Arjun Tiwari

Photodamage of iron–sulphur clusters in photosystem I induces non-photochemical energy dissipation

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.

Superoxide oxidase and reductase activity of cytochrome b559 in photosystem II

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)(*-)) was observed, as confirmed by EPR spin-trapping spectroscopy. The observations that the addition of enzymatic (superoxide dismutase) and non-enzymatic (cytochrome c, alpha-tocopherol and Trolox) O(2)(*-) 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)(*-), respectively. Redox changes in cyt b(559) by an exogenous source of O(2)(*-) reconfirmed the superoxide oxidase and reductase activity of cyt b(559). Furthermore, the light-induced conversion of IP to HP(red) form of cyt b(559) was completely inhibited at pH>8 and by chemical modification of the imidazole ring of histidine residues using diethyl pyrocarbonate. We proposed that a change in the environment around the heme iron, induced by the protonation and deprotonation of His(22) residue generates a favorable condition for the oxidation and reduction of O(2)(*-), respectively.

Heat-induced changes in photosystem I activity as measured with different electron donors in isolated spinach thylakoid membranes.

Heat-induced changes in photosystem I (PSI) have been studied in terms of rates of oxygen consumption using various donors (DCPIPH2, TMPDred and DADred), formation of photo-oxidized P700 and changes in Chl a fluorescence emission at 77 K. Linear heating of thylakoid membranes from 35 degrees C to 70 degrees C caused an enhancement in PSI-mediated electron transfer rates (DCPIPH2-->MV) up to 55 degrees C. However, no change was observed in PSI rates when other electron donors were used (TMPDred and DADred). 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 approximately 50-55 degrees C without DCMU treatment. The steady-state signal of the photo-oxidized P700 was studied in the presence of DCPIPH2 and TMPDred as electron donors in DCMU-treated control and in 50 degrees 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.

Low pH-induced regulation of excitation energy between the two photosystems

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.

Differential response of chloride binding sites to elevated temperature: a comparative study in spinach thylakoids and PSII-enriched membranes

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 Cl− in the PSII complex. For this, Cl−-sufficient membranes and low-Cl− membranes were used. The presence of Cl− in 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 Cl− in PSII. Electron paramagnetic resonance study of manganese (Mn) release per reaction center/YD+ showed that there was little loss of Mn2+ 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 Cl− causing 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 high-affinity (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).

Differential mechanism of light-induced and oxygen-dependent restoration of the high-potential form of cytochrome b559 in Tris-treated Photosystem II 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.

Inactivation of iron-sulfur cluster biogenesis regulator SufR in Synechocystis sp. PCC 6803 induces unique iron-dependent protein-level responses

BACKGROUND Iron-sulfur (Fe-S) clusters are protein-bound cofactors associated with cellular electron transport and redox sensing, with multiple specific functions in oxygen-evolving 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. GENERAL SIGNIFICANCE The study provides new insight into the regulation of Fe-S cluster biogenesis via suf operon, and the associated wide-ranging protein-level changes in photosynthetic cyanobacteria.

Water-splitting manganese complex controls light-induced redox changes of cytochrome b559 in photosystem II.

The effect of water-splitting Mn complex on light-induced redox changes of cytochrome b559 (cyt b559) 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 b559 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.

Interaction between photosynthetic electron transport and chloroplast sinks triggers protection and signalling important for plant productivity

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 plants, can negatively impact metabolism and signalling, and thereby threaten plant growth and stress tolerance. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’.

RCD1 Coordinates Chloroplastic and Mitochondrial Electron Transfer through Interaction with ANAC Transcription Factors

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 interacts in vivo with ANAC013 and ANAC017 transcription factors, which are the mediators of the ROS-related mitochondrial complex retrograde signa and suppresses activity of ANAC013 and ANAC017. Inactivation of RCD1 leads 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 including chloroplast signaling by 3’-phosphoadenosine 5’-phosphate (PAP). Sensitivity of RCD1 to organellar ROS provides feedback control of nuclear gene expression.