Laboratories

Grażyna Dobrowolska, PhD, DSc, Prof.

Laboratory of Plant Protein Phosphorylation

Research Scope

Our laboratory investigates the response of plants to environmental stresses, especially drought, salinity, and extreme temperatures. Our studies focus on annexins and protein kinases that are involved in stress signal transduction pathways. We seek to elucidate the role of these proteins and mechanisms of the regulation of their activity.

Research

Main Scientific Achievements

  • We identified several cellular partners of SnRK2 - their substrates as well as their regulators.
  • We described a novel mechanism of the inhibition of SnRK2 activity by plant-specific calcium-binding protein SCS (SnRK2 interacting calcium sensor).
  • We showed that SnRK2.10 in Arabidopsis mitigates the adverse effects of salinity by protecting photosynthetic machinery.
  • We found that annexin 1 in Arabidopsis is involved in the interplay between light and jasmonate signaling.
  • We identified 23 genes that encode CDPKs in the Solanum tuberosum genome.

Research Description

The main scientific topic of our laboratory is the plant response to environmental stress. Our interest focuses on annexins and protein kinases that are involved in the stress response, mainly the plant-specific SNF1-related kinases type 2 (SnRK2s) and calcium-dependent protein kinases (CDPKs).

  • SnRK2s: mechanisms of regulation of its activity and role in the plant response to abiotic stress

SnRK2s are considered key signaling elements that are involved in plant development and responses to harsh environmental conditions, primarily osmotic stress that is triggered by drought and salinity. Based on phylogenetic analysis, SnRK2s have been classified into three groups. This classification correlates closely with their sensitivity to abscisic acid (ABA). Group 1 consists of SnRK2s that are not activated by ABA. Group 2 consists of kinases that are not or only weakly activated by ABA. Group 3 consists of kinases that are strongly activated by the phytohormone. Numerous data show an important role of ABA-activated SnRK2s in plant acclimation to environmental stress. They are involved in regulation of the expression of various stress-response genes and the activity of several proteins that are engaged in plant tolerance to harmful conditions. The activity of SnRK2s is rigorously controlled in plant cells. The mechanism of the regulation of ABA-activated SnRK2s has been extensively studied. Autophosphorylation or phosphorylation by Raf1-like kinases activates these kinases, whereas clade A phosphoprotein phosphatases 2C (PP2Cs) are their major negative regulators. Significantly less is known about the role and mechanisms of regulation of kinases that belong to group 1 of the SnRK2 family. Therefore, the main scientific interest of our group focuses on ABA-nonactivated SnRK2s. In our research, we use the model plant Arabidopsis thaliana. We found that clade A PP2Cs (ABI1, PP2CA) and okadaic acid-sensitive phosphatases of the PPP family dephosphorylate and inhibit some group 1 SnRK2s. Moreover, we identified a novel negative regulator of SnRK2s that appears to be a plant-specific calcium-binding protein. Our studies of the role of ABA-nonactivated SnRK2s revealed that Arabidopsis SnRK2.4 and SnRK2.10 are involved in regulating plant responses to cadmium and salinity stress through the regulation of reactive oxygen species homeostasis. Our recent results indicate that SnRK2.10 confers plant resistance to salinity by protecting photosynthetic machinery. Using an integrated approach, by combining phosphoproteomic, biochemical, molecular biology, and plant physiology tools, we identified several potential targets of ABA-nonactivated SnRK2s (e.g., dehydrins [ERD10 and ERD14] and RNA- and DNA-binding proteins). We showed that the phosphorylation of ERD14 by SnRK2s impacts its subcellular localization. We are currently studying the phosphorylation of selected RNA- and DNA-binding proteins that are potential SnRK2 targets to establish the role of group 1 SnRK2s in the plant response to different stresses (i.e., salinity, drought, and extreme temperatures) and leaf senescence. In this part, our research focuses on the involvement of SnRK2s in the regulation of gene expression at the transcriptional and posttranscriptional levels.

  • Integration of stress signals in plants: light and osmotic stress perception

This part of our research focuses on the interplay between light perception via photoreceptors and the response to stress factors that evoke osmotic stress. We have been studying the involvement of light and photoreceptors in the plant response to osmotic stress and vice versa and the influence of osmotic stress (and protein kinases that are activated in the response to osmotic stress) on light signaling. We found that light-induced chloroplast movements are inhibited by salt stress, and we are studying the role of SnRK2 in this process. We also study novel roles of PLASTID MOVEMENT IMPAIRED 1 (PMI1), a protein that was initially identified as a regulator of light responses. Our research suggests that PMI1 may play a role in SnRK2-mediated responses.

  • CDPKs: role in stress signaling

Three model plants have been chosen for studies of the role of CDPKs in the stress response: Zea mays, Arabidopsis thaliana, and Solanum tuberosum. Our studies have shown that the maize CDPK ZmCPK11 participates in local and systemic responses to wounds and salinity. Under stress conditions, this kinase regulates the expression of several stress-responsive genes. The activation of ZmCPK11 in wound signaling is mediated by phosphatidic acid that is produced by phospholipase D. Moreover, wound-induced ZmCPK11 transcript levels are regulated by DNA demethylation. ZmCPK11 improves salt tolerance in Arabidopsis by preventing damage to photosystem II and regulating sodium and potassium homeostasis.

Our bioinformatics studies showed the presence of 23 genes that encode CDPKs in the Solanum tuberosum genome. Based on phylogenetic analysis, CDPKs have been divided into four subfamilies. Our studies showed that the identified CDPK genes were expressed in all plant organs but with different expression patterns. The expression of some of them was enhanced in response to abiotic stress (e.g., injury, drought, and salinity) or Phytophthora infestans infection. Our current research on CDPKs focuses on establishing the role of selected potato CDPKs and homologues of ZmCPK11 in Arabidopsis, CPK4 and CPK11, in the plant response to environmental stress, especially P. infestans.

  • Annexins: role in high-light stress signaling

Annexins are the superfamily of calcium and phospholipid binding proteins that are present in nearly all eukaryotes. In plants, their physiological role is still vague. Using comparative transcriptomic profiling we found that in Arabidopsis, annexin 1 is involved in the interplay between light and jasmonate signaling.

Methodology

  • DNA cloning, construction of plasmids, site-directed mutagenesis
  • Production of proteins in bacterial and plant expression systems
  • Protein purification
  • Analysis of proteins, DNA, and RNA by electrophoresis
  • Protein immuno-analysis: Western blot, immunoprecipitation
  • Protein-protein interaction: BiFC, yeast two-hybrid, pull-down assays, co-immunoprecipitation
  • Protein-RNA/DNA interaction: EMSA
  • Gene expression analysis: qPCR
  • Transcriptome analysis: library preparation and data analysis
  • Stable plant transformation, obtaining transgenic and gene-edited Arabidopsis using CRISPR-Cas9
  • Transient transformation of thaliana protoplasts and N. benthamiana leaves
  • Protein phosphorylation: analysis of protein kinase activity in solution and in-gel assays, preparation of samples for LC-MS/MS analysis
  • Analysis of reactive oxygen species and reactive nitrogen species accumulation in plant tissue using various methods and monitoring the activity of enzymes/proteins that are involved in the regulation of reactive oxygen species production and scavenging
  • Analysis of selected metabolites (e.g., proline)
  • Physiological assays of the stress response (e.g., seed germination, root growth, seedling survival, etc.).
  • Equipment: FPLCs systems: AKTA FPLC and NGC Discover 10 Pro (Pharmacia, Bio-Rad), Step One Plus Real-Time PCR System (Applied Biosystems), Backman Ultracentrifuge.

Selected Publications

    • Two SnRK2-Interacting Calcium Sensor Isoforms Negatively Regulate SnRK2 Activity by Different Mechanisms.Tarnowski K, Klimecka M, Ciesielski A, Goch G, Kulik A, Fedak H, Poznański J, Lichocka M, Pierechod M, Engh RA, Dadlez M, Dobrowolska G, Bucholc M. Plant Physiol. 2020. doi: 10.1104/pp.19.00900.
    • Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1-related protein kinase 2.10 in response to osmotic stress. Maszkowska J, Dębski J, Kulik A, Kistowski M, Bucholc M, Lichocka M, Klimecka M, Sztatelman O, Szymańska KP, Dadlez M, Dobrowolska G. Plant Cell Environ. 2019. doi: 10.1111/pce.13465.
    • Phosphatase ABI1 and okadaic acid-sensitive phosphoprotein phosphatases inhibit salt stress-activated SnRK2.4 kinase. Krzywińska E, Bucholc M, Kulik A, Ciesielski A, Lichocka M, Dębski J, Ludwików A, Dadlez M, Rodriguez PL, Dobrowolska G. BMC Plant Biol. 2016. doi: 10.1186/s12870-016-0817-1.
    • SNF1-related protein kinases type 2 are involved in plant responses to cadmium stress. Kulik A, Anielska-Mazur A, Bucholc M, Koen E, Szymanska K, Zmienko A, Krzywinska E, Wawer I, McLoughlin F, Ruszkowski D, Figlerowicz M, Testerink C, Sklodowska A, Wendehenne D, Dobrowolska G. Plant Physiol. doi: 10.1104/pp.112.194472.
    • SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor. Bucholc M, Ciesielski A, Goch G, Anielska-Mazur A, Kulik A, Krzywińska E, Dobrowolska G. J Biol Chem. 2011. doi: 10.1074/jbc.M110.115535.

Collaborations

    • David Wendehenne, Agroecologie, AgroSup Dijion, CNRS, INRA, Universisty of Bourgone, France
    • Christa Testerink, Laboratory of Plant Physiology, Plant Science Group, Wageningen Universisty and Research, The Netherlands
    • Dorothee Staiger, Faculty of Biology, RNA Biology and Molecular Physiology Group, Bielefeld Universisty, Germany
    • Zofia Szweykowska-Kulińska and Artur Jarmołowski – Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz Universisty, Poland
    • Justyna Łabuz, Laboratory of Photobiology, Małopolska Centre of Biotechnology, Jagiellonian University, Poland
    • Katarzyna Banaś, Departament of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Poland
    • Joanna Grzyb, Department of Biophysics, Faculty of Biotechnology, University of Wroclaw, Poland
    • Katarzyna Gieczewska, Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, Poland
    • Maciej Garstka and Radosław Mazur, Department of Metabolic Regulation, Faculty of Biology, University of Warsaw, Poland

Prizes and Awards

    • Anna Kulik. Fellowship for Outstanding Young Scientists. 2015. Ministry of Science and Higher Education, Poland

Publications

KOEN E., SZYMANSKA K., KLINGUER A., DOBROWOLSKA G., BESSON-BARD A., WENDEHENNE D., Nitric oxide and glutathione impact the expression of iron uptake-and iron transport-related genes as well as the content of metals in A. thaliana plants grown under iron deficiency. Plant Signaling and Behavior (2012) 7(10): 1246-1250
KULIK A., ANIELSKA-MAZUR A., BUCHOLC M., KOEN E., SZYMANSKA K., ŻMIEŃKO A., KRZYWIŃSKA E., WAWER I., McLOUGHLIN F., RUSZKOWSKI D., FIGLEROWICZ M., TESTERINK C., SKŁODOWSKA A., WENDEHENNE D., DOBROWOLSKA G., SNF1-related protein kinases type 2 are involved in plant responses to cadium stress. Plant Physiology (2012) 160(2):868-883 IF 6.535
KULIK A., WAWER I., KRZYWIŃSKA E., BUCHOLC M., DOBROWOLSKA G., SnRK2 protein kinases-key regulators of plant response to abiotic stresses. OMICS: A Journal of Integrative Biology (2011) 15(12): 859-872 IF 1.944
KLIMECKA M.M., SZCZEGIELNIAK J., GODECKA L., LEWANDOWSKA-GNATOWSKA E., DOBROWOLSKA G., MUSZYNSKA G., Regulation of wound-responsive calcium-dependent protein kinase from maize (ZmCPK11) by phosphatidic acid. Acta Biochimica Polonica (2011) 58(4): 589-595 IF 1.234
BUCHOLC M., CIESIELSKI A., GOCH G., ANIELSKA-MAZUR A., KULIK A., KRZYWIŃSKA E., DOBROWOLSKA G., SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor. Journal of Biological Chemistry (2011) 286(5): 3429-41 IF 5.328
WAWER I., BUCHOLC M., ASTIER J., ANIELSKA-MAZUR A., DAHAN J., KULIK A., WYSLOUCH-CIESZYNSKA A., ZARĘBA-KOZIOŁ M., KRZYWIŃSKA E., DADLEZ M., DOBROWOLSKA G., WENDEHENNE D., Regulation of Nicotiana tabacum osmotic stress-activated protein kinase and its cellular partner GAPDH by nitric oxide in response to salinity. Biochemical Journal (2010) 429(1): 73-83 IF 5,155