ANAC102 predominantly expresses a nuclear protein and acts as a negative regulator of methyl viologen-induced retrograde signaling.

Plants, being sessile organisms, constantly need to respond to environmental stresses, often leading to the accumulation of reactive oxygen species (ROS). While ROS can be harmful, they also act as messengers guiding plant growth and stress responses. Because chloroplasts are sensitive to environmental changes and are both a source and target of ROS during stress conditions, they are important in conveying environmental changes to the nucleus, where acclimation responses are coordinated to maintain organellar and overall cellular homeostasis. ANAC102 has previously been established as a regulator of ß-cyclocitral-mediated chloroplast-to-nucleus signaling, protecting plants against photooxidative stress. However, debates persist about where ANAC102 is located - in chloroplasts or in the nucleus. Our study, utilizing the genomic ANAC102 sequence driven by its native promoter, establishes ANAC102 primarily as a nuclear protein, lacking a complete N-terminal chloroplast-targeting peptide. Moreover, our research reveals the sensitivity of plants overexpressing ANAC102 to severe superoxide-induced chloroplast oxidative stress. Transcriptome analysis unraveled ANAC102's dual role in negatively and positively regulating genome-wide transcriptional responses to chloroplast oxidative stress. Through the integration of published data and our own study, we constructed a comprehensive transcriptional network, which suggests that ANAC102 exerts direct and indirect control over transcriptional responses through downstream transcription factor networks, providing deeper insights into the ANAC102-mediated regulatory landscape during oxidative stress.

FRIENDLY is required for efficient dark-induced mitophagy and controlled senescence in Arabidopsis.

Mitochondria play essential roles in plant metabolism, supporting both development and stress responses. To maintain a healthy pool of mitochondria, several quality control systems are in place. Selected degradation of mitochondria by autophagy -mitophagy- has been extensively studied in yeast and animals, but information on mitophagy components in plants is limited. The 'Friendly Mitochondria' (FRIENDLY; FMT) protein, homologous to 'clustered mitochondria protein homolog' CLU in animals, was recently suggested to mediate mitophagy of depolarized mitochondria. In this study, we evaluated the role of FMT in carbon starvation- and dark senescence-induced mitophagy in Arabidopsis. Using mitophagy flux assays, we show that loss of FMT results in decreased mitophagy during dark-induced senescence. Mitophagy induced by inhibition of Target of Rapamycin (TOR) signalling is also partially impaired in fmt mutants. The FMT protein is mostly localised in the cytosol, but we show that during darkness FMT is redistributed into speckles, some of which associate with mitochondria. Fmt mutants were initially identified for their abnormal mitochondrial morphology, with mitochondria often forming clusters. We found that dark senescence strongly increases the number and size of mitochondrial clusters in fmt mutants. In agreement with a role for FMT in mitophagy, we show that fmt mutants show accelerated senescence phenotypes comparable to autophagy 11 (atg11) mutants, but less prominent than in atg5 mutants. Furthermore, FMT prevents excessive dark-induced cell death and hydrogen peroxide production, and supports mitophagy and greening in etiolated seedlings. Our findings thus indicate that FMT contributes to mitophagy and provide evidence that mitophagy is required for controlled senescence and prevention of accelerated cell death. We propose that FMT mediates efficient mitophagy by preventing mitochondrial clustering, thereby allowing mitochondria to be captured more effectively by autophagosomes, rather than by acting as a direct mitophagy receptor.

Carbon starvation, senescence and specific mitochondrial stresses, but not nitrogen starvation and general stresses, are major triggers for mitophagy in Arabidopsis.

Selective degradation of mitochondria by autophagy (mitophagy) is thought to play an important role in mitochondrial quality control, but our understanding of which conditions induce mitophagy in plants is limited. Here, we developed novel reporter lines to monitor mitophagy in plants and surveyed the rate of mitophagy under a wide range of stresses and developmental conditions. Especially carbon starvation induced by dark-incubation causes a dramatic increase in mitophagy within a few hours, further increasing as dark-induced senescence progresses. Natural senescence was also a strong trigger of mitophagy, peaking when leaf yellowing became prominent. In contrast, nitrogen starvation, a trigger of general autophagy, does not induce strong increases in mitophagy. Similarly, general stresses such as hydrogen peroxide, heat, UV-B and hypoxia did not appear to trigger substantial mitophagy in plants. Additionally, we exposed plants to inhibitors of the mitochondrial electron transport chain, mitochondrial translation and protein import. Although short-term treatments did not induce high mitophagy rates, longer term exposures to uncoupling agent and inhibitors of mitochondrial protein import/translation could clearly increase mitophagic flux. These findings could further be confirmed using confocal microscopy. To validate that mitophagy is mediated by the autophagy pathway, we showed that mitophagic flux is abolished or strongly decreased in atg5/AuTophaGy 5 and atg11 mutants, respectively. Finally, we observed high rates of mitophagy in etiolated seedlings, which remarkably was completely repressed within 6 h after light exposure. In conclusion, we propose that dark-induced carbon starvation, natural senescence and specific mitochondrial stresses are key triggers of mitophagy in plants.Abbreviations: AA: antimycin A; ATG: AuToPhagy related; ConA: concanamycin A; DIS: dark-induced senescence; Dox: doxycycline; FCCP: carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; GFP: green fluorescent protein; IDH1: isocitrate dehydrogenase 1; MB: MitoBlock-6; Mito-GFP: transgenic Arabidopsis line expressing a mitochondrially targeted protein fused to GFP; mtETC: mitochondrial electron transport chain; OXPHOS: oxidative phosphorylation; PQC: protein quality control; TOM20: Translocase of Outer Membrane 20.

Selective 13C labelling reveals the electronic structure of flavocoenzyme radicals.

Flavocoenzymes are nearly ubiquitous cofactors that are involved in the catalysis and regulation of a wide range of biological processes including some light-induced ones, such as the photolyase-mediated DNA repair, magnetoreception of migratory birds, and the blue-light driven phototropism in plants. One of the factors that enable versatile flavin-coenzyme biochemistry and biophysics is the fine-tuning of the cofactor's frontier orbital by interactions with the protein environment. Probing the singly-occupied molecular orbital (SOMO) of the intermediate radical state of flavins is therefore a prerequisite for a thorough understanding of the diverse functions of the flavoprotein family. This may be ultimately achieved by unravelling the hyperfine structure of a flavin by electron paramagnetic resonance. In this contribution we present a rigorous approach to obtaining a hyperfine map of the flavin's chromophoric 7,8-dimethyl isoalloxazine unit at an as yet unprecedented level of resolution and accuracy. We combine powerful high-microwave-frequency/high-magnetic-field electron-nuclear double resonance (ENDOR) with 13C isotopologue editing as well as spectral simulations and density functional theory calculations to measure and analyse 13C hyperfine couplings of the flavin cofactor in DNA photolyase. Our data will provide the basis for electronic structure considerations for a number of flavin radical intermediates occurring in blue-light photoreceptor proteins.

Intracellular Protein-Lipid Interactions Studied by Rapid-Scan Electron Paramagnetic Resonance Spectroscopy.

Protein-membrane interactions play key roles in essential cellular processes; studying these interactions in the cell is a challenging task of modern biophysical chemistry. A prominent example is the interaction of human α-synuclein (αS) with negatively charged membranes. It has been well-studied in vitro, but in spite of the huge amount of lipid membranes in the crowded environment of biological cells, to date, no interactions have been detected in cells. Here, we use rapid-scan (RS) electron paramagnetic resonance (EPR) spectroscopy to study αS interactions with negatively charged vesicles in vitro and upon transfection of the protein and lipid vesicles into model cells, i.e., oocytes of Xenopus laevis. We show that protein-vesicle interactions are reflected in RS spectra in vitro and in cells, which enables time-resolved monitoring of protein-membrane interaction upon transfection into cells. Our data suggest binding of a small fraction of αS to endogenous membranes.

Rapid-Scan Electron Paramagnetic Resonance of Highly Resolved Hyperfine Lines in Organic Radicals.

X-band (ca. 9 GHz) fluid solution rapid-scan electron paramagnetic resonance spectra are reported for radicals with multiline spectra and resolution of hyperfine lines as narrow as 30 mG. Highly-resolved spectra of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy, diphenylnitroxide, galvinoxyl, and perylene cation radical with excellent signal-to-noise are shown, demonstrating the capabilities of the rapid-scan technique to characterize very small, well-resolved hyperfine couplings. To acquire high resolution spectra the signal bandwidth must be less than the resonator bandwidth. Signal bandwidth is inversely proportional to linewidth and proportional to scan rate. Resonator bandwidth is inversely proportional to resonator Q. Proper selection of scan rate and resonator Q is needed to achieve resolution of closely-spaced narrow EPR lines.

The transcription factor ANAC017 is a key regulator of mitochondrial proteotoxic stress responses in plants.

Impaired mitochondrial translation or reduced mitochondrial protein import can lead to imbalances in mitochondrial protein composition. Such mitochondrial proteotoxic stresses can trigger a nuclear transcriptional response commonly described as the mitochondrial unfolded protein response (UPRmt). Despite extensive studies of UPRmt pathways in animal and fungal systems, very little is known about how the UPRmt is regulated in plants. Through comparison of Arabidopsis thaliana whole-genome transcriptome data, it was found that most genes induced by mitochondrial ribosome inhibitor doxycycline are also induced by Complex III inhibitor antimycin A. We demonstrate that transcriptional responses to a wide range of mitochondrial proteotoxic stress-triggers are regulated by the transcription factor ANAC017, which was shown to reside in the endoplasmic reticulum (ER). By contrast, no consistent evidence was found for genes that are specifically induced by doxycycline but not antimycin A. Furthermore, ANAC017 gain- and loss-of-function mutants showed marked resistance or susceptibility, respectively, to mitochondrial stress-inducing treatments, demonstrating the physiological importance of ANAC017 during mitochondrial proteotoxic stress. Finally, it was shown that ethylene signalling promotes mitochondria-to-nucleus signalling, most likely independently of ANAC017. Overall, this study shows that in plants, the UPRmt is largely overlapping with, and perhaps identical to, 'classical' mitochondrial retrograde signalling, and is mediated by ER-anchored transcription factor ANAC017. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Distance measurements in the F0F1-ATP synthase from E. coli using smFRET and PELDOR spectroscopy.

Fluorescence resonance energy transfer in single enzyme molecules (smFRET, single-molecule measurement) allows the measurement of multicomponent distance distributions in complex biomolecules similar to pulsed electron-electron double resonance (PELDOR, ensemble measurement). Both methods use reporter groups: FRET exploits the distance dependence of the electric interaction between electronic transition dipole moments of the attached fluorophores, whereas PELDOR spectroscopy uses the distance dependence of the interaction between the magnetic dipole moments of attached spin labels. Such labels can be incorporated easily to cysteine residues in the protein. Comparison of distance distributions obtained with both methods was carried out with the H+-ATPase from Escherichia coli (EF0F1). The crystal structure of this enzyme is known. It contains endogenous cysteines, and as an internal reference two additional cysteines were introduced (EF0F1-γT106C-εH56C). These positions were chosen to allow application of both methods under optimal conditions. Both methods yield very similar multicomponent distance distributions. The dominating distance distribution (> 50%) is due to the two cysteines introduced by site-directed mutagenesis and the distance is in agreement with the crystal structure. Two additional distance distributions are detected with smFRET and with PELDOR. These can be assigned by comparison with the structure to labels at endogenous cysteines. One additional distribution is detected only with PELDOR. The comparison indicates that under optimal conditions smFRET and PELDOR result in the same distance distributions. PELDOR has the advantage that different distributions can be obtained with ensemble measurements, whereas FRET requires single-molecule techniques.

The retrograde signaling protein GUN1 regulates tetrapyrrole biosynthesis.

The biogenesis of the photosynthetic apparatus in developing seedlings requires the assembly of proteins encoded on both nuclear and chloroplast genomes. To coordinate this process there needs to be communication between these organelles, but the retrograde signals by which the chloroplast communicates with the nucleus at this time are still essentially unknown. The Arabidopsis thaliana genomes uncoupled (gun) mutants, that show elevated nuclear gene expression after chloroplast damage, have formed the basis of our understanding of retrograde signaling. Of the 6 reported gun mutations, 5 are in tetrapyrrole biosynthesis proteins and this has led to the development of a model for chloroplast-to-nucleus retrograde signaling in which ferrochelatase 1 (FC1)-dependent heme synthesis generates a positive signal promoting expression of photosynthesis-related genes. However, the molecular consequences of the strongest of the gun mutants, gun1, are poorly understood, preventing the development of a unifying hypothesis for chloroplast-to-nucleus signaling. Here, we show that GUN1 directly binds to heme and other porphyrins, reduces flux through the tetrapyrrole biosynthesis pathway to limit heme and protochlorophyllide synthesis, and can increase the chelatase activity of FC1. These results raise the possibility that the signaling role of GUN1 may be manifested through changes in tetrapyrrole metabolism, supporting a role for tetrapyrroles as mediators of a single biogenic chloroplast-to-nucleus retrograde signaling pathway.

A Simple Method for Quantification of Protochlorophyllide in Etiolated Arabidopsis Seedlings.

Etiolated seedlings accumulate the chlorophyll biosynthesis intermediate protochlorophyllide (Pchlide) and measuring Pchlide can be important for characterizing photomorphogenic mutants that may be affected in chloroplast development. In this chapter we outline a simple and sensitive method for quantifying Pchlide in extracts of Arabidopsis seedlings using fluorescence spectroscopy. This method can be easily adapted to study chloroplast development in a wide range of plant species.

Global analysis of complex PELDOR time traces.

Pulsed electron-electron double resonance (PELDOR, alternatively called DEER for double electron-electron resonance) pulse sequences allow for the detection of echo decay curves that are modulated by dipole-dipole-coupling frequencies of interacting electron spins. With increasing distance between them, the echo decay needs to be monitored over a progressively extended time period. However, since the echo intensity typically falls off exponentially with increasing time, this might be problematic with respect to the minimum signal-to-noise ratio required for a sound data analysis. In this contribution we present the new PELDOR analysis tool GloPel (Global analysis of PELDOR data), an open-source Python-based application, that allows to extract improved-quality distance distributions from PELDOR data for which no ideal signal-to-noise ratio can be achieved for a very long observation window. By using Tikhonov regularization, GloPel allows for the simultaneous analysis of two time traces acquired for a sample in two different observation time windows, thus taking advantage of both, the typically high signal-to-noise ratio of the time trace acquired at early times of the echo decay, and the best possible background function fitted for the decay at later times, which is in most cases superimposed with considerable noise. In this way, short distances are not overseen in the higher noise of the longer time traces while long distances are not artificially shortened by limiting the observation time window of the experiment. Following our suggested data acquisition procedure, a significant reduction of the measurement time may also be achieved.

Synthesis, Characterisation and Reactions of Truly Cationic NiI -Phosphine Complexes.

The recently published purely metallo-organic NiI salt [Ni(cod)2 ][Al(ORF )4 ] (1, cod=1,5-cyclooctadiene, RF =C(CF3 )3 ) provides a starting point for a new synthesis strategy leading to NiI phosphine complexes, replacing cod ligands by phosphines. Clearly visible colour changes indicate reactions within minutes, while quantum chemical calculations (PBE0-D3(BJ)/def2-TZVPP) approve exergonic reaction enthalpies in all performed ligand exchange reactions. Hence, [Ni(dppp)2 ][Al(ORF )4 ] (2, dppp=1,3-bis(diphenylphosphino)propane), [Ni(dppe)2 ][Al(ORF )4 ] (3, dppe=1,3-bis(diphenyl-phosphino)ethane), three-coordinate [Ni(PPh3 )3 ][Al(ORF )4 ] (4) and a remarkable two-coordinate NiI phosphine complex [Ni(PtBu3 )2 ][Al(ORF )4 ] (5) were characterised by single crystal X-ray structure analysis. EPR studies were performed, confirming a nickel d9 -configuration in complexes 2, 4 and 5. This result is supported by additional magnetization measurements of 4 and 5. Further investigations by cyclic voltammetry indicate relatively high oxidation potentials for these NiI compounds between 0.7 and 1.7 V versus Fc/Fc+ . Screening reactions with O2 and CO gave first insights on the reaction behaviour of the NiI phosphine complexes towards small molecules with formation of mixed phosphine-CO-NiI complexes and oxidation processes yielding new NiI and/or NiII derivatives. Moreover, 4 reacted with CH2 Cl2 at RT to give a dimeric NiII ylide complex (4 c). As CH2 Cl2 is a rather stable alkyl halide with relatively high C-Cl bond energies, 4 appears to be a suitable reagent for more general C-Cl bond activation reactions.

Intersubunit distances in full-length, dimeric, bacterial phytochrome Agp1, as measured by pulsed electron-electron double resonance (PELDOR) between different spin label positions, remain unchanged upon photoconversion.

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion.

Mechanism of the Ti(III)-Catalyzed Acyloin-Type Umpolung: A Catalyst-Controlled Radical Reaction.

The titanium(III)-catalyzed cross-coupling between ketones and nitriles provides an efficient stereoselective synthesis of α-hydroxyketones. A detailed mechanistic investigation of this reaction is presented, which involves a combination of several methods such as EPR, ESI-MS, X-ray, in situ IR kinetics, and DFT calculations. Our findings reveal that C-C bond formation is turnover-limiting and occurs by a catalyst-controlled radical combination involving two titanium(III) species. The resting state is identified as a cationic titanocene-nitrile complex and the beneficial effect of added Et3N·HCl on yield and enantioselectivity is elucidated: chloride coordination initiates the radical coupling. The results are fundamental for the understanding of titanium(III)-catalysis and of relevance for other metal-catalyzed radical reactions. Our conclusions might apply to a number of reductive coupling reactions for which conventional mechanisms were proposed before.

[Ni(cod)2][Al(OR(F))4], a Source for Naked Nickel(I) Chemistry.

The straightforward synthesis of the cationic, purely organometallic Ni(I) salt [Ni(cod)2](+)[Al(OR(F))4](-) was realized through a reaction between [Ni(cod)2] and Ag[Al(OR(F))4] (cod = 1,5-cyclooctadiene). Crystal-structure analysis and EPR, XANES, and cyclic voltammetry studies confirmed the presence of a homoleptic Ni(I) olefin complex. Weak interactions between the metal center, the ligands, and the anion provide a good starting material for further cationic Ni(I) complexes.

Role of phytochromes A and B in the regulation of cell death and acclimatory responses to UV stress in Arabidopsis thaliana.

Plants coordinate their responses to various biotic and abiotic stresses in order to optimize their developmental and acclimatory programmes. The ultimate response to an excessive amount of stress is local induction of cell death mechanisms. The death of certain cells can help to maintain tissue homeostasis and enable nutrient remobilization, thus increasing the survival chances of the whole organism in unfavourable environmental conditions. UV radiation is one of the environmental factors that negatively affects the photosynthetic process and triggers cell death. The aim of this work was to evaluate a possible role of the red/far-red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) and their interrelations during acclimatory responses to UV stress. We showed that UV-C treatment caused a disturbance in photosystem II and a deregulation of photosynthetic pigment content and antioxidant enzymes activities, followed by increased cell mortality rate in phyB and phyAB null mutants. We also propose a regulatory role of phyA and phyB in CO2 assimilation, non-photochemical quenching, reactive oxygen species accumulation and salicylic acid content. Taken together, our results suggest a novel role of phytochromes as putative regulators of cell death and acclimatory responses to UV.

Strategy for Enhancement of (13)C-Photo-CIDNP NMR Spectra by Exploiting Fractional (13)C-Labeling of Tryptophan.

The photo-CIDNP effect has proven to be useful to strongly enhance NMR signals of photochemically active proteins simply by irradiation with light. The evolving characteristic patterns of enhanced absorptive and emissive NMR lines can be exploited to elucidate the photochemistry and photophysics of light-driven protein reactions. In particular, by the assignment of (13)C NMR resonances, redox-active amino acids may be identified and thereby electron-transfer pathways unraveled, in favorable cases, even with (13)C at natural abundance. If signal enhancement is weak, uniform (13)C isotope labeling is traditionally applied to increase the signal strength of protein (13)C NMR. However, this typically leads to cross relaxation, which transfers light-induced nuclear-spin polarization to adjacent (13)C nuclei, thereby preventing an unambiguous analysis of the photo-CIDNP effect. In this contribution, two isotope labeling strategies are presented; one leads to specific but ubiquitous (13)C labeling in tryptophan, and the other is based on fractional isotope labeling affording sets of isotopologs with low probability of next-neighbor isotope accumulation within individual tryptophan molecules. Consequently, cross relaxation is largely avoided while the signal enhancement by (13)C enrichment is preserved. This results in significantly simplified polarization patterns that are easier to analyze with respect to the generation of light-generated nuclear-spin polarization.

One protein, two chromophores: comparative spectroscopic characterization of 6,7-dimethyl-8-ribityllumazine and riboflavin bound to lumazine protein.

We investigated the lumazine protein from Photobacterium leiognathi in complex with its biologically active cofactor, 6,7-dimethyl-8-ribityllumazine, at different redox states and compared the results with samples containing a riboflavin cofactor. Using anaerobic photoreduction, we were able to record optical absorption kinetics from both cofactors in similar protein environments. It could be demonstrated that the protein is able to stabilize a neutral ribolumazine radical with ∼35% yield. The ribolumazine radical state was further investigated by W-band continuous-wave EPR and X-band pulsed ENDOR spectroscopy. Here, both the principal values of the g-tensor and an almost complete mapping of the proton hyperfine couplings (hfcs) could be obtained. Remarkably, the g-tensor's principal components are similar to those of the respective riboflavin-containing protein; however, the proton hfcs show noticeable differences. Comparing time-resolved optical absorption and fluorescence data from ribolumazine-containing samples, solely fluorescence but no signs of any intermediate radical or a triplet state could be identified. This is in contrast to lumazine protein samples containing the riboflavin cofactor, for which a high yield of the photogenerated triplet state and some excited flavin radical could be detected using time-resolved spectroscopy. These results clearly demonstrate that ribolumazine is a redox-active molecule and could, in principle, be employed as a cofactor in other enzymatic reactions.