Seedlings Lacking the PTM Protein Do Not Show a genomes uncoupled (gun) Mutant Phenotype.

The ptm mutant of Arabidopsis does not show a genomes uncoupled mutant phenotype and PTM is therefore unlikely to function in chloroplast-to-nucleus signaling as previously reported

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

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

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’

Plastid-to-nucleus retrograde signalling during chloroplast biogenesis does not require ABI4

Multiple abi4 alleles fail to show a deficiency in chloroplast-to-nucleus retrograde signalling indicating that, contrary to contemporary models, ABI4 is not a component of this signalling pathway

Light-generated paramagnetic intermediates in BLUF domains

Blue-light sensitive photoreceptory BLUF domains are flavoproteins, which regulate various, mostly stress-related processes in bacteria and eukaryotes. The photoreactivity of the flavin adenine dinucleotide (FAD) cofactor in three BLUF domains from Rhodobacter sphaeroides, Synechocystis sp. PCC 6803 and Escherichia coli have been studied at low temperature using timeresolved electron paramagnetic resonance. Photoinduced flavin triplet states and radical-pair species have been detected on a microsecond time scale. Differences in the electronic structures of the FAD cofactors as reflected by altered zero-field splitting parameters of the triplet states could be correlated with changes in the amino-acid composition of the various BLUF domains’ cofactor binding pockets. For the generation of the light-induced, spin-correlated radical-pair species in the BLUF domain from Synechocystis sp. PCC 6803, a tyrosine residue near the flavin’s isoalloxazine moiety plays a critical role