Lea Vojta

Tethering of ferredoxin:NADP+ oxidoreductase to thylakoid membranes is mediated by novel chloroplast protein TROL

Working in tandem, two photosystems in the chloroplast thylakoid membranes produce a linear electron flow from H(2)O to NADP(+). Final electron transfer from ferredoxin to NADP(+) is accomplished by a flavoenzyme ferredoxin:NADP(+) oxidoreductase (FNR). Here we describe TROL (thylakoid rhodanese-like protein), a nuclear-encoded component of thylakoid membranes that is required for tethering of FNR and sustaining efficient linear electron flow (LEF) in vascular plants. TROL consists of two distinct modules; a centrally positioned rhodanese-like domain and a C-terminal hydrophobic FNR binding region. Analysis of Arabidopsis mutant lines indicates that, in the absence of TROL, relative electron transport rates at high-light intensities are severely lowered accompanied with significant increase in non-photochemical quenching (NPQ). Thus, TROL might represent a missing thylakoid membrane docking site for a complex between FNR, ferredoxin and NADP(+). Such association might be necessary for maintaining photosynthetic redox poise and enhancement of the NPQ.

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

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.

Energy conductance from thylakoid complexes to stromal reducing equivalents

Working in synchrony, photosynthetic charge separation, electron transfer, and redox reactions generate proton motif force necessary for the synthesis of ATP and funneling of electrons toward stromal reducing equivalent NADPH. The last step of electron transfer from ferredoxin to NADP+ is catalyzed by ubiquitous flavin adenine dinucleotide (FAD)-binding enzyme, ferredoxin-NADP+ oxidoreductase (FNR). Apart from this most notable activity, FNR has been implicated in various other thylakoid energy transduction pathways ranging from cyclic electron flow around photosystem I (PSI) to regulation and management of oxidative stress. Different supramolecular complexes of FNR have so far been described, but until recently, specific FNR interacting partners have eluded detection. Two proteins, TROL (thylakoid rhodanese-like) and Tic62 (62 kDa component of the translocon at the inner envelope of chloroplasts) have been characterized and shown to form dynamic complexes with FNR. Being multi-pass membrane protein, TROL qualifies for the long-sought membrane anchor of FNR. Both TROL and Tic62 can also be found at chloroplast inner envelope, revisiting the notion of electron transfer chain specific for this compartment. Additionally, Tic62 in complex with FNR can be found in chloroplast stroma, implicating its role in formation and stabilization of FNR dimmers. Tethering of FNR to Tic62/TROL is accomplished via a conserved Ser/Pro-rich motif which forms type II alpha-helix and presumably mediates the interaction in pH-dependent manner. Inactivation of TROL leads to changes in efficiency of electron transfer and induction of non-photochemical quenching. TROL-deficient plants have changed nuclear gene expression with up-regulation of NADPH-dependent malic enzyme, which can form NADPH in an alternative pathway. Thus, NADPH synthesis, mediated by FNR-TROL interaction, may be the source element in metabolic retrograde signal-transduction pathway linking light reactions with nuclear gene expression. This review will focus on recent developments in the field of FNR/TROL-Tic62 mediated electron transfer and will explore future perspectives in the research of these important elements of photosynthetic energy transduction.

Data supporting the absence of FNR dynamic photosynthetic membrane recruitment in trol mutants

In photosynthesis, the flavoenzyme ferredoxin:NADP+ oxidoreductase (FNR) catalyses the final electron transfer from ferredoxin to NADP+, which is considered as the main pathway of high-energy electron partitioning in chloroplasts (DOI: 10.1111/j.1365-313X.2009.03999.x[1], DOI: 10.1038/srep10085[2]). Different detergents and pH treatments of photosynthetic membranes isolated from the Arabidopsis wild-type (WT) and the loss-of-function mutants of the thylakoid rhodanase-like protein TROL (trol), pre-acclimated to either dark, growth-light, or high-light conditions, were used to probe the strength of FNR-membrane associations. Detergents β-DM (decyl-β-D-maltopyranoside) or β-DDM (n-dodecyl-β-D-maltopyranoside) were used to test the stability of FNR binding to the thylakoid membranes, and to assess different membrane domains containing FNR. Further, the extraction conditions mimicked pH status of chloroplast stroma during changing light regimes. Plants without TROL are incapable of the dynamic FNR recruitment to the photosynthetic membranes.

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

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.

The outer membrane Omp85-like protein P39 influences metabolic homeostasis in mature Arabidopsis thaliana

The Omp85 proteins form a large membrane protein family in bacteria and eukaryotes. Omp85 proteins are composed of a C-terminal β-barrel-shaped membrane domain and one or more N-terminal polypeptide transport-associated (POTRA) domains. However, Arabidopsis thaliana contains two genes coding for Omp85 proteins without a POTRA domain. One gene is designated P39, according to the molecular weight of the encoded protein. The protein is targeted to plastids and it was established that p39 has electrophysiological properties similar to other Omp85 family members, particularly to that designated as Toc75V/Oep80. We analysed expression of the gene and characterised two T-DNA insertion mutants, focusing on alterations in photosynthetic activity, plastid ultrastructure, global expression profile and metabolome. We observed pronounced expression of P39, especially in veins. Mutants of P39 show growth aberrations, reduced photosynthetic activity and changes in plastid ultrastructure, particularly in the leaf tip. Further, they display global alteration of gene expression and metabolite content in leaves of mature plants. We conclude that the function of the plastid-localised and vein-specific Omp85 family protein p39 is important, but not essential, for maintenance of metabolic homeostasis of full-grown A. thaliana plants. Further, the function of p39 in veins influences the functionality of other plant tissues. The link connecting p39 function with metabolic regulation in mature A. thaliana is discussed.

Rapid transient expression of human granulocyte-macrophage colony-stimulating factor in two industrial cultivars of tobacco (Nicotiana tabacum L.) by agroinfiltration

Highlights • Successful expression of human cytokine GM-CSF in two industrial tobacco cultivars is achieved.• Rapid cloning in two binary destination vectors is accomplished by using Gateway approach.• Agrobacterial infiltration procedure is optimized and shown to require the surface tension lowering agent Silwet L-77.• Accumulation of recombinant protein was confirmed by using high affinity monoclonal hemagglutinin tag antibodies.• Production of hGM-CSF has been achieved without plant codon usage optimization.

Electron Transfer Routes in Oxygenic Photosynthesis: Regulatory Mechanisms and New Perspectives

Light-driven photosynthetic charge separation supplies power for the synthesis of chemical energy equivalents, ATP and NADPH, which consequently fuel metabolic biochemical reactions required by plant. This fundamental process of energy conversion is exceptionally complex and involves light harvesting, water splitting, proton pumping, and electron partitioning mechanisms which have to be poised in order to efficiently synthesize chemical energy equivalents. Photosynthetic membranes possess enormous physiological versatility, in particular the ability to manage short- and long-term changes in the light environment. Various mechanisms can regulate the flow and partitioning of excitation energy between the two photosystems, and others can convert excess excitation energy into thermal energy. Once photodamage has occurred, a regulated protein turnover can re-establish function, or can acclimatize the photosynthetic machinery to seasonal changes. A large number of regulatory and supporting enzymes are involved in these physiological processes. These include, for instance, protein kinases and phosphatases, chaperones, a substantial number of proteases, and diverse other protein components that are required for the biogenesis of multisubunit complexes. Numerous studies have contributed to the understanding of protein phosphorylation involved in regulation of photosynthesis. Several thylakoid (and chloroplast) kinases, STN7, TAK, and phosphateses PPH1/TAP38 have been identified, and shown to be involved in photosynthetic regulatory pathways. Regulatory signals can be of different origin, but redox-dependent inputs have been implicated as major triggers. Various auxiliary proteins have been linked to these processes, with TLP40 lumenal immunofilin being one of them. Further, our understanding of different photosynthetic electron transport chains has substantially increased. We now know that three major pathways, linear, cyclic and pseudo-cyclic, are necessary for poised and sustained synthesis of ATP and NADPH. Also, alternative routes of electron partitioning have been recognized, raising the possibility of further regulation governed by switchable energy partitioning. An example of such novel mechanism is the dynamic binding and release of ferredoxin NADP+ oxidoreductase (FNR) from the thylakoid rhodanese-like protein TROL. Here, we review the above outlined regulatory mechanisms and aim to suggest direction for further research on these topics.