Department of Biophysics

Steady state and time-resolved fluorescence properties of chlorin p 6 , a potential drug for photodynamic therapy, have been investigated as functions of pH. A decrease in pH of the medium has been shown to cause protonation of the ionizable carboxylic acid side chain, leading to an increase in hydrophobicity and consequent aggregation. The aggregates dissociate on further protonation. The dissociation is explained in terms of formation of cations and their mutual repulsion. A synchronous fluorescence spectroscopic study revealed the presence of two anionic forms in equilibrium at physiological pH, with a shift in the equilibrium on slight decrease in the pH. The anionic nature of chlorin p 6 in aqueous solutions at physiological pH has been confirmed by complexation with surfactants. The nature of the charge on the headgroups of the surfactants has been found to govern the formation of chlorin-surfactant complexes.

This study provides evidence for the superoxide oxidase and the superoxide reductase activity of cytochrome b 559 (cyt b 559 ) in PSII. It is reported that in Tris-treated PSII membranes upon illumination, both the intermediate potential (IP) and the reduced high potential (HP red ) forms of cyt b 559 exhibit superoxide scavenging activity and interconversion between IP and HP red form. When Tris-treated PSII membranes were illuminated in the presence of spin trap EMPO, the formation of superoxide anion radical (O 2 U − ) was observed, as confirmed by EPR spin-trapping spectroscopy. The observations that the addition of enzymatic (superoxide dismutase) and non-enzymatic (cytochrome c, α-tocopherol and Trolox) O 2 U − scavengers prevented the light-induced conversion of IP ↔ HP red cyt b 559 confirmed that IP and HP red cyt b 559 are reduced and oxidized by O 2 U − , respectively. Redox changes in cyt b 559 by an exogenous source of O 2 U − reconfirmed the superoxide oxidase and reductase activity of cyt b 559 .

Exclusion of UV (280-380 nm) radiation from the solar spectrum can be an important tool to assess the impact of ambient UV radiation on plant growth and performance of crop plants. The effect of exclusion of UV-B and UV-A from solar radiation on the growth and photosynthetic components in soybean (Glycine max) leaves were investigated. Exclusion of solar UV-B and UV-

A study of heat effects was performed in thylakoids and photosystem II (PSII)-enriched membranes isolated from spinach in relation to Cl --induced activation of PSII catalyzed oxygen evolution and the retention of Clin the PSII complex. For this, Cl -sufficient membranes and low-Clmembranes were used. The presence of Clin the reaction medium did accelerate oxygen evolution, which remained unaffected by heat treatment up to 40°C in PSII membranes and up to 42.5°C in thylakoids. Heat resistance of Cl --induced activation of oxygen evolution was found to be independent of the presence of 'bound Cl -' in the preparations. However, the functional stability of the PSII complex during heat treatment showed a marked dependence on the presence of bound Clin PSII. Electron paramagnetic resonance study of manganese (Mn) release per reaction center/Y D + showed that there was little loss of Mn 2+ up to 42°C in our preparations, although the PSII activity was significantly lowered. These observations together with data from steady state chlorophyll a fluorescence imply that the site of action of Clcausing direct activation of oxygen evolution was different from the site of primary heat damage. A differential response of chloride binding sites to heat stress was observed. The highaffinity (tightly bound, slow exchanging) site of chloride is affected earlier (~37°C) while low-affinity (loosely bound, fast exchanging) site gets affected at higher temperatures (42.5°C in thylakoids and 40°C in the case of PSII-enriched membranes).

The effect of illumination and molecular oxygen on the redox and the redox potential changes of cytochrome b 559 (cyt b 559 ) has been studied in Tris-treated spinach photosystem II (PSII) membranes. It has been demonstrated that the illumination of Tris-treated PSII membranes induced the conversion of the intermediate-potential (IP) to the reduced high-potential (HP Fe2+ ) form of cyt b 559 , whereas the removal of molecular oxygen resulted in the conversion of the IP form to the oxidized high-potential (HP Fe3+ ) form of cyt b 559 . Light-induced conversion of cyt b 559 from the IP to the HP form was completely inhibited above pH 8 or by the modification of histidine ligand that prevents its protonation. Interestingly, no effect of high pH or histidine modification was observed during the conversion of the IP to the HP form of cyt b 559 after the removal of molecular oxygen. These results indicate that conversion from the IP to the HP form of cyt b 559 proceeds via different mechanisms. Under illumination, conversion of the IP to the HP form of cyt b 559 depends primarily on the protonation of the histidine residue, whereas under anaerobic conditions, the conversion of the IP to the HP form of cyt b 559 is driven by higher hydrophobicity of the environment around the heme iron resulting from the absence of molecular oxygen.

The effect of water-splitting Mn complex on lightinduced redox changes of cytochrome b 559 (cyt b 559 ) was studied in spinach photosystem II (PSII) membranes. Photoreduction of the heme iron in the intact PSII membranes was completely suppressed by DCMU, whereas photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were unaffected by DCMU. Interesingly, photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were completely diminished by exogenous superoxide dismutase (SOD), whereas no effect of SOD on photoreduction of the heme iron was observed in the intact PSII membranes. The current work shows that the light-induced redox changes of cyt b 559 proceed via a different mechanism in the both types of PSII membranes. In the intact PSII membranes, photoreduction of the heme iron is mediated by plastoquinol. However, in the Mn-depleted PSII membranes, photoreduction and photooxidation of the heme iron are mediated by superoxide anion radical formed in PSII. Keywords Cytochorome b 559 . Photosystem II . Redox potential . Water-splitting manganese complex Abbreviations cyt b 559 cytochorome b 559 Em midpoint redox potential HP high-potential form of cyt b 559 IP intermediate potential form of cyt b 559 LP low-potential form of cyt b 559 MES 2-[N-Morpholino]ethanesulfonic acid PSII photosystem II SOD superoxide dismutase SOO superoxide oxidase SOR superoxide reductase Q A primary quinone electron acceptor of PSII Q B

The present study for the first time describes a close relationship between a change in the states of Cyt b559, a damage to Mn complex and a rapid reduction of tyrosine D (Y D ) as a function of temperature in spinach thylakoid membranes. Measurements of the EPR signal of dark stable tyrosine D in heat-treated thylakoid membranes showed a gradual decay of the oxidized state of tyrosine D with the progression of temperature. Simultaneously, it leads to the conversion of high-potential Cytochrome b559 into its low-potential form. We have speculated a possible involvement of Cytochrome b559 in the primary reduction events of tyrosine D in dark at high temperature. However, rapid reduction of tyrosine D may also be due to the disassembly of the Mn clock, which causes exposure of Y D to the lumen and thereby its reduction by some unknown factor. These conclusions are supported by the measurements of Mn 2+ release and thermoluminescence curves of various charge pairs in heat-treated thylakoid membranes. The results reveal an important aspect on the role of Cyt b559 in PS II during temperature stress.

Heat-induced changes in photosystem I (PSI) have been studied in terms of rates of oxygen consumption using various donors (DCPIPH 2 , TMPD red and DAD red ), formation of photo-oxidized P700 and changes in Chl a fluorescence emission at 77 K. Linear heating of thylakoid membranes from 35 • C to 70 • C caused an enhancement in PSI-mediated electron transfer rates (DCPIPH 2 →MV) up to 55 • C. However, no change was observed in PSI rates when other electron donors were used (TMPD red and DAD red ). Similarly, Chl a fluorescence emission spectra at 77 K of heat-treated thylakoid membranes did not show any increase in peak at 735 nm, however, a significant decrease was observed as a function of temperature in the peaks at 685 and 694 nm. In DCMU-treated control thylakoid membranes maximum photo-oxidized P700 was generated at g = 2.0025. In heat-treated thylakoid membranes maximum intensity of photo-oxidized P700 signal was observed at ∼50-55 • C without DCMU treatment. The steady-state signal of the photo-oxidized P700 was studied in the presence of DCPIPH 2 and TMPD red as electron donors in DCMU-treated control and in 50 • C treated thylakoid membranes. We present here the first of such comparative study of PSI activity in terms of the rates of oxygen consumption and re-reduction kinetics of photo-oxidized P700 in the presence of different electron donors. It appears that the formation of the P700 + signal in heat-treated thylakoid membranes is due to an inhibited electron supply from PSII and not due to spillover or antenna migration.

Earlier studies have proposed that low pH causes state transitions in spinach thylakoid membranes. Several Arabidopsis mutants (stn7 incapable in phosphorylation of LHC II, stn8 incapable in phosphorylation of PSII core proteins, stn7 stn8 double mutant and npq4 lacking PsbS and hence qE) were used to investigate the mechanisms involved in low pH induced changes in the thylakoid membrane. We propose that protonation of PsbS at low pH is involved in enhancing energy spillover to PS I.

The photosynthetic light reactions provide energy that is consumed and stored in electron sinks, the products of photosynthesis. A balance between light reactions and electron consumption in the chloroplast is vital for plants, and is protected by several photosynthetic regulation mechanisms. Photosystem I (PSI) is particularly susceptible to photoinhibition when these factors become unbalanced, which can occur in low temperatures or in high light. In this study we used the pgr5 Arabidopsis mutant that lacks ΔpH-dependent regulation of photosynthetic electron transport as a model to study the consequences of PSI photoinhibition under high light. We found that PSI damage severely inhibits carbon fixation and starch accumulation, and attenuates enzymatic oxylipin synthesis and chloroplast regulation of nuclear gene expression after high light stress. This work shows that modifications to regulation of photosynthetic light reactions, which may be designed to improve yield in crop plant...

Background Iron-sulfur (Fe-S) clusters are protein-bound cofactors associated with cellular electron transport and redox sensing, with multiple specific functions in oxygenevolving photosynthetic cyanobacteria. The aim here was to elucidate protein-level effects of the transcriptional repressor SufR involved in the regulation of Fe-S cluster biogenesis in the cyanobacterium Synechocystis sp. PCC 6803. Methods The approach was to quantitate 94 pre-selected target proteins associated with various metabolic functions using SRM in Synechocystis. The evaluation was conducted in response to sufR deletion under different iron conditions, and complemented with EPR analysis on the functionality of the photosystems I and II as well as with RT-qPCR to verify the effects of SufR also on transcript level. Results The results on both protein and transcript levels show that SufR acts not only as a repressor of the suf operon when iron is available but also has other direct and indirect functions in the cell, including maintenance of the expression of pyruvate:ferredoxin oxidoreductase NifJ and other Fe-S cluster proteins under iron sufficient conditions. Furthermore, the results imply that in the absence of iron the suf operon is repressed by some additional regulatory mechanism independent of SufR. Conclusions The study demonstrates that Fe-S cluster metabolism in Synechocystis is stringently regulated, and has complex interactions with multiple primary functions in the cell, including photosynthesis and central carbon metabolism.

Mitochondrial retrograde signals control expression of nuclear mitochondrial dysfunction stimulon (MDS) genes. Although MDS gene products mostly affect mitochondrial functions, they also influence production of reactive oxygen species (ROS) and redox status of chloroplasts. To study this inter-organellar interaction, we analysed the response of the Arabidopsis MDS-overexpressor mutantrcd1to methyl viologen (MV), which catalyses electron transfer from Photosystem I (PSI) to molecular oxygen, generating ROS in Mehler’s reaction. The response of plants to MV was investigated by imaging chlorophyll fluorescence in aerobic and hypoxic environments, and by membrane inlet mass spectrometry. Hypoxic treatment abolished the effect of MV on photosynthetic electron transfer inrcd1, but not in wild type. A similar reaction to hypoxia was observed in other MDS-activating lines and treatments. This suggests that MDS gene products contribute to oxygen depletion at the PSI electron-acceptor side. I...

Signaling from chloroplasts and mitochondria, both dependent on reactive oxygen species (ROS), merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). ROS produced in the chloroplasts affect the abundance, thiol redox state and oligomerization of RCD1. RCD1 directly interactsin vivowith ANAC013 and ANAC017 transcription factors, which are the mediators of the ROS-related mitochondrial complex III retrograde signa and suppresses activity of ANAC013 and ANAC017. Inactivation ofRCD1leads to increased expression of ANAC013 and ANAC017-regulated genes belonging to the mitochondrial dysfunction stimulon (MDS), including genes for mitochondrial alternative oxidases(AOXs).Accumulating AOXs and other MDS gene products alter electron transfer pathways in the chloroplasts, leading to diminished production of chloroplastic ROS and increased protection of photosynthetic apparatus from ROS damage. RCD1-dependent regulation affects chloroplastic and mitochondrial retrograde signaling incl...

In plant science, 2,4-dinitrophenylether of iodonitrothymol (DNP-INT) is frequently used as an alternative to 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) to examine the capacity of plastoquinol and semiquinone to reduce O2. DNP-INT is considered an effective inhibitor of the photosynthetic electron transfer chain (PETC) through its binding at the Q0 site of Cyt-b6f. The binding and action of DNP-INT has been previously characterized spectroscopically in purified Cyt-b6f complex reconstituted with Plastocyanin, PSII membranes and plastoquinone, as well as in isolated thylakoids based on its property to block MV-mediated O2 consumption. Contrary to the conclusions made from these experiments, we observed clear reduction of P700+ in samples incubated with DNP-INT during our recent investigation into the sites of oxygen consumption in isolated thylakoids. Therefore, we carried out an extensive investigation of DNP-INT’s chemical efficacy in isolated thylakoids and intact l...

Reactive oxygen species (ROS) are generated in electron transport processes of living organisms in oxygenic environments. Chloroplasts are plant bioenergetics hubs where imbalances between photosynthetic inputs and outputs drive ROS generation upon changing environmental conditions. Plants have harnessed various site-specific thylakoid membrane ROS products into environmental sensory signals. Our current understanding of ROS production in thylakoids suggests that oxygen (O2) reduction takes place at numerous components of the photosynthetic electron transfer chain (PETC). To refine models of site- specific O2 reduction capacity of various PETC components in isolated thylakoids, the stoichiometry of oxygen production and consumption reactions, associated with H2O2 accumulation, was quantified using membrane inlet mass spectrometry and specific inhibitors. Combined with P700 spectroscopy and electron paramagnetic resonance spin trapping, we demonstrate that electron flow to PSI is ess...

Reactive oxygen species (ROS) are generated in electron transport processes of living organisms in oxygenic environments. Chloroplasts are plant bioenergetics hubs where imbalances between photosynthetic inputs and outputs drive ROS generation upon changing environmental conditions. Plants have harnessed various site-specific thylakoid membrane ROS products into environmental sensory signals. Our current understanding of ROS production in thylakoids suggests that oxygen (O2) reduction takes place at numerous components of the photosynthetic electron transfer chain (PETC). To refine models of site-specific O2 reduction capacity of various PETC components in isolated thylakoids of Arabidopsis thaliana, we quantified the stoichiometry of oxygen production and consumption reactions associated with hydrogen peroxide (H2O2) accumulation using membrane inlet mass spectrometry and specific inhibitors. Combined with P700 spectroscopy and electron paramagnetic resonance spin trapping, we demo...

Photosystem I (PSI) uses light energy and electrons supplied by photosystem II (PSII) to reduce NADP + to NADPH. PSI is very tolerant of excess light but extremely sensitive to excess electrons from PSII. It has been assumed that PSI is protected from photoinhibition by strict control of the intersystem electron transfer chain (ETC). Here we demonstrate that the iron-sulphur (FeS) clusters of PSI are more sensitive to high light stress than previously anticipated, but PSI with damaged FeS clusters still functions as a non-photochemical photoprotective energy quencher (PSI-NPQ). Upon photoinhibition of PSI, the highly reduced ETC further triggers thylakoid phosphorylation-based mechanisms that increase energy flow towards PSI. It is concluded that the sensitivity of FeS clusters provides an additional photoprotective mechanism that is able to downregulate PSII, based on PSI quenching and protein phosphorylation.

The Arabidopsis mutant rcd1 is tolerant to methyl viologen (MV). MV enhances the Mehler reaction, i.e. electron transfer from Photosystem I (PSI) to O 2 , generating reactive oxygen species (ROS) in the chloroplast. To study the MV tolerance of rcd1 , we first addressed chloroplast thiol redox enzymes potentially implicated in ROS scavenging. NADPH-thioredoxin oxidoreductase type C (NTRC) was more reduced in rcd1 . NTRC contributed to the photosynthetic and metabolic phenotypes of rcd1 , but did not determine its MV tolerance. We next tested rcd1 for alterations in the Mehler reaction. In rcd1 , but not in the wild type, the PSI-to-MV electron transfer was abolished by hypoxic atmosphere. A characteristic feature of rcd1 is constitutive expression of mitochondrial dysfunction stimulon (MDS) genes that affect mitochondrial respiration. Similarly to rcd1 , in other MDS-overexpressing plants hypoxia also inhibited the PSI-to-MV electron transfer. One possible explanation is that the MD...

In natural growth habitats, plants face constant, unpredictable changes in light conditions. To avoid damage to the photosynthetic apparatus on thylakoid membranes in chloroplasts, and to avoid wasteful reactions, it is crucial to maintain a redox balance both within the components of photosynthetic electron transfer chain and between the light reactions and stromal carbon metabolism under fluctuating light conditions. This requires coordinated function of the photoprotective and regulatory mechanisms, such as non‐photochemical quenching (NPQ) and reversible redistribution of excitation energy between photosystem II (PSII) and photosystem I (PSI). In this paper, we show that the NADPH‐dependent chloroplast thioredoxin system (NTRC) is involved in the control of the activation of these mechanisms. In plants with altered NTRC content, the strict correlation between lumenal pH and NPQ is partially lost. We propose that NTRC contributes to downregulation of a slow‐relaxing constituent o...