Arabidopsis transcription factor ANAC102 predominantly expresses a nuclear protein and acts as a negative regulator of methyl viologen-induced oxidative stress responses.

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 second messengers guiding plant growth and stress responses. Because chloroplasts are sensitive to environmental changes and are both a source and a 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 a dual role of ANAC102 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.

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.

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.

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'.

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.