Topology of TROL protein in thylakoid membranes of Arabidopsis thaliana.

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.

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 Omp85-type outer membrane protein p36 of Arabidopsis thaliana evolved by recent gene duplication.

Proteins of the Omp85 family are involved in the insertion of ß-barrel shaped outer membrane proteins in bacteria and mitochondria, and-at least-in the transfer of preproteins across the chloroplast outer envelope. In general these proteins consist of up to five N-terminal "polypeptide transport associated" (POTRA) domains and a C-terminal, membrane embedded ß-barrel domain. In Arabidopsis thaliana two plastidic gene families coding for Omp85-like proteins exist, namely the Toc75-III and the Toc75-V/Oep80 sub-family. The latter is composed of three genes, of which two do not contain POTRA domains. These are annotated as P39 and P36. However, P36 resulted from a very recent gene duplication of P39 and appears to be specific to Arabidopsis thaliana. Furthermore, we show that P39 is specifically expressed in vein tissues, while P36 is expressed at early and late developmental stages. T-DNA insertion in P36 causes a mild phenotype with reduced starch accumulation in chloroplasts of sepals pointing towards a yet to be described plastid function.

Thylakoid Rhodanese-like Protein-Ferredoxin:NADP+ Oxidoreductase Interaction Is Integrated into Plant Redox Homeostasis System.

In vascular plants, the final photosynthetic electron transfer from ferredoxin (Fd) to NADP+ is catalyzed by the flavoenzyme ferredoxin:NADP+ oxidoreductase (FNR). FNR is recruited to thylakoid membranes via an integral membrane protein TROL (thylakoid rhodanese-like protein) and the membrane associated protein Tic62. We have previously demonstrated that the absence of TROL triggers a very efficient superoxide (O2•-) removal mechanism. The dynamic TROL-FNR interaction has been shown to be an apparently overlooked mechanism that maintains linear electron flow before alternative pathway(s) is(are) activated. In this work, we aimed to further test our hypothesis that the FNR-TROL pair could be the source element that triggers various downstream networks of chloroplast ROS scavenging. Tandem affinity purification followed by the MS analysis confirmed the TROL-FNR interaction and revealed possible interaction of TROL with the thylakoid form of the enzyme ascorbate peroxidase (tAPX), which catalyzes the H2O2-dependent oxidation of ascorbate and is, therefore, the crucial component of the redox homeostasis system in plants. Further, EPR analyses using superoxide spin trap DMPO showed that, in comparison with the wild type, plants overexpressing TROL (TROL OX) propagate more O2•- when exposed to high light stress. This indicates an increased sensitivity to oxidative stress in conditions when there is an excess of membrane-bound FNR and less free FNR is found in the stroma. Finally, immunohistochemical analyses of glutathione in different Arabidopsis leaf cell compartments showed highly elevated glutathione levels in TROL OX, indicating an increased demand for this ROS scavenger in these plants, likely needed to prevent the damage of important cellular components caused by reactive oxygen species.

Virus and Virus-like Pathogens in the Grapevine Virus Collection of Croatian Autochthonous Grapevine Cultivars.

Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses (arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leafroll-associated viruses 1, 2, and 3, and grapevine viruses A and B), a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of "flavescence dorée" phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of "flavescence dorée", makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years.

Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae.

Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H(2) O(2) ) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress.

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.

Qualitative and quantitative dataset of TROL protein interaction with C3 and C4 ferredoxin: NADP+ oxidoreductases.

Last step of electron transport from ferredoxin to NADP+ in photosynthesis light reactions catalyses ferredoxin: NADP+ oxidoreductase (FNR). FNR is present as soluble protein in stroma, but also bound to the protein complexes on the membrane with thylakoid rhodanase-like protein (TROL) and translocon on the inner envelope chloroplast membrane (Tic62), which have identical C terminal FNR binding domain [1,2]. During the electron transport, FNR anchored by TROL protein transfers electrons on NADP+ and forms NADPH which is then used in Calvin cycle as reducing agent. TROL is an integral membrane protein [3] with an inactive rhodanase-like domain (RHO) facing stroma which, as proposed earlier [4], could bind a small ligand leading to releasing or binding of FNR. FNR-TROL protein complex is necessary for optimal photosynthetic electron flow [1]. It has been shown that C4 plant maize FNR isomers have different N-terminal structure which determines binding affinity to protein complexes and different ratios of bound and unbound FNR in bundle sheath and mesophyll cells, depending on preferable photosynthetic electron transport [5]. Mutant Arabidopsis plant that contain maize FNR1 protein showed influence on dynamic association of FNR and change in excitation balance between photosystems which then influenced photo induced electron transport and finally photosynthesis [5]. In order to determine the influence of maize FNR1 on photosynthesis in C3 plants and difference in interaction strength with TROL, we preformed Yeast two hybrid screening, x-alpha-gal assay and ß-galactosidase assay.

Complex lumenal immunophilin AtCYP38 influences thylakoid remodelling in Arabidopsis thaliana.

Investigations of the luminal immunophilin AtCYP38 (cyclophilin 38) in Arabidopsis thaliana (At), the orthologue of the complex immunophilin TLP40 from Spinacia oleracea, revealed its involvement in photosystem II (PSII) repair and assembly, biogenesis of PSII complex, and cellular signalling. However, the main physiological roles of AtCYP38 and TLP40 are related to regulation of thylakoid PP2A-type phosphatase involved in PSII core protein dephosphorylation, and chaperone function during protein folding. Here we further investigate physiological roles of AtCYP38 and analyse the ultrastructure of chloroplasts from cyp38-2 plants. Transmission electron microscopy followed by quantitative micrography revealed modifications in thylakoid stacking. We also confirm that the depletion of AtCYP38 influences PSII performance, which leads to stunted phenotype of cyp38-2 plants.

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 ß-DM (decyl-ß-D-maltopyranoside) or ß-DDM (n-dodecyl-ß-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.

Chloroplast immunophilins.

Immunophilins occur in almost all living organisms. They are ubiquitously expressed proteins including cyclophilins, FK506/rapamycin-binding proteins, and parvulins. Their functional significance in vascular plants is mostly related to plant developmental processes, signalling, and regulation of photosynthesis. Enzymatically active immunophilins catalyse isomerization of proline imidic peptide bonds and assist in rapid folding of nascent proline-containing polypeptides. They also participate in protein trafficking and assembly of supramolecular protein complexes. Complex immunophilins possess various additional functional domains associated with a multitude of molecular interactions. A considerable number of immunophilins act as auxiliary and/or regulatory proteins in highly specialized cellular compartments, such as lumen of thylakoids. In this review, we present a comprehensive overview of so far identified chloroplast immunophilins that assist in specific assembly/repair processes necessary for the maintenance of efficient photosynthetic energy conversion.

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

We report the production of hGM-CSF cytokine in leaves of industrial tobacco cultivars DH-17 and DH-27 by using Agrobacterium-mediated transient expression. We prove the concept that very high biomass industrial tobacco plants are suitable platforms for rapid, low cost production of foreign proteins. Successful transient expression of the GM-CSF was achieved in less than three months, opening the possibility for future applications of this approach in rapid response production of various proteins of non-plant origin in industrial tobacco.

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.

Degradation of chloroplast DNA during natural senescence of maple leaves.

The fate of chloroplast DNA (cpDNA) during plastid development and conversion between various plastid types is still not very well understood. This is especially true for the cpDNA found in plastids of naturally senescing leaves. Here, we describe changes in plastid nucleoid structure accompanied with cpDNA degradation occurring during natural senescence of the free-growing deciduous woody species Acer pseudoplatanus L. Natural senescence was investigated using three types of senescing leaves: green (G), yellow-green (YG) and yellow (Y). The extent of senescence was evaluated at the level of photosynthetic pigment degradation, accumulation of starch and plastid ultrastructure. Determination of cpDNA amount was carried out by in planta visualization with 4,6-diamidino-2-phenylindole, by Southern hybridization, and by dot-blot using an rbcL gene probe. During natural senescence, plastid nucleoids undergo structural rearrangements accompanied by an almost complete loss of cpDNA. Furthermore, senescence-associated protein components exhibiting strong binding to an ∼10kbp rbcL-containg cpDNA fragment were identified. This interaction might be important for rbcL expression and Rubisco degradation during the course of natural senescence in trees.

Changes in photosynthetic performance and antioxidative strategies during maturation of Norway maple (Acer platanoides L.) leaves.

Different structural and functional changes take place during leaf development. Since some of them are highly connected to oxidative metabolism, regulation of reactive oxygen species (ROS) abundance is required. Most of the reactive oxygen species ROS in plant cells are produced in chloroplasts as a result of highly energetic reactions of photosynthesis. The aim of our study was to examine the changes in concentration of oxidative stress parameters (TBARS - thiobarbituric acid-reacting substances and protein carbonyls) as well as antioxidative strategies during development of maple (Acer platanoides L.) leaves in the light of their enhanced photosynthetic performance. We reveal that biogenesis of the photosynthetic apparatus during maple leaf maturation corresponded with oxidative damage of lipids, but not proteins. In addition, antioxidative responses in young leaves differed from that in older leaves. Young leaves had high values of non-photochemical quenching (NPQ) and catalase (CAT, EC 1.11.1.6) activity which declined during the maturation process. Developing leaves were characterized by an increase in TBARS level, the content of non-enzymatic antioxidants as well as ascorbate peroxidase activity (APX, EC 1.11.1.11), while the content of protein carbonyls decreased with leaf maturation. Fully developed leaves had the highest lipid peroxidation level accompanied by a maximum in ascorbic acid content and superoxide dismutase activity (SOD, EC1.15.1.1). These observations imply completely different antioxidative strategies during leaf maturation enabling them to perform their basic function.

Subcellular distribution of glutathione and cysteine in cyanobacteria.

Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria.

Transcriptional control of photosynthesis genes: the evolutionarily conserved regulatory mechanism in plastid genome function.

Chloroplast sensor kinase (CSK) is a bacterial-type sensor histidine kinase found in chloroplasts--photosynthetic plastids--in eukaryotic plants and algae. Using a yeast two-hybrid screen, we demonstrate recognition and interactions between: CSK, plastid transcription kinase (PTK), and a bacterial-type RNA polymerase sigma factor-1 (SIG-1). CSK interacts with itself, with SIG-1, and with PTK. PTK also interacts directly with SIG-1. PTK has previously been shown to catalyze phosphorylation of plastid-encoded RNA polymerase (PEP), suppressing plastid transcription nonspecifically. Phospho-PTK is inactive as a PEP kinase. Here, we propose that phospho-CSK acts as a PTK kinase, releasing PTK repression of chloroplast transcription, while CSK also acts as a SIG-1 kinase, blocking transcription specifically at the gene promoter of chloroplast photosystem I. Oxidation of the photosynthetic electron carrier plastoquinone triggers phosphorylation of CSK, inducing chloroplast photosystem II while suppressing photosystem I. CSK places photosystem gene transcription under the control of photosynthetic electron transport. This redox signaling pathway has its origin in cyanobacteria, photosynthetic prokaryotes from which chloroplasts evolved. The persistence of this mechanism in cytoplasmic organelles of photosynthetic eukaryotes is in precise agreement with the CoRR hypothesis for the function of organellar genomes: the plastid genome and its primary gene products are Co-located for Redox Regulation. Genes are retained in plastids primarily in order for their expression to be subject to this rapid and robust redox regulatory transcriptional control mechanism, whereas plastid genes also encode genetic system components, such as some ribosomal proteins and RNAs, that exist in order to support this primary, redox regulatory control of photosynthesis genes. Plastid genome function permits adaptation of the photosynthetic apparatus to changing environmental conditions of light quantity and quality.

Abrupt telomere shortening in normal human fibroblasts.

Aging is one of the most basic properties of living organisms. Abundant evidence supports the idea that cell senescence underlies organismal aging in higher mammals. Therefore, examining the molecular mechanisms that control cell and replicative senescence is of great interest for biology and medicine. Several discoveries strongly support telomere shortening as the main molecular mechanism that limits the growth of normal cells. Although cultures gradually approach their growth limit, appearance of individual senescent cells is sudden and stochastic. A theoretical model of abrupt telomere shortening has been proposed in order to explain this phenomenon, but until now there was no reliable experimental evidence supporting this idea. Here, we have employed novel methodology to provide evidence for the generation of extrachromosomal circular telomeric DNA as a result of abrupt telomere shortening in normal human fibroblasts. This mechanism ensures heterogeneity in growth potential among individual cells, which is crucial for gradual progression of the aging process.

Differential accumulation of plastid preprotein translocon components during spruce (Picea abies L. Karst.) needle development.

We demonstrate that basic components of the plastid protein-import apparatus originally found in pea, Toc34, Toc159, and Tic110, are also conserved in evolutionarily younger gymnosperms. We show that multiple isoforms of the preprotein receptor Toc34 differentially accumulate in various stages of needle development, while the amounts of Toc159 drastically decrease during chloroplast morphogenesis. Spruce Toc34 and Toc159 receptors are able to recognise and interact with the angiosperm precursor of the Rubisco small subunit. Young proplastids found in closed buds contain a highly elevated number of protein translocation complexes equipped with only two types of outer envelope receptors, Toc159 and a 30-kDa Toc34-related protein. Photosystem II (PSII) can already be assembled in a fully functional complex at this very early stage of needle development, suggesting that no additional receptor isoforms are needed for translocation of all necessary PSII components. We conclude that the accumulation of evolutionarily conserved plastid preprotein translocation components is differentially regulated during spruce needle development.