POG OP 6.1:
The different ways in which plants (try to) keep a happy mitochondrial pool
Prof. Olivier Van Aken | Lund University | Sweden
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.
POG OP 6.2:
Mitochondrial retrograde signaling regulates hypoxia tolerance in rice
Prof. Dr. Su-May Yu | Academia Sinica | Taiwan
Mitochondria, as the powerhouse in eukaryotic cells, are affected by various biotic and abiotic stresses. Our recent studies indicate that maintaining functional and flexible mitochondrial metabolisms via retrograde regulation of nuclear genes facilitates plant adaption to stressful environments. Alternative oxidase (AOX) is a non-ATP generating terminal oxidase in the plant mitochondrial electron transport chain. We showed that AOX expression is induced and it functions in mitochondria for metabolic and signaling homeostasis in response to hypoxia in rice. We observed that H2O2 activates the expression of AOX and genes encoding enzymes involved in H2O2 homeostasis in mitochondria, as well as genes that regulate sugar production, glycolysis, and ethylene biosynthesis essential for rice seedling development under hypoxia/submergence. We also identified many protein kinases and transcription factors that could be involved in hypoxia signaling. We demonstrated that AOX is necessary and sufficient for germination and seedling development, as well as H2O2 accumulation, in rice under both aerobic and anaerobic conditions. Moreover, ectopic expression of AOX1 enhances root aerenchyma development and lateral root emergence that enhance root system under submergence, as well as photosynthesis rates and grain yield in the field under regular and reduced irrigation conditions. Our discoveries reveal new insights into a unique regulatory mechanism for maintaining ROS homeostasis and regulating seedling development in rice under submergence, and highlight genes involved in ROS homeostasis as potential targets for crop improvement.
Chloroplasts lacking class I glutaredoxins – Unraveling the function of GRXC5 in Physcomitrium patens
Finja Bohle* | Department of Biology, Technical University Kaiserslautern | Germany
Oxidative stress is known to induce post-translational protein modifications on cysteines such as protein S-glutathionylation. Protein S-glutathionylation is reversible and catalysed by class I glutaredoxins (GRX) belonging to a family of small oxidoreductases. Four classes of GRX are described so far, based on the active site motif. Plastids contain members of class I (GRXC5, GRXS12) and class II glutaredoxins. Class I glutaredoxins are known to be involved in protein (de)-glutathionylation while class II glutaredoxins play a role in iron sulfur cluster coordination. Phylogenetic analysis revealed that GRXC5 is the ancestral isoform and the only plastidial class I glutaredoxin in Physcomitrium patens. However, the exact function and impact of class I glutaredoxins on plastid redox processes in vivo is still unknown. Here we show that P. patens plants lacking plastid class I glutaredoxin are still viable and show alterations in stromal redox dynamics. We generated knock-out lines of GRXC5 in P. patens and introduced plastid-targeted redox-sensitive GFP2 (roGFP2) as model target for protein S-glutathionylation, into WT and mutant background (∆grxc5). Using plate-reader based fluorometry assays, we found altered light-dependent roGFP2 dynamics compared to WT. Moreover, after oxidative challenge, the ability to deglutathionylate stromal roGFP2 was largely impaired in ∆grxc5 while plant growth under control and tested abiotic stress conditions was not distinguishable from WT. Our results suggest that P. patens ∆grxc5 plants can maintain growth without glutaredoxins catalyzing protein deglutathionylation in plastids under the tested conditions, even though removal of S-glutathionylation is retarded upon stress. Future challenges include to identify GRXC5 target proteins and to further assess S-glutathionylation dynamics in vivo.
Redox properties of the plant atypical thioredoxin DCC1
Linda De Bont | Université de Lorraine, INRAE, IAM, F-54000 Nancy, France
The redox state of cysteinyl residues is mainly controlled by proteins such as thioredoxins, glutaredoxins and protein-disulfide isomerases (Zaffagnini et al., 2019). These thiol-disulfide oxidoreductases are present in all kingdoms of life and carry a common thioredoxin-fold. They typically use a pair of reactive cysteines present in a CXXC motif to perform thiol–disulfide exchange reactions and control the activity or folding of their target proteins. Many uncharacterized proteins possess one or several conserved CXXC motif. In Arabidopsis thaliana and other terrestrial plants, three proteins are formed by a DUF393 domain (Pfam database), that includes a conserved N-terminal DXXCXXC motif, which led to the name DCC proteins (Ginalski et al., 2004). Some representatives are also present in algae and some archaea and bacteria. The DCC proteins are predicted to adopt a thioredoxin-fold, but with a C-terminal extension. In order to characterize the biochemical and structural properties of these atypical thioredoxins, the sequence encoding the mature form of Arabidopsis thaliana and Populus trichocarpa DCC1 proteins were expressed as recombinant proteins in E. coli, the proteins purified and their redox characteristics analyzed, .i.e. activity profiling, susceptibility to oxidative modifications, interactions with other oxidoreductases.
Zaffagnini M, Fermani S, Marchand CH, Costa A, Sparla F, Rouhier N, Geigenberger P, Lemaire SD, Trost P. Redox Homeostasis in Photosynthetic Organisms: Novel and Established Thiol-Based Molecular Mechanisms. Antioxid Redox Signal. 2019 Jul 20;31(3):155-210.
Ginalski K, Kinch L, Rychlewski L, Grishin NV. DCC proteins: a novel family of thiol-disulfide oxidoreductases. Trends Biochem Sci. 2004 Jul;29(7):339-42.
An interactomics and chemical genomics-based approach for the elucidation of mitochondrial retrograde signaling pathways
Prof Dr Inge De Clercq | VIB-UGent Center for Plant Systems Biology, Ghent University | Belgium
We have previously identified a mitochondria-to-nucleus signaling pathway mediated by the NAC transcription factors ANAC013 and ANAC017 that are anchored to the ER membranes under basal conditions. Upon stresses that perturb the mitochondrial and chloroplastic function, these NAC transcription factors are proteolytically released from the ER membranes and translocated to the nucleus. In the nucleus, ANAC013/17 regulate (oxidative) stress responsive gene expression to protect plant cells against organellar-induced oxidative stresses. However, little is known on how the activity of these NAC transcription factors is controlled at the posttranslational level. To identify regulatory proteins, including proteases that cleave the NACs at their transmembrane domain, we apply different approaches based on interactomics, proximity labelling and chemical genomics using ANAC013 and ANAC017 as baits. We identified a novel interactor that is essential for the ANAC013/17’s function in retrograde signaling and defense against organellar-induced oxidative stress.