, down-regulation of photosynthesis, the establishment of anaerobiosis and expression of active hydrogenase. Photosystem II (PSII) plays dual role in H2 production because it supplies electrons but the evolved O2 inhibits the hydrogenase. Here, we show that upon sulphur deprivation, the ascorbate content in C. reinhardtii increases about 50-fold, reaching the mM range; at this concentration, ascorbate inactivates the Mn-cluster of PSII, and afterwards, it can donate electrons to tyrozin Z(+) at a slow rate. This stage is followed by donor-side-induced photoinhibition, leading to the loss of charge separation activity in PSII and reaction centre degradation. The time point at which maximum ascorbate concentration is reached in the cell is critical for the establishment of anaerobiosis and initiation of H2 production. We also show that ascorbate influenced H2 evolution via altering the photosynthetic electron transport rather than hydrogenase activity and starch degradation.
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http://dx.doi.org/10.1111/pce.12701DOI Listing
July 2016

Identification of the Elusive Chloroplast Ascorbate Transporter Extends the Substrate Specificity of the PHT Family.

Mol Plant 2015 May 18;8(5):674-6. Epub 2015 Feb 18.

Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, PO Box 521, Szeged H-6701, Hungary.

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http://dx.doi.org/10.1016/j.molp.2015.02.006DOI Listing
May 2015

Photosynthetic complex stoichiometry dynamics in higher plants: biogenesis, function, and turnover of ATP synthase and the cytochrome b6f complex.

J Exp Bot 2015 May 24;66(9):2373-400. Epub 2014 Dec 24.

Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

During plant development and in response to fluctuating environmental conditions, large changes in leaf assimilation capacity and in the metabolic consumption of ATP and NADPH produced by the photosynthetic apparatus can occur. To minimize cytotoxic side reactions, such as the production of reactive oxygen species, photosynthetic electron transport needs to be adjusted to the metabolic demand. The cytochrome b6f complex and chloroplast ATP synthase form the predominant sites of photosynthetic flux control. Accordingly, both respond strongly to changing environmental conditions and metabolic states. Usually, their contents are strictly co-regulated. Thereby, the capacity for proton influx into the lumen, which is controlled by electron flux through the cytochrome b6f complex, is balanced with proton efflux through ATP synthase, which drives ATP synthesis. We discuss the environmental, systemic, and metabolic signals triggering the stoichiometry adjustments of ATP synthase and the cytochrome b6f complex. The contribution of transcriptional and post-transcriptional regulation of subunit synthesis, and the importance of auxiliary proteins required for complex assembly in achieving the stoichiometry adjustments is described. Finally, current knowledge on the stability and turnover of both complexes is summarized.
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http://dx.doi.org/10.1093/jxb/eru495DOI Listing
May 2015

Inducible Repression of Nuclear-Encoded Subunits of the Cytochrome b6f Complex in Tobacco Reveals an Extraordinarily Long Lifetime of the Complex.

Plant Physiol 2014 Aug 24;165(4):1632-1646. Epub 2014 Jun 24.

Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany

The biogenesis of the cytochrome bf complex in tobacco (Nicotiana tabacum) seems to be restricted to young leaves, suggesting a high lifetime of the complex. To directly determine its lifetime, we employed an ethanol-inducible RNA interference (RNAi) approach targeted against the essential nuclear-encoded Rieske protein (PetC) and the small M subunit (PetM), whose function in higher plants is unknown. Young expanding leaves of both PetM and PetC RNAi transformants bleached rapidly and developed necroses, while mature leaves, whose photosynthetic apparatus was fully assembled before RNAi induction, stayed green. In line with these phenotypes, cytochrome bf complex accumulation and linear electron transport capacity were strongly repressed in young leaves of both RNAi transformants, showing that the M subunit is as essential for cytochrome bf complex accumulation as the Rieske protein. In mature leaves, all photosynthetic parameters were indistinguishable from the wild type even after 14 d of induction. As RNAi repression of PetM and PetC was highly efficient in both young and mature leaves, these data indicate a lifetime of the cytochrome bf complex of at least 1 week. The switch-off of cytochrome bf complex biogenesis in mature leaves may represent part of the first dedicated step of the leaf senescence program.
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http://dx.doi.org/10.1104/pp.114.243741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4119044PMC
August 2014

Photosynthetic complex stoichiometry dynamics in higher plants: environmental acclimation and photosynthetic flux control.

Front Plant Sci 2014 13;5:188. Epub 2014 May 13.

Max Planck Institute of Molecular Plant Physiology Potsdam-Golm, Germany.

The composition of the photosynthetic apparatus of higher plants is dynamically adjusted to long-term changes in environmental conditions such as growth light intensity and light quality, and to changing metabolic demands for ATP and NADPH imposed by stresses and leaf aging. By changing photosynthetic complex stoichiometry, a long-term imbalance between the photosynthetic production of ATP and NADPH and their metabolic consumption is avoided, and cytotoxic side reactions are minimized. Otherwise, an excess capacity of the light reactions, relative to the demands of primary metabolism, could result in a disturbance of cellular redox homeostasis and an increased production of reactive oxygen species, leading to the destruction of the photosynthetic apparatus and the initiation of cell death programs. In this review, changes of the abundances of the different constituents of the photosynthetic apparatus in response to environmental conditions and during leaf ontogenesis are summarized. The contributions of the different photosynthetic complexes to photosynthetic flux control and the regulation of electron transport are discussed.
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http://dx.doi.org/10.3389/fpls.2014.00188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026699PMC
June 2014

Chlorophyll a fluorescence: beyond the limits of the Q(A) model.

Photosynth Res 2014 May 1;120(1-2):43-58. Epub 2013 Mar 1.

Institute of Plant Biology, Biological Research Center Szeged, Hungarian Academy of Sciences, Szeged, 6701, Hungary,

Chlorophyll a fluorescence is a non-invasive tool widely used in photosynthesis research. According to the dominant interpretation, based on the model proposed by Duysens and Sweers (1963, Special Issue of Plant and Cell Physiology, pp 353-372), the fluorescence changes reflect primarily changes in the redox state of Q(A), the primary quinone electron acceptor of photosystem II (PSII). While it is clearly successful in monitoring the photochemical activity of PSII, a number of important observations cannot be explained within the framework of this simple model. Alternative interpretations have been proposed but were not supported satisfactorily by experimental data. In this review we concentrate on the processes determining the fluorescence rise on a dark-to-light transition and critically analyze the experimental data and the existing models. Recent experiments have provided additional evidence for the involvement of a second process influencing the fluorescence rise once Q(A) is reduced. These observations are best explained by a light-induced conformational change, the focal point of our review. We also want to emphasize that-based on the presently available experimental findings-conclusions on α/ß-centers, PSII connectivity, and the assignment of FV/FM to the maximum PSII quantum yield may require critical re-evaluations. At the same time, it has to be emphasized that for a deeper understanding of the underlying physical mechanism(s) systematic studies on light-induced changes in the structure and reaction kinetics of the PSII reaction center are required.
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http://dx.doi.org/10.1007/s11120-013-9806-5DOI Listing
May 2014

The physiological roles and metabolism of ascorbate in chloroplasts.

Physiol Plant 2013 Jun 6;148(2):161-75. Epub 2012 Dec 6.

Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, P.O. Box 521, H-6701, Hungary.

Ascorbate is a multifunctional metabolite in plants. It is essential for growth control, involving cell division and cell wall synthesis and also involved in redox signaling, in the modulation of gene expression and regulation of enzymatic activities. Ascorbate also fulfills crucial roles in scavenging reactive oxygen species, both enzymatically and nonenzymatically, a well-established phenomenon in the chloroplasts stroma. We give an overview on these important physiological functions and would like to give emphasis to less well-known roles of ascorbate, in the thylakoid lumen, where it also plays multiple roles. It is essential for photoprotection as a cofactor for violaxanthin de-epoxidase, a key enzyme in the formation of nonphotochemical quenching. Lumenal ascorbate has recently also been shown to act as an alternative electron donor of photosystem II once the oxygen-evolving complex is inactivated and to protect the photosynthetic machinery by slowing down donor-side induced photoinactivation; it is yet to be established if ascorbate has a similar role in the case of other stress effects, such as high light and UV-B stress. In bundle sheath cells, deficient in oxygen evolution, ascorbate provides electrons to photosystem II, thereby poising cyclic electron transport around photosystem I. It has also been shown that, by supporting linear electron transport through photosystem II in sulfur-deprived Chlamydomonas reinhardtii cells, in which oxygen evolution is largely inhibited, externally added ascorbate enhances hydrogen production. For fulfilling its multiple roles, Asc has to be transported into the thylakoid lumen and efficiently regenerated; however, very little is known yet about these processes.
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http://dx.doi.org/10.1111/ppl.12006DOI Listing
June 2013

The chl a fluorescence intensity is remarkably insensitive to changes in the chlorophyll content of the leaf as long as the chl a/b ratio remains unaffected.

Biochim Biophys Acta 2012 May 9;1817(5):770-9. Epub 2012 Feb 9.

Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary.

The effects of changes in the chlorophyll (chl) content on the kinetics of the OJIP fluorescence transient were studied using two different approaches. An extensive chl loss (up to 5-fold decrease) occurs in leaves suffering from either an Mg(2+) or SO(4)(2-) deficiency. The effects of these treatments on the chl a/b ratio, which is related to antenna size, were very limited. This observation was confirmed by the identical light intensity dependencies of the K, J and I-steps of the fluorescence rise for three of the four treatments and by the absence of changes in the F(685 nm)/F(695 nm)-ratio of fluorescence emission spectra measured at 77K. Under these conditions, the F(0) and F(M)-values were essentially insensitive to the chl content. A second experimental approach consisted of the treatment of wheat leaves with specifically designed antisense oligodeoxynucleotides that interfered with the translation of mRNA of the genes coding for chl a/b binding proteins. This way, leaves with a wide range of chl a/b ratios were created. Under these conditions, an inverse proportional relationship between the F(M) values and the chl a/b ratio was observed. A strong effect of the chl a/b ratio on the fluorescence intensity was also observed for barley Chlorina f2 plants that lack chl b. The data suggest that the chl a/b ratio (antenna size) is a more important determinant of the maximum fluorescence intensity than the chl content of the leaf.
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http://dx.doi.org/10.1016/j.bbabio.2012.02.003DOI Listing
May 2012

Synthetic antisense oligodeoxynucleotides to transiently suppress different nucleus- and chloroplast-encoded proteins of higher plant chloroplasts.

Plant Physiol 2011 Dec 6;157(4):1628-41. Epub 2011 Oct 6.

Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, H-6701 Szeged, Hungary.

Selective inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) is widely applied in gene function analyses; however, experiments with ODNs in plants are scarce. In this work, we extend the use of ODNs in different plant species, optimizing the uptake, stability, and efficiency of ODNs with a combination of molecular biological and biophysical techniques to transiently inhibit the gene expression of different chloroplast proteins. We targeted the nucleus-encoded phytoene desaturase (pds) gene, encoding a key enzyme in carotenoid biosynthesis, the chlorophyll a/b-binding (cab) protein genes, and the chloroplast-encoded psbA gene, encoding the D1 protein. For pds and psbA, the in vivo stability of ODNs was increased by phosphorothioate modifications. After infiltration of ODNs into juvenile tobacco (Nicotiana benthamiana) leaves, we detected a 25% to 35% reduction in mRNA level and an approximately 5% decrease in both carotenoid content and the variable fluorescence of photosystem II. In detached etiolated wheat (Triticum aestivum) leaves, after 8 h of greening, the mRNA level, carotenoid content, and variable fluorescence were inhibited up to 75%, 25%, and 20%, respectively. Regarding cab, ODN treatments of etiolated wheat leaves resulted in an up to 59% decrease in the amount of chlorophyll b, a 41% decrease of the maximum chlorophyll fluorescence intensity, the cab mRNA level was reduced to 66%, and the protein level was suppressed up to 85% compared with the control. The psbA mRNA and protein levels in Arabidopsis (Arabidopsis thaliana) leaves were inhibited by up to 85% and 72%, respectively. To exploit the potential of ODNs for photosynthetic genes, we propose molecular design combined with fast, noninvasive techniques to test their functional effects.
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http://dx.doi.org/10.1104/pp.111.185462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327186PMC
December 2011

Thermoluminescence and P700 redox kinetics as complementary tools to investigate the cyclic/chlororespiratory electron pathways in stress conditions in barley leaves.

Physiol Plant 2012 Jan 25;144(1):83-97. Epub 2011 Oct 25.

Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, G Bonchev Str., Bl. 21, Sofia 1113, Bulgaria.

Cyclic electron flow around photosystem I drives additional proton pumping into the thylakoid lumen, which enhances the protective non-photochemical quenching and increases ATP synthesis. It involves several pathways activated independently. In whole barley leaves, P700 oxidation under far-red illumination and subsequent P700(+) dark reduction kinetics provide a major probe of the activation of cyclic pathways. Two 'intermediate' and 'slow' exponential reduction phases are always observed and they become faster after high light illumination, but dark inactivation of the Benson-Calvin cycle causes the emergence of both a transient in the P700 oxidation and a 'fast' phase in the P700(+) reduction. We investigate here the afterglow (AG) thermoluminescence emission as another tool to detect the activation of cyclic electron pathways from stroma reductants to the acceptor side of photosystem II. This transfer is activated by warming, yielding an AG band at about 45°C. However, treatments that accelerate the 'intermediate' and 'slow' P700(+) reduction phases (brief anoxia, hexose infiltration, fast dehydration of excised leaves) also produced a downshift of this AG band. This pathway ascribable to NADPH dehydrogenase (NDH) would be triggered by a deficit in ATP, while the 'fast' reduction phase corresponding to the ferredoxin plastoquinone reductase pathway is triggered by an overreduction of the photosystem I acceptor pool and is undetected in thermoluminescence. Contrastingly, slow dehydration of unwatered plants did not cause faster reduction of P700(+) nor temperature downshift of the AG band, that is no induction of the NDH pathway, whereas an increased intensity of the AG band indicated a strong NADPH + ATP assimilatory potential.
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http://dx.doi.org/10.1111/j.1399-3054.2011.01519.xDOI Listing
January 2012

Evidence for a fluorescence yield change driven by a light-induced conformational change within photosystem II during the fast chlorophyll a fluorescence rise.

Biochim Biophys Acta 2011 Sep 7;1807(9):1032-43. Epub 2011 Jun 7.

Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary.

Experiments were carried out to identify a process co-determining with Q(A) the fluorescence rise between F(0) and F(M). With 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), the fluorescence rise is sigmoidal, in its absence it is not. Lowering the temperature to -10°C the sigmoidicity is lost. It is shown that the sigmoidicity is due to the kinetic overlap between the reduction kinetics of Q(A) and a second process; an overlap that disappears at low temperature because the temperature dependences of the two processes differ. This second process can still relax at -60°C where recombination between Q(A)(-) and the donor side of photosystem (PS) II is blocked. This suggests that it is not a redox reaction but a conformational change can explain the data. Without DCMU, a reduced photosynthetic electron transport chain (ETC) is a pre-condition for reaching the F(M). About 40% of the variable fluorescence relaxes in 100ms. Re-induction while the ETC is still reduced takes a few ms and this is a photochemical process. The fact that the process can relax and be re-induced in the absence of changes in the redox state of the plastoquinone (PQ) pool implies that it is unrelated to the Q(B)-occupancy state and PQ-pool quenching. In both +/-DCMU the process studied represents ~30% of the fluorescence rise. The presented observations are best described within a conformational protein relaxation concept. In untreated leaves we assume that conformational changes are only induced when Q(A) is reduced and relax rapidly on re-oxidation. This would explain the relationship between the fluorescence rise and the ETC-reduction.
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http://dx.doi.org/10.1016/j.bbabio.2011.05.022DOI Listing
September 2011

The physiological role of ascorbate as photosystem II electron donor: protection against photoinactivation in heat-stressed leaves.

Plant Physiol 2011 May 28;156(1):382-92. Epub 2011 Feb 28.

Institute of Plant Biology, Biological Research Center Szeged, Hungarian Academy of Sciences, H-6701 Szeged, Hungary.

Previously, we showed that ascorbate (Asc), by donating electrons to photosystem II (PSII), supports a sustained electron transport activity in leaves in which the oxygen-evolving complexes were inactivated with a heat pulse (49°C, 40 s). Here, by using wild-type, Asc-overproducing, and -deficient Arabidopsis (Arabidopsis thaliana) mutants (miox4 and vtc2-3, respectively), we investigated the physiological role of Asc as PSII electron donor in heat-stressed leaves (40°C, 15 min), lacking active oxygen-evolving complexes. Chlorophyll-a fluorescence transients show that in leaves excited with trains of saturating single-turnover flashes spaced 200 ms apart, allowing continual electron donation from Asc to PSII, the reaction centers remained functional even after thousands of turnovers. Higher flash frequencies or continuous illumination (300 μmol photons m(-2) s(-1)) gradually inactivated them, a process that appeared to be initiated by a dramatic deceleration of the electron transfer from Tyr(Z) to P680(+), followed by the complete loss of charge separation activity. These processes occurred with half-times of 1.2 and 10 min, 2.8 and 23 min, and 4.1 and 51 min in vtc2-3, the wild type, and miox4, respectively, indicating that the rate of inactivation strongly depended on the Asc content of the leaves. The recovery of PSII activity, following the degradation of PSII proteins (D1, CP43, and PsbO), in moderate light (100 μmol photons m(-2) s(-1), comparable to growth light), was also retarded in the Asc-deficient mutant. These data show that high Asc content of leaves contributes significantly to the ability of plants to withstand heat-stress conditions.
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http://dx.doi.org/10.1104/pp.110.171918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091034PMC
May 2011

Experimental evidence for ascorbate-dependent electron transport in leaves with inactive oxygen-evolving complexes.

Plant Physiol 2009 Mar 14;149(3):1568-78. Epub 2009 Jan 14.

Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary.

Previously, we showed that in barley (Hordeum vulgare) leaves with heat-inactivated oxygen-evolving complexes, photosystem II (PSII) has access to a large pool of alternative electron donors. Based on in vitro data, we proposed that this donor was ascorbate, yet this hypothesis has not been substantiated in vivo. In this paper, with the aid of chlorophyll a fluorescence induced by short (5-ms) light pulses and 820-nm absorbance transient measurements on wild-type and ascorbate-deficient (vtc2-1) mutant leaves of Arabidopsis (Arabidopsis thaliana), we show that in heat-treated leaves the rate of electron donation to PSII as well as the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive electron transport toward photosystem I depend on the ascorbate content of the leaves: upon ascorbate treatment, the donation half-time in the wild type and the mutant decreased from 25 to 22 ms and from 55 to 32 ms, respectively. Thermoluminescence measurements show that Tyr(Z)(+) is involved in the electron transfer from ascorbate to PSII. These data and the similar ascorbate dependencies of the heat-treated and the tris(hydroxymethyl)aminomethane-treated thylakoid membranes, with maximal donation half-times of about 16 ms, show that ascorbate is capable of supporting a sustained electron transport activity in leaves containing inactivated oxygen-evolving complexes. This alternative electron transport appears to be ubiquitous in the plant kingdom and is present in the green alga Chlamydomonas reinhardtii, and its rate depends on the physiological state of the plants and on environmental conditions. Our data suggest that ascorbate, as an alternative PSII electron donor, plays a physiological role in heat-stressed plants.
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http://dx.doi.org/10.1104/pp.108.132621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649403PMC
March 2009

Role of phosphatidylglycerol in the function and assembly of Photosystem II reaction center, studied in a cdsA-inactivated PAL mutant strain of Synechocystis sp. PCC6803 that lacks phycobilisomes.

Biochim Biophys Acta 2008 Sep 10;1777(9):1184-94. Epub 2008 Jun 10.

Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary.

To analyze the role of phosphatidylglycerol (PG) in photosynthetic membranes of cyanobacteria we used two mutants of Synechocystis sp. PCC6803: the PAL mutant which has no phycobilisomes and shows a high PSII/PSI ratio, and a mutant derived from it by inactivating its cdsA gene encoding cytidine 5'-diphosphate diacylglycerol synthase, a key enzyme in PG synthesis. In a medium supplemented with PG the PAL/DeltacdsA mutant cells grew photoautotrophically. Depletion of PG in the medium resulted (a) in an arrest of cell growth and division, (b) in a slowdown of electron transfer from the acceptor Q(A) to Q(B) in PSII and (c) in a modification of chlorophyll fluorescence curve. The depletion of PG affected neither the redox levels of Q(A) nor the S(2) state of the oxygen-evolving manganese complex, as indicated by thermoluminescence studies. Two-dimensional PAGE showed that in the absence of PG (a) the PSII dimer was decomposed into monomers, and (b) the CP43 protein was detached from a major part of the PSII core complex. [(35)S]-methionine labeling confirmed that PG depletion did not block de novo synthesis of the PSII proteins. We conclude that PG is required for the binding of CP43 within the PSII core complex.
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http://dx.doi.org/10.1016/j.bbabio.2008.06.003DOI Listing
September 2008

A non-invasive assay of the plastoquinone pool redox state based on the OJIP-transient.

Photosynth Res 2007 Jul-Sep;93(1-3):193-203. Epub 2007 May 9.

Laboratory of Bioenergetics, University of Geneva, Chemin des Embrouchis 10, CH-1254, Jussy, Geneva, Switzerland.

The plastoquinone (PQ) pool of the photosynthetic electron transport chain becomes reduced under anaerobic conditions. Here, anaerobiosis was used as a tool to manipulate the PQ-pool redox state in darkness and to study the effects of the PQ-redox state on the Chl-a fluorescence (OJIP) kinetics in pea leaves (Pisum sativum L.). It is shown that the F(J) (fluorescence intensity at 3 ms) is linearly related to the area above the OJ-phase (first 3 ms) representing the reduction of the acceptor side of photosystem II (PSII) and F(J) is also linearly related to the area above the JI-phase (3-30 ms) that parallels the reduction of the PQ-pool. This means that F(J) depends on the availability of oxidized PQ-molecules bound to the Q(B)-site. The linear relationships between F(J) and the two areas indicate that F(J) is not sensitive to energy transfer between PSII-antennae (connectivity). It is further shown that a approximately 94% reduced PQ-pool is in equilibrium with a approximately 19% reduction of Q(A) (primary quinone acceptor of PSII). The non-linear relationship between the initial fluorescence value (F(20 micros)) and the area above the OJ-phase supports the idea that F(20 mus )is sensitive to connectivity. This is reinforced by the observation that this non-linearity can be overcome by transforming the F(20 micros)-values into [Q(A) (-)]-values. Based on the F(J)-value of the OJIP-transient, a simple method for the quantification of the redox state of the PQ-pool is proposed.
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http://dx.doi.org/10.1007/s11120-007-9179-8DOI Listing
November 2007

Photosynthetic electron transport activity in heat-treated barley leaves: the role of internal alternative electron donors to photosystem II.

Biochim Biophys Acta 2007 Apr 3;1767(4):295-305. Epub 2007 Mar 3.

Laboratory of Bioenergetics, University of Geneva, Chemin des Embrouchis 10, CH-1254 Jussy, Geneva, Switzerland.

Electron transport processes were investigated in barley leaves in which the oxygen-evolution was fully inhibited by a heat pulse (48 degrees C, 40 s). Under these circumstances, the K peak (approximately F(400 micros)) appears in the chl a fluorescence (OJIP) transient reflecting partial Q(A) reduction, which is due to a stable charge separation resulting from the donation of one electron by tyrozine Z. Following the K peak additional fluorescence increase (indicating Q(A)(-) accumulation) occurs in the 0.2-2 s time range. Using simultaneous chl a fluorescence and 820 nm transmission measurements it is demonstrated that this Q(A)(-) accumulation is due to naturally occurring alternative electron sources that donate electrons to the donor side of photosystem II. Chl a fluorescence data obtained with 5-ms light pulses (double flashes spaced 2.3-500 ms apart, and trains of several hundred flashes spaced by 100 or 200 ms) show that the electron donation occurs from a large pool with t(1/2) approximately 30 ms. This alternative electron donor is most probably ascorbate.
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http://dx.doi.org/10.1016/j.bbabio.2007.02.019DOI Listing
April 2007

Dark recovery of the Chl a fluorescence transient (OJIP) after light adaptation: the qT-component of non-photochemical quenching is related to an activated photosystem I acceptor side.

Biochim Biophys Acta 2006 Jul 4;1757(7):787-97. Epub 2006 May 4.

Bioenergetics Laboratory, University of Geneva, Chemin des Embrouchis 10, Jussy, Switzerland.

The dark recovery kinetics of the Chl a fluorescence transient (OJIP) after 15 min light adaptation were studied and interpreted with the help of simultaneously measured 820 nm transmission. The kinetics of the changes in the shape of the OJIP transient were related to the kinetics of the qE and qT components of non-photochemical quenching. The dark-relaxation of the qE coincided with a general increase of the fluorescence yield. Light adaptation caused the disappearance of the IP-phase (20-200 ms) of the OJIP-transient. The qT correlated with the recovery of the IP-phase and with a recovery of the re-reduction of P700(+) and oxidized plastocyanin in the 20-200 ms time-range as derived from 820 nm transmission measurements. On the basis of these observations, the qT is interpreted to represent the inactivation kinetics of ferredoxin-NADP(+)-reductase (FNR). The activation state of FNR affects the fluorescence yield via its effect on the electron flow. The qT therefore represents a form of photochemical quenching. Increasing the light intensity of the probe pulse from 1800 to 15000 mumol photons m(-2) s(-1) did not qualitatively change the results. The presented observations imply that in light-adapted leaves, it is not possible to 'close' all reaction centers with a strong light pulse. This supports the hypothesis that in addition to Q(A) a second modulator of the fluorescence yield located on the acceptor side of photosystem II (e.g., the occupancy of the Q(B)-site) is needed to explain these results. Besides, some of our results indicate that in pea leaves state 2 to 1 transitions may contribute to the qI-phase.
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http://dx.doi.org/10.1016/j.bbabio.2006.04.019DOI Listing
July 2006