Energy Conductance from Thylakoid Complexes to Stromal Reducing Equivalents

Carić

Cesar

et al. 2015

Sci Rep

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In photosynthesis, final electron transfer from ferredoxin to NADP+ is accomplished by the flavo enzyme ferredoxin:NADP+ oxidoreductase (FNR). FNR is recruited to thylakoid membranes via integral membrane thylakoid rhodanase-like protein TROL. We address the fate of electrons downstream of photosystem I when TROL is absent. We have employed electron paramagnetic resonance (EPR) spectroscopy to study free radical formation and electron partitioning in TROL-depleted chloroplasts. DMPO was used to detect superoxide anion (O2.−) formation, while the generation of other free radicals was monitored by Tiron. Chloroplasts from trol plants pre-acclimated to different light conditions consistently exhibited diminished O2.− accumulation. Generation of other radical forms was elevated in trol chloroplasts in all tested conditions, except for the plants pre-acclimated to high-light. Remarkably, dark- and growth light-acclimated trol chloroplasts were resilient to O2.− generation induced by methyl-viologen. We propose that the dynamic binding and release of FNR from TROL can control the flow of photosynthetic electrons prior to activation of the pseudo-cyclic electron transfer pathway.

Section: Discussionmentioning

confidence: 85%

mentioning

confidence: 99%

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TROL-FNR interaction reveals alternative pathways of electron partitioning in photosynthesis

Carić

Cesar

et al. 2015

Sci Rep

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“…A). As we proposed earlier (Vojta et al , Vojta and Fulgosi ), inactive RHO domain could bind a small ligand, a molecule similar or analogous to plastoquinone, which would trigger the transmembrane signal necessary for the binding or release of FNR. This assumption remains to be investigated.…”

Section: Discussionmentioning

confidence: 76%

Topology of TROL protein in thylakoid membranes of Arabidopsis thaliana

Fulgosi

2019

Physiologia Plantarum

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Thylakoid rhodanase‐like protein (TROL) is a nuclear‐encoded protein of thylakoid membranes required for tethering of ferredoxin:nicotinamide adenine dinucleotide phosphate (NADPH) oxydoreductase (FNR). It has been proposed that the dynamic interaction of TROL with flavoenzyme FNR, influenced by environmental light conditions, regulates the fate of photosynthetic electrons, directing them either to NADPH synthesis or to other acceptors, including reactive oxygen species detoxification pathways. Inside the chloroplasts, TROL has a dual localization: an inner membrane precursor form and a thylakoid membrane mature form, which has been confirmed by several large‐scale chloroplast proteomics studies, as well as protein import experiments. Unlike the localization, the topology of TROL in the membranes, which is a prerequisite for further studies of its properties and function, has not been experimentally confirmed yet. Thermolysin was proven to be a valuable protease to probe the surface of chloroplasts and membranes in general. By treating the total chloroplast membranes using increasing protease concentration, sequential degradation of TROL was observed, indicating protected polypeptides of TROL and possible domain orientation. To further substantiate the obtained results, TROL‐overexpressing Arabidopsis line (OX) and line in which the central rhodanase‐like domain (RHO) has been partially deleted (ΔRHO), were used as well. While OX line showed the same degradation pattern of TROL as the wild‐type, surprisingly, TROL from ΔRHO membranes was not detectable even at the lowest protease concentration applied, indicating the importance of this domain to the integrity of TROL. In conclusion, TROL is a polytopic protein with a stroma‐exposed C‐terminal FNR‐binding region, and the thylakoid lumen‐located RHO domain.

“…The C-terminus of the protein resides in the cytosol and consists of a single hydrophobic FNR-binding region, ITEP (highly conserved module of TROL necessary for establishing high-affinity interaction with FNR), and a region upstream of the ITEP domain, designated PEPE (Pro-Val-Pro repeat-rich region), which is followed by a possible PVP hinge, proposed to introduce flexibility to the FNR-binding region. In our previous research we proposed that the TROL–FNR interaction is dynamic [ 2 , 3 ], in which binding and release of FNR from TROL can regulate the flow of photosynthetic electrons before the pseudo-cyclic electron transfer pathway becomes activated [ 4 ]. By studying Arabidopsis trol mutants we proposed that the TROL–FNR interaction is the bifurcating point between electron-conserving and electron-dissipating pathways [ 4 ].…”

Section: Introductionmentioning

confidence: 99%

Effects of TROL Presequence Mutagenesis on Its Import and Dual Localization in Chloroplasts

Čuletić

Fulgosi

2018

IJMS

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Thylakoid rhodanase-like protein (TROL) is involved in the final step of photosynthetic electron transport from ferredoxin to ferredoxin: NADP+ oxidoreductase (FNR). TROL is located in two distinct chloroplast compartments—in the inner envelope of chloroplasts, in its precursor form; and in the thylakoid membranes, in its fully processed form. Its role in the inner envelope, as well as the determinants for its differential localization, have not been resolved yet. In this work we created six N-terminal amino acid substitutions surrounding the predicted processing site in the presequence of TROL in order to obtain a construct whose import is affected or localization limited to a single intrachloroplastic site. By using in vitro transcription and translation and subsequent protein import methods, we found that a single amino acid exchange in the presequence, Ala67 to Ile67 interferes with processing in the stroma and directs the whole pool of in vitro translated TROL to the inner envelope of chloroplasts. This result opens up the possibility of studying the role of TROL in the chloroplast inner envelope as well as possible consequence/s of its absence from the thylakoids.