POG OP 5.1:
New insights into the redox regulation of autophagy in the single-cell microalga Chlamydomonas reinhardtii
Dr. M. Esther Perez-Perez | Consejo Superior de Investigaciones Científicas (CSIC) | Spain
Autophagy is a major catabolic process by which eukaryotic cells degrade and recycle intracellular material including protein aggregates or dysfunctional organelles to maintain cellular homeostasis and to cope with stress. A hallmark of autophagy is the formation of autophagosomes, double-membrane vesicles in which the cargo that will be finally degraded in the vacuole is engulfed. The ATG8 lipidation system, which includes the ubiquitin-like ATG8 protein, the ATG4 protease and the E1- and E2-activating enzymes ATG7 and ATG3, is essential for autophagosome formation since these proteins catalyze the conjugation of ATG8 to the lipid phosphatidylethanolamine. ATG8 lipidation has been widely used as a molecular autophagy marker in many eukaryotes, including the model microalga Chlamydomonas reinhardtii. Our previous studies have demonstrated that autophagy is a redox-regulated process in Chlamydomonas. Indeed, there is a strong connection between different ROS-generating stress conditions and the activation of autophagy in this microalga. We have previously shown that ATG4 is regulated by the intracellular redox potential through the formation of a disulfide bond. At present, we are investigating whether other ATG proteins respond to redox signals and the underlying mechanism of this redox regulation. Moreover, we are analyzing an atg8 mutant strain under ROS-generating and autophagy-activating conditions. Our results indicate that this mutant is hypersensitive to oxidative stress an displays an altered global response to chloroplast damage in Chlamydomonas.
POG OP 5.2:
Ascorbate peroxidase 2 of Chlamydomonas reinhardtii is involved in the regulation of the plastocyanin levels
Anna Caccamo | InBios/Phytosystem, University of Liège | Belgium
In the green microalga Chlamydomonas reinhardtii, APX2 is one of the four ascorbate peroxidase isoforms. These H2O2–scavenging enzymes use ascorbate for the reduction of H2O2. APX2 from C. reinhardtii and APX6 from A. thaliana, its orthologous, belong to a new class, named Ascorbate Peroxidase-Related (APX-R). The APX-R enzymes lack the essential amino acids to bind ascorbate and in vitro studies confirmed that AtAPX6 does not bind ascorbate, but several aromatic compounds . In silico analyses showed that APX2 might reside in the lumen of the thylakoid. However, no differences were observed during growth in null apx2 mutants. The photosynthetic activity at increasing light intensities was only impacted when apx2 mutant cells were grown under phototrophic condition in low light. This was accompanied by a faster P700 oxidation upon a sudden increase of light and a slower re-reduction rate, a phenotype observed under all tested growth conditions. Furthermore, no H2O2 increase was detected in the apx2 mutants when they were transferred from low light to high light, suggesting that the lower photosynthetic activity would rather be due to regulation at the intersystem electron carriers than to oxidative stress damage. We then analysed soluble extracts with spectroscopic and mass spectrometry techniques, which showed a reduced levels of PC and the presence of the pre-apo-plastocyanin form in the apx2 mutants. In addition, a functional replacement of PC by cytochrome c6 under Cu2+ deficient conditions restored the wild-type phenotype of the electron transport to Photosystem I, confirming a specific role of APX2 on PC. Additionally, predicted structures with AlphaFold2 indicated a high probability of an interacting complex APX2:PC.
Our results suggest that APX2 might be involved in the regulation of the PC levels, which questions the role of APX2 during photosynthesis.
Funded by FNRS-FWO EOS Project 30829584
 Lazzarotto et al., (2021). Antioxidants, 10(1):65
Influence of nitrogen availability and form on sensitivity to heat and light stress in coral endosymbionts
Dr Stephane Roberty | University of Liège, Liège, Belgium | Belgium
The trophic and structural foundations of coral reef ecosystems rely on the mutualistic relationship existing between Scleractinia and dinoflagellates of the Family Symbiodiniaceae. In this endosymbiosis, the photosynthetic algae reside in a symbiosome within the gastrodermal cells of the coral host, allowing reciprocal and complimentary transfers of highly energetic compounds and efficient recycling of growth-limiting nutrients in an oligotrophic environment.
Coral reef cover is declining globally because of anthropogenic stresses. Among these, high sea surface temperature accompanied by high levels of solar irradiance are known to cause coral bleaching, a phenomenon during which the host loses most of its symbionts and becomes physiologically and nutritionally compromised. Data accumulated to date indicate that the disruption of metabolic homeostasis between host and symbiont and the dysregulation of redox homeostasis are responsible for the collapse of the symbiosis.
Local-scale stressors, such as nutrient loading and sedimentation can also exacerbate the impacts of thermal stress on corals. Although numerous studies have investigated the interactive effects of elevated temperature and nitrogen availability on reef-building corals, results documented to date are inconsistent, with some showing an increase and others a decrease in the susceptibility to bleaching. In this study, we investigated the effects of nitrogen source (no nitrogen, 500 µM NH4+ or 500 µM NO3-) and temperature stress combined with light stress in Symbiodinium microadriaticum, isolated from the coral Stylophora pistillata. After 24h of treatment, we observed a significant impact of light and temperature stress on photosynthesis (lower Fv/Fm and max rETRPSII) and on photoprotective mechanisms (increased DPS of the xanthophylls but lower NPQ). This treatment also significantly increased the intra- and extracellular reactive species production. Nitrogen-deprived cells appeared more sensitive to stress and displayed a higher NPQ, DPS, and production of intracellular reactive species compared to cells grown with NO3- and NH4+.
POG OP 5.4:
Redox-sensitive fluorescent biosensors detect the symbiotic bacteria Sinorhizobium meliloti intracellular redox changes under free-living and symbiotic lifestyles
Prof. Pierre Frendo | Université Côte d'Azur, INRAE, CNRS, ISA, Sophia-Antipolis, France | France
Reactive Oxygen Species such as hydrogen peroxide (H2O2) are key signaling molecules that control the setup and functioning of Rhizobium-Legume symbiosis. This interaction results in the formation of a new organ, the root nodule, in which bacteria enter the host cells and differentiate into atmospheric nitrogen (N2)-fixing bacteroids. The interaction between Sinorhizobium meliloti and Medicago truncatula is a genetic model to study N2-fixing symbiosis. In previous work, several S. meliloti mutants, impaired in antioxidant defense, showed altered symbiotic properties, emphasizing the importance of redox-based regulation in the bacterial partner. However, direct measurements of S. meliloti intracellular redox state have never been performed. Here, we measured dynamic changes of intracellular H2O2 and glutathione redox potential by expressing roGFP2-Orp1 and Grx1-roGFP2 biosensors in S. meliloti. Kinetic analyses of redox changes under free-living conditions showed that these biosensors are suitable to monitor the bacterial redox state in real-time, after H2O2 challenge and in different genetic backgrounds. In planta, flow cytometry and confocal imaging experiments allowed the determination of sensor oxidation state in nodule bacteria. These cellular studies establish the existence of an oxidative shift in the redox status of S. meliloti during bacteroid differentiation, opening up new avenues for in vivo studies of redox dynamics during N2-fixing symbiosis.