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Department of Plant Biochemistry

Head: Professor Grażyna Muszyńska

The activity of the Department is focused on elucidation of the mechanisms of plant responses to osmotic stress, wounding, sulfur deficiency and the pathogen infection. The role of plant specific protein kinases in stress signal transduction is the subject of study of research groups of G. Muszyńska and G. Dobrowolska. Group led by A. Sirko is interested on the characterization of regulatory elements involved in plant responses to sulfur deficiency and heavy metal stresses, as well as in molecular farming problems. Studies on signalling pathways (phospholipases and protein kinases) in different types of interactions between Solanum genotypes and Phytophthora infestans (late blight) and cytogenetic analysis of hybrids of potato varying in resistance to this pathogen are conducted by B. Wielgat group. The role of phospholipase A2 in lipid metabolism as the response on Phytophthora infestans elicitor and elaboration of cytogenetic markers for pathogen resistance are investigated (B. Wielgat). The results of E. Kraszewska group study on Nudix hydrolase (AtNUDT7) suggest that this protein acts as the effector of protein G-dependent signal transduction pathway.  


Plant protein phosphorylation

Group leader: Prof. Grażyna Muszyńska

Staff: Dr. Jadwiga Szczegielniak, Ph.D. students: M.Sc. Elżbieta Lewandowska-Gnatowska, M.Sc. Lidia Szymona

 

Reversible protein phosphorylation is an essential component of plant stress signal transduction pathways dedicated to defense and recovery. We have demonstrated that expression of ZmCPK11, a member of the Calcium Dependent Protein Kinases (CDPKs) family, is induced by mechanical wounding both locally at the injury site, and systemically in distant unwounded maize leaves. The rapid, within minutes, activation of ZmCPK11 and stimulation of ZmCPK11 mRNA accumulation both locally and systemically indicate its participation in early stages of local and systemic responses to wounding. The plant alarmone jasomonic acid, its methyl ester, and its precursor, linolenic acid, induced the expression and activity of ZmCPK11 in maize leaves, which indicates that this protein kinase is involved in jasmonic acid-dependent wound signaling.

Analysis of the sequence of plant CDPKs has suggested that ZmCPK11 homologs are present only in monocots. To verify this suggestion the homolog of ZmCPK11 was cloned in barley. The clone was designated HvCDPK12, and assigned the accession no EU246600. The HvCDPK12 activity and its transcript level increased after leaf wounding, indicating that maize and barley CDPKs are structural and functional homologs.

 

Current topics:
- The role of protein phosphorylation in plant stresses
- Function of calcium depedent protein kinases in monocotyledones


Research grants:
2008-2011 "The role of maize ZmCPK11 in wound signal transduction pathways", (Ministry of Science and Higher Education)
2009-2012 "Structure and function of the gene encoding ZmCPK11", (Ministry of Science and Higher Education)

  

Selected publications:

  1. Łebska M., Szczegielniak J., Dobrowolska G., Cozza G., Moro S., Muszyńska G. A novel splicing variant encoding putative catalytic α subunit of maize protein kinase CK2. Physiol. Plant. (2009) 136: 251-263
  2. Bretner M., Najda-Bernatowicz A., Łebska M., Muszyńska G., Kilianowicz A., Sapota A. New inhibitors of protein kinase CK2, analogues of benzimidazolne and benzotriazole. Mol. Cell Biochem. (2008) 316: 87-89
  3. Kilmecka M.M., Muszyńska G. Structure and function of plant calcium-dependent protein kinases. Acta Biochimica Polonica54: 219-233
  4. Miernyk J.A., Szurmak B., Towar-Mendez A., Randall D.D., Muszyńska G. Is there a signal transduction pathway that links events at the plasma membrane to the phosphorylation state of the mitochondria pyruvate dehydrogenase complex? Physiol. Plant. (2007) 129: 104-113
  5. Szczegielniak J., Klimecka M., Liwosz A., Ciesielski A., Kaczanowski S., Dobrowolska G., Harmon A.C., Muszyńska G. A wound-responsive and phospholipid-regulated maize Calcium-Dependent Protein Kinase. Plant Physiology (2005) 139: 1970-1983.

Tissue and cell cultures of potato: pathogenesis and plants transformation

 Group leader: Prof. Bernard Wielgat

Staff: Dr. Urszula Maciejewska, Dr. Lidia Polkowska-Kowalczyk, Dr. Anna Szczerbakowa, Dr. Krzysztof Olszak, M.Sc. Justyna Tarwacka

 

Our studies are focused on cytogenetic analysis of the wild Solanum species and their interspecific hybrids with cultivated potato varying in resistance to Phytophthora infestans, including determination of chromosome number, genome size and composition, as well as in vitro stability of combined hybrid genomes. The effects of changes in hybrid ploidy, caused by colchicin treatment, on hybrid resistance and fertility are also under investigation.

    The early defense processes in response to P. infestans are studied in somatic hybrids, as well as in parental genotypes. The processes concern production of reactive oxygen species (ROS), the activity of antioxidant enzymes (ascorbate peroxidase, glutathione reductase, glutathione-S-transferase), phospholipases, protein kinases and lipid metabolism.


Research grants:
2008-2011 "Studies on singnalling pathways in different types of interactions between Solanum genotypes and
                 Phytophthora infestans",
(Ministry of Science and Higher Education)
2007-2010 "Production and molecular analysis of interspecific somatic hybrids of Solanum as new source of resistance
                 to Phytophthora infestans for the cultivated potato", (Ministry of Science and Higher Education)

2007-2010 "Introgression of the genes of resistance to Phytophthora infestans from Solanum michoacanum (Bitter.)
                 Rydb. into cultivated potato S. tuberosum L. and development of the respective PCR markers for their
                 application in selection", (Ministry of Science and Higher Education)

  

Selected publications:

  1. Polkowska-Kowalczyk L., Montillet J-L., Agnel J-P., Triantaphylidès C., Wielgat B., Maciejewska U. Changes in the initial phase of lipid peroxidation induced by elicitor from Phytophthora infestans in Solanum species. J Plant Physiol. (2008) 165: 1929-1939
  2. Polkowska-Kowalczyk L., Wielgat B., Maciejewska U. Changes in the antioxidant status in leaves of Solanum species in response to elicitor from Phytophthora infestans. J Plant Physiol. (2007) 164: 1268-1277
  3. Bołtowicz D., Szczerbakowa A., Wielgat B. RAPD analysis of the interspecific somatic hybrids Solanum bulbocastanum (+)
    S. tuberosum
    . Cell Mol Biol Lett (2005) 10: 151-162
  4. Szczerbakowa A., Bołtowicz D., Lebecka R., Radomski P., Wielgat B. Characteristics of the interspecific somatic hybrids
    S. pinnatisectum
    (+) S. tuberosum H-8105. Acta Physiol Plant (2005) 27: 265-273
  5. Montillet J-L., Garnier L., Cacas J-L., Montané M-H., Douki T., Bessoule J-J., Polkowska-Kowalczyk L., Maciejewska U., Agnel J-P., Vial A., Triantaphylidès C The upstream oxylipin profile of Arabidopsis thaliana: a tool to scan for oxidative stresses.  Plant J (2004) 40: 439-451
  6. Polkowska-Kowalczyk L., Wielgat B., Maciejewska U. The elicitor-induced oxidative processes in leaves of Solanum species with differential polygenic resistance to Phytophthora infestansJ Plant Physiol. (2004) 161: 913-920
  7. Szczerbakowa A., Bołtowicz B., Wielgat B. Interspecific somatic hybrids Solanum bulbocastanum (+) S. tuberosum H-8105. Acta Physiol Plant (2003a) 25: 365-373
  8. Szczerbakowa A., Maciejewska U., Zimnoch-Guzowska E., Wielgat B. Somatic hybrids Solanum nigrum (+) S. tuberosum: morphological assessment and verification of hybridity. Plant Cell Rep (2003b) 21: 577-584
  9. Zimnoch-Guzowska E., Lebecka R., Kryszczuk A., Maciejewska U., Szczerbakowa A., Wielgat B. Resistance to Phytophthora infestans in somatic hybrids of Solanum nigrum L. and diploid potato. Theor Appl Genet (2003) 107: 43-48

 


Expression of genes encoding proteins of biotechnological significance in plants; Sulfur metabolism in plants; Heavy metals tolerance and accumulation

Group leader: Prof. Agnieszka Sirko

Staff: Dr. Anna Góra-Sochacka, Dr. Małgorzata Lewandowska (currently abroad), Dr. Anna Wawrzyńska, M.Sc. Jolanta Kamińska (Ph.D. student), M.Sc. Grzegorz Moniuszko (Ph.D. student), M.Sc. Patrycja Redkiewicz (Ph.D. student), M.Sc. Katarzyna Zientara, (Ph.D. student), Magdalena Dydo, Dawid Głów, Rafał Gwozdecki, Agata Kurzyk, Róża Sawicka, Anna Stachyra, Przemysław Surowiecki

 

The scientific activity of our group has been focused since several years on the following subjects: plant-based production of proteins that could have potential therapeutic or biotechnological applications (molecular farming approach), regulatory aspects of plant response to sulfur deficit, sulfur metabolism in plants and its relation to heavy metals tolerance and accumulation by plants.

Plant-based systems (either transient or based on a stable genetic transformation) might facilitate inexpensive production of various therapeutic proteins in the contained environment. Efficient production of human IL-2 and murine GM-CSF in tobacco plants was recently achieved in our laboratory. Their biological activity has been confirmed using appropriate cell lines which are growth and propagation dependent on these cytokines. Currently, we focus on engineering the cytokines expression cassettes in order to increase yields of the recombinant proteins.

One of the important aims of our research is characterization of plant response to sulfur (S) deficiency stress. Our interest is focused particularly on genes encoding potential regulatory proteins. Knowledge of the regulatory elements can facilitate future manipulation of S metabolism in plants in order to increase production of desired S-containing compounds, such as glutathione or secondary metabolites. We investigate the function of several novel genes induced by S deficit, which encode good candidates for such regulatory factors. We also explore the possibility of modification of the plant cysteine synthase complex to maximize the activity of each of its component enzymes, SAT and OAS-TL.

Cadmium enters the human and animal body mostly through the food chain after being accumulated in plant tissues. In plants, Cd tolerance is strongly related to S nutrition and metabolism. In order to understand the mechanisms of Cd toxicity in plants we monitor its influence on gene expression and investigate the role of selected transporters and chelators in Cd tolerance and accumulation.

More information: http://www.ibb.waw.pl/~sirkolab/

Current projects:

  1. Plant-based production of mammalian cytokines and other therapeutic proteins.
  2. Veterinary vaccine against avian influenza and biosensors for detection of AIV (in collaboration).
  3. Identification and characteristics of plant genes induced during sulfur deficit
  4. Regulation of sulfur flux in plants on various levels (in collaboration).
  5. Unbiased approach to monitor changes in gene expression in tobacco plants exposed to cadmium.
  6. Analysis of tobacco plants transformed with genes encoding metal transporters and metal chelators from Thlaspi caerlulescens.

Research grants:
2008-2011 "Low alkaloid transgenic tobacco plants as a source of recombinant IL-2 and GMCSF protein and their fusion
                 with vaccine antigen", (Ministry of Science and Higher Education)
2007-2011 "Molecular approaches towards control of sulfur flux in plants through selective deregulation of cysteine
                 synthase complexes", (DFG-Ministry of Science and Higher Education)
2008-2011 "Biotechnology Center for therapeutic proteins. Packet of innovative biofarmaceuticals for humans and animals
                  therapies and prophylaxes", (Structural Funds, POIG, Ministry of Science and Higher Education)
2008-2011 "Role of the selected proteins encoded by the genes induced in plants in response to sulfur deficit",
                 (Ministry of Science and Higher Education)
2008-2010 "Influence of modification of the selected metals chelators and transporters on accumulation of cadmium
                 and zinc in tobacco", (Ministry of Science and Higher Education)

  

Selected publications:

  1. Wawrzyńska A, Lewandowska M, Sirko A. 2010. Nicotiana tabacum EIL2 directly regulates expression of at least one tobacco gene induced by suphur starvation J Exp Bot – in press
  2. Góra-Sochacka A, Redkiewicz P, Napiórkowska B, Gaganidze D, Brodzik R, Sirko A. 2010. Recombinant Mouse GM-CSF is glycosylated in transgenic tobacco and maintains its biological activity. J Interf Cytok Res 30(3) – in press
  3. Zientara K, Wawrzyńska A, Łukomska J, Lopez Moya RJ, Liszewska F, Assunção AGL, Aarts MGM, Sirko A. 2009 Activity of the AtMRP3 promoter in transgenic Arabidopsis thaliana and Nicotiana tabacum plants is increased by cadmium, nickel, arsenic, cobalt and lead but not by zinc and iron. J Biotechnol 139: 258-263
  4. Góra-Sochacka A, Redkiewicz P, Napiórkowska B, Sirko A. 2009. Wykorzystanie systemów roślinnych do produkcji rekombinowanych cytokin. Postępy Biochemii 55: 85-94
  5. Lewandowska M, Bajda A, Świeżewska E, Sirko A. 2009. Influence of short term sulfur starvation on photosynthesis-related compounds and processes in tobacco. In Sulfur Metabolism in Plants. Regulatory Aspects, Significance of Sulfur in the Food Chain, Agriculture and the Environment, Sirko A, DeKok LJ, Haneklaus S, Hawkesford M, Rennenberg H, Saito K, Schnug E, Stulen I, (Eds.) Margraf Publishers GmbH Scientific Books, Weikersheim, Germany, ISBN 978-3-8236-1547-7; 978-90-5782-215-5; pp. 79-83
  6. Lewandowska M, Sirko A. 2009. Identification of additional genes regulated by sulfur shortage in tobacco. In Sulfur Metabolism in Plants. Regulatory Aspects, Significance of Sulfur in the Food Chain, Agriculture and the Environment, Sirko A, DeKok LJ, Haneklaus S, Hawkesford M, Rennenberg H, Saito K, Schnug E, Stulen I, (Eds.) Margraf Publishers GmbH Scientific Books, Weikersheim, Germany, ISBN 978-3-8236-1547-7; 978-90-5782-215-5; pp. 73-77
  7. Bandurska K, Brodzik R, Spitsin S, Kohl T, Portocarrero C, Smirnov Y, Pogrebnyak N, Sirko A, Koprowski H, Golovkin M. 2008. Plant-produced hepatitis B core protein chimera carrying anthrax protective antigen domain-4. Hybridoma 27: 241-247; doi:10.1089/hyb.2008.0008
  8. Antosiewicz MD, Sirko A, Sowiński P. 2008. Trace elements transport in plants. In: Trace Elements: Environmental Contamination and Quality of Life (Ed. Prasad MNV). John Wiley & Sons, Inc. ISBN: 978-0-470-18095-2, pp. 413-448
  9. Lewandowska M, Sirko A. 2008. Current advances in understanding plant response to sulfur-deficiency stress. Acta Biochim Polon 55: 457-471
  10. Moniuszko G. Sirko A. Metabolizm siarki w roślinach i jego regulacja. 2008. Postępy Biochemii 54: 402-411

 


Osmotic stress signal transduction in plants

Group leader: Assoc. Prof. Grażyna Dobrowolska

Staff: Dr. Maria Bucholc, M.Sc. Anna Kulik (Ph.D. student), M.Sc. Ewa Krzywińska (Ph.D. student)

 

Our laboratory investigates plant responses to abiotic stress, especially to osmotic stress triggered by drought and salinity.

Our interest is focused on protein kinases, which belong to the SNF1-related kinases type 2 (SnRK2) family. It is known that SnRK2s are plant specific enzymes, involved in plant adaptive responses to osmotic stress and in abscisic acid-regulated plant development. Our studies on this project started several years ago, when we had identified a protein kinase rapidly activated in response to osmotic stress in tobacco cells. We named the kinase NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase) and classified it to the SnRK2 family. We analyzed the mechanism of NtOSAK activation and its role in plant response to stress. The results indicate that in response to osmotic stress NtOSAK is regulated by phosphorylation of two specific serine residues in the kinase activation loop. Moreover, nitric oxide plays a key role in regulation of the kinase activity. Our recent data show that besides osmotic stress NtOSAK is activated also by wounding and oxidative and heavy metal stresses. In order to determine the kinase function in planta we have constructed transgenic tobacco plants with altered NtOSAK expression and activity. In parallel, we analyze the function of the closest homologues of NtOSAK in Arabidopsis thaliana, SnRK2.4 and SnRK2.10. For these studies we obtained Arabidopsis mutant plants, knockouts, as well as plants overexpressing each of the kinases. Our preliminary data obtained by the transgenic approach suggest that studied kinases: NtOSAK and SnRK2.10 play a regulatory role in salinity and heavy metal stress tolerance.

Presently, we have been using an integrated approach, by combining biochemical, molecular biology, proteomic, bioinformatics, and transgenic plants tools, to discover elements of SnRK2 transduction pathway(s), as well as the main substrates phosphorylated and regulated by selected members of the SnRK2 family.  We have found one potential negative regulator of SnRK2s, which appears to be a plant specific calcium sensor. The function of this protein and several others possible regulators of SnRK2’s activity in connection to plant growth, development and response to abiotic stress is under investigation.


Current projects:

  1. The role of SnRK2 kinases in plant response to salinity, drought, wounding and oxidative and heavy metal stresses.
  2. Interrelation between nitric oxide and SnRK2 in stress signal transduction in plants.

Research grants:
2007-2010 "The role of the SnRK2 (SNF1-related protein kinase 2) family members in plant response to abiotic stress,
                 Analysis of function and regulation of kinase activity in response to stress", (Ministry of Science and
                 Higher Education)
2009-2012 "The role of the SnRK2 (SNF1-related protein kinase 2) in regulation of plant sensitivity to water deficit and
                  salinity", (Ministry of Science and Higher Education)

  

Selected publications:

  1. Mikołajczyk M, Awotunde OS, Muszyńska G, Klessig DF, Dobrowolska G (2000) Osmotic stress induces rapid activation of a SIP kinase and a homolog of protein kinase ASK1 in tobacco cells. Plant Cell 12: 165-178
  2. Kelner A, Pękala I, Kaczanowski S, Muszyńska G, Hardie DG, Dobrowolska G (2004) Biochemical characterization of the tobacco 42-kD protein kinase activated by osmotic stress. Plant Physiol 136(2): 3255-65
  3. Burza AM, Pękala I, Sikora J, Siedlecki P, Małagocki P, Bucholc M, Koper L, Zielenkiewicz P, Dadlez M, Dobrowolska G (2006) Nicotiana tabacum Osmotic Stress-activated Kinase is regulated by phosphorylation on Ser-154 and Ser-158 in the kinase activation loop. J Biol Chem 281(45): 34299-34311
  4. Lamotte O, Courtois C, Dobrowolska G, Besson A, Pugin A, Wendehenne D (2006) Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells. Free Radic Biol Med  40(8):1369-76
  5. Courtois C, Besson A, Dahan J, Bourque S, Dobrowolska G, Pugin A and Wendehenne D (2008) Nitric oxide signalling in plants: interplays with Ca2+ and protein kinases” J Exp Bot 59(2): 155-163
  6. Besson A, Courtois C, Gauthier A, Dahan J, Dobrowolska G, Jeandroz S, Pugin A and Wendehenne D (2008) Nitric oxide in plants: production and cross-talk with Ca2+ signalling Molecular Plant 1(2): 218-228

Characterization of the chosen proteins involved in cellular metabolism of Arabidopsis thaliana

Group leader: Dr. Elżbieta Kraszewska

Staff: Prof. Jerzy Buchowicz, Katarzyna Tracz, Agata Lipko (M.Sc. student), Marta Modzelan (M.Sc. student)


Range of interests: biochemistry, structure, participation in signal transduction pathways, proteasome degradation of Nudix proteins.

Previously, we have established that the AtNUDT7 Nudix hydrolase, a key regulator of the basal cellular defence response, interacts with two regulatory proteins, 14.3.3 and RACK1A (Receptor for Activated C Kinase 1). Structurally and functionally RACK proteins resemble the β subunit of a signal transducing G protein. Hence, we tested the ability of the RACK1A protein to interact with the members of the Arabidopsis G complex. Pull-down and yeast two- hybrid experiments have shown that contrary to the canonical β subunit, RACK1A does not bind the α subunit. However, the interactions of RACK1A with both canonical γ subunits were observed, suggesting that RACK1A can substitute for the β subunit in the transducing βγ complex. The possibility that the AtNUDT7 protein acts as a down- stream effector of the RACK1A/ γ complex is under investigations.

Further analyses of the AtNUDT7 mutated protein have revealed that proper dimer formation is required not only for the catalytic activity of the hydrolase but also for its interactions with protein partners, indicating that the biologically active form of ATNUDT7 is the dimer.

Preliminary results indicate that the cellular level of the AtNUDT7 protein is controlled by 26S proteasome.

During a study on the other Arabidopsis Nudix hydrolase, the AtNUDT13 protein, it was established that this ApnA hydrolase is targeted to mitochondria in yeast and plant cells.

Study of some Nudix proteins from plant pathogenic bacteria Pseudomonas syringae DC 3000 are also conducted.


Research grants:
2008-2011 "Function of the AtNUDT7 Nudix protein in the plant response to stress", (Ministry of Science
                 and Higher Education)
2006-2008 "Molecular characterization of AtNUDT7 from Arabidopsis thaliana-the protein that participates in signal
                  transduction during abiotic stress", (Ministry of Science and Higher Education)
2004-2007 "Participation of the Ku70 and Nudix proteins in the light signal transduction and plant response to abiotic
                  stress", (Ministry of Science and Higher Education)
1999-2002 "Plant homologues of the bacterial MutT protein", (Grant Nr. 4PW)

 

Selected publications:

  1. Olejnik K., Płochocka D., Grynberg M., Goch G., Gruszecki W.I., Basińska T., Kraszewska E.  Mutational analysis of AtNUDT7 Nudix hydrolase reveals residues required for protein quarternary structure formation and activity. Acta Biochim. Pol. (2009) 56, 291-300.
  2. Kraszewska E.  The plant Nudix hydrolase family. (Review). Acta Biochim. Pol. (2008) 55, 663-671.
  3. Szurmak B., Wysłouch-Cieszyńska A., Wszelaka-Rylik M., Bal W., Dobrzanska M. A diadenosine 5’, 5’’-P(1)P(4) tetraphosphate (Ap4A) hydrolase from Arabidopsis thaliana that is activated preferentially by Mn2+ ions. Acta Biochim. Pol. (2008) 55, 151-160.
  4. Olejnik K., Murcha M.W., Whelan J., Kraszewska E.  Cloning and characterization of AtNUDT13, a novel mitochondrial Arabidopsis thaliana Nudix hydrolase specific for long-chain diadenosine polyphosphates. FEBS J. (2007) 274, 4877-4885.
  5. Olejnik K., Kraszewska  E.  Cloning and characterization of an Arabidopsis thaliana Nudix hydrolase homologous to the mammalian GFG protein. Biochim. Biophys. Acta (2005) 1752, 133-141.
  6. Dobrzanska M., Szurmak B., Wysłouch-Cieszynska A. Kraszewska E.  Cloning and characterization of the first member of the Nudix family from Arabidopsis thaliana. J. Biol. Chem. (2002) 277, 50482-50486.