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Multiple spots of Chlamydomonas reinhardtii cells on agar plates

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Identification and characterization of the mechanisms governing singlet oxygen-induced chloroplast retrograde signalling using model organism Chlamydomonas reinhardtii

Project acronym: ROS signalling

Principal Investigator (PI)

Pawel Brzezowski, Ph.D., Assistant Professor

Curriculum vitae of the PI

  • 2023-present, Assistant Professor, Principal Investigator, Department of Plant Physiology and Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Cracow, Poland

Research dedicated to the mechanisms governing intracellular and singlet oxygen-triggered retrograde signalling. Project title, “Identification and characterization of the mechanisms governing singlet oxygen-induced chloroplast retrograde signalling using model organism Chlamydomonas reinhardtii”

The research is part of the project No. 2022/47/P/NZ1/01947 within the POLONEZ BIS programme co-funded by the National Science Centre and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 945339.

  • 2022-2023, Senior Scientist I, Selvita Services Sp. z o.o., Kraków, Poland
  • 2017-2021, AG Pflanzenphysiologie, Institut für Biologie, Lebenswissenschaftliche Fakultät, Humboldt Universität zu Berlin, Germany, Wissenschaftlicher Mitarbeiter/Postdoctoral researcher
  • 2016-2017, Laboratoire de Bioénergétique et Biotechnologie des Bactéries et Microalgues, Institut de Biosciences et Biotechnologies, Aix-Marseille University, CEA Cadarache, Saint-Paul-lez-Durance, France, Ingénieur Chercheur/Postdoctoral researcher
  • 2012-2015, AG Pflanzenphysiologie, Institut für Biologie, Lebenswissenschaftliche Fakultät, Humboldt Universität zu Berlin, Germany, Wissenschaftlicher Mitarbeiter/Postdoctoral researcher
  • 2006-2012, Doctor of Philosophy, Algal Functional Genomics, Department of Biology, College of Arts and Science, University of Saskatchewan, Saskatoon, SK, Canada, Natural Sciences, Biology
  • 2002-2004, Master of Science, Plant Physiology, Biology, Natural Sciences Department, University of Wroclaw, Poland, Natural Sciences, Biology (Botany, Plant Physiology)
  • 1999-2002, Bachelor of Science, Biology, Natural Sciences Department, University of Wroclaw, Poland, Natural Sciences, Biology

Funding of the project

The logo of the polonez bis programme

This research is part of the project No. 2022/47/P/NZ1/01947 within the POLONEZ BIS programme co-funded by the National Science Centre and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 945339

The flag of the European Union

 

logo of the National Science Centre

Description of the project

Endosymbiotic processes were the defining events in the evolution of the eukaryotic organisms. As current chloroplasts and mitochondria evolved in this course, the development of an efficient communication system between the organelle and nucleus was required. Such signalling system is one of the most crucial factors for any “symbiotic consortium” to function properly. Information exchange between chloroplasts, mitochondria, and nucleus takes place by means of anterograde (“forward”, nucleus-to-organelle) and retrograde (“backward”, organelle-to-nucleus) signalling pathways. This bidirectional communication is necessary for coordination of organelles’ development, function, and adjustments to changing environmental conditions. There is evidence that chloroplasts as well as mitochondria can exert an effect on nuclear gene expression. One of the retrograde signalling pathways was proposed to involve reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2) and singlet oxygen (1O2). Although some progress has been made in deciphering the involvement of H2O2, our knowledge regarding the mechanisms governing 1O2-induced chloroplast retrograde signalling is scarce and fragmentary.

The aim of the project presented here is elucidation of these mechanisms. With the intention to search for the mechanisms responsible for conveying the 1O2 information from the chloroplast to the nucleus, a signalling Reporter (sigRep) strain of Chlamydomonas reinhardtii was produced, which allowed us to monitor the efficiency of the 1O2-signalling in this unicellular organism. Subsequent random mutagenesis performed on sigRep resulted in isolation of a series of mutants disrupted in 1O2-signalling. These mutants were named genomes uncoupled Singlet Oxygen Signalling (gunSOS). We discovered that one of these mutants, gunSOS1, showed significantly altered expression of several genes, followed by altered content of certain metabolites. Among metabolites accumulating in gunSOS1 relative to sigRep, fumarate is a well-recognized oncometabolite in mammalian cells, associated with development of tumors. 2-oxoglutarate was shown to regulate expression of enzyme involved in nitrogen assimilation in Nicotiana tabacum, while myo-inositol is a building block for several molecules also involved in the signalling. We decided to test the effect of these metabolites on 1O2-signalling by providing them exogenously to the cell cultures. Fumarate, 2-oxogluatarate, and myo-inositol, always had a concentration-dependent suppressing effect on 1O2-signalling in sigRep, with no change in gunSOS1 and wild type, relative to their respective non-treated controls. Among the metabolites, which showed decreased content in gunSOS1 relative to sigRep were mannose 6-phosphate and glucose 6-phosphate, which are mainly involved in starch and glucose metabolism. Exogenous application of the sugar phosphates had a positive effect on 1O2-signalling in sigRep, but no effect could be observed in gunSOS1 and wild type. However, aconitate was another metabolite deficient in gunSOS1, relative to sigRep. Addition of aconitate to the cell cultures increased the efficiency of the 1O2-signalling in sigRep, but most importantly rescued 1O2-signal transduction in gunSOS1. Furthermore, addition of aconitate rescued low expression of the proteins required for the 1O2-signalling.

Thus, the main goal of the research presented here is to explain the mechanisms behind observed positive effect of aconitate on 1O2-signalling. There is no information in the literature about direct involvement of aconitate in any of the known signalling pathways, but increasing amount of reports points to the role of aconitase in stress responses. Aconitase is an enzyme interconverting citrate and isocitrate via cis-aconitate in the processes taking place in mitochondria. The expression level of aconitase in gunSOS1 was significantly decreased relative to sigRep, but increased expression was observed after addition of aconitate. I hypothesize that not aconitate, but aconitase is the key player in chloroplast 1O2-signalling in C. reinhardtii, while its localization in this organism depends on the redox balance between different compartments of the cell. Aconitase in plants and animals is susceptible to posttranslational modifications. The possibility of such modification and their role in aconitase function and involvement in 1O2-signalling will be examined in C. reinhardtii, by means of modifications of the possible active amino acid residues, followed by biochemical analyses.

The results of the project will expand our understanding of the 1O2-signalling and its role in regulation of growth, development, and response to various stresses via exerted changes on nuclear gene expression. Additionally, because of the apparent cross-talk and dependence of 1O2-signalling on other processes and signalling pathways, it is also expected that our knowledge regarding important signalling systems will also improve, which will open new routes in study of regulation of gene expression, development, and response to various stresses.

Recommended literature

  • Al Youssef, W.A., Feil, R., Saint-Sorny, M., Johnson, X., Lunn, J.E., Grimm, B., and Brzezowski, P. (2023). Singlet oxygen-induced signalling depends on the metabolic status of the Chlamydomonas reinhardtii cell. Communications biology 6, 529.
  • Bradley, D., and Beltrao, P. (2019). Evolution of protein kinase substrate recognition at the active site. PLoS Biol. 17, e3000341.
  • Brzezowski, P., Wilson, K.E., and Gray, G.R. (2012). The PSBP2 protein of Chlamydomonas reinhardtii is required for singlet oxygen-dependent signaling. Planta 236, 1289-1303.
  • Cairo, G., Recalcati, S., Pietrangelo, A., and Minotti, G. (2002). The iron regulatory proteins: targets and modulators of free radical reactions and oxidative damage. Free radical biology & medicine 32, 1237-1243.
  • Castro, L., Tórtora, V., Mansilla, S., and Radi, R. (2019). Aconitases: Non-redox Iron-Sulfur Proteins Sensitive to Reactive Species. Accounts of chemical research 52, 2609-2619.
  • Lushchak, O.V., Piroddi, M., Galli, F., and Lushchak, V.I. (2014). Aconitase post-translational modification as a key in linkage between Krebs cycle, iron homeostasis, redox signaling, and metabolism of reactive oxygen species. Redox Rep 19, 8-15.
  • Meng, X., Li, L., Pascual, J., Rahikainen, M., Yi, C., Jost, R., He, C., Fournier-Level, A., Borevitz, J., Kangasjärvi, S., Whelan, J., and Berkowitz, O. (2022). GWAS on multiple traits identifies mitochondrial ACONITASE3 as important for acclimation to submergence stress. Plant Physiol.
  • Moeder, W., Del Pozo, O., Navarre, D.A., Martin, G.B., and Klessig, D.F. (2007). Aconitase plays a role in regulating resistance to oxidative stress and cell death in Arabidopsis and Nicotiana benthamiana. Plant Mol. Biol. 63, 273-287.
  • Pascual, J., Rahikainen, M., Angeleri, M., Alegre, S., Gossens, R., Shapiguzov, A., Heinonen, A., Trotta, A., Durian, G., Winter, Z., Sinkkonen, J., Kangasjärvi, J., Whelan, J., and Kangasjärvi, S. (2021). ACONITASE 3 is part of the ANAC017 transcription factor-dependent mitochondrial dysfunction response. Plant Physiol. 186, 1859-1877.
  • Shao, N., Duan, G.Y., and Bock, R. (2013). A mediator of singlet oxygen responses in Chlamydomonas reinhardtii and Arabidopsis identified by a luciferase-based genetic screen in algal cells. Plant Cell 25, 4209-4226.
  • Ternette, N., Yang, M., Laroyia, M., Kitagawa, M., O'Flaherty, L., Wolhulter, K., Igarashi, K., Saito, K., Kato, K., Fischer, R., Berquand, A., Kessler, B.M., Lappin, T., Frizzell, N., Soga, T., Adam, J., and Pollard, P.J. (2013). Inhibition of mitochondrial aconitase by succination in fumarate hydratase deficiency. Cell reports 3, 689-700.
  • Wakao, S., Chin, B.L., Ledford, H.K., Dent, R.M., Casero, D., Pellegrini, M., Merchant, S.S., and Niyogi, K.K. (2014). Phosphoprotein SAK1 is a regulator of acclimation to singlet oxygen in Chlamydomonas reinhardtii. eLife 3, e02286.

Project news, seminars, and social media

Project location

Jagiellonian University

The Department of Plant Biochemistry was established in 1965 as one of three units in the newly established Department of Biochemistry and Biophysics. In 1986, the name was changed to the current name, Department of Plant Physiology and Biochemistry.
Department heads:
1965-1971 prof. Ignacy Reifer
1971-1997 prof. dr. hab. Stanisław Więckowski
1997-2017 prof. dr hab. Kazimierz Strzałka
since 2017 prof. Ph.D. Jerzy Kruk

Contact: Department of Plant Physiology and Biochemistry, Faculty of Biochemistry and Biotechnology, Jagiellonian University,  Gronostajowa St. 7, 30-387 Kraków phone: 12 664 65 19

PI Contact details

Pawel Brzezowski, Ph.D., Assistant Professor
Department of Plant Physiology and Biochemistry
Faculty of Biochemistry, Biophysics and Biotechnology
Jagiellonian University
Gronostajowa 7, 30-387 Kraków
Poland
email: pawel.brzezowski@uj.edu.pl, chlamydoplast@gmail.com
tel. +48 12 664 6372
Rm A221 (4.1.23)