PN-II-RU-TE-2014-4-2555

Ctr. 221/01/10/2015

Team:

Dr. Augustin C. Mot – project director

Dr. Cristina Bischin – research assistant

PhD student Cristina Mircea – research assistant

PhD student Anca D. Farcas – research assistant

Chim. Galaba Naumova-Letia – chemist

 

Implementation period: October 2015 – September 2017

 

 

Nonsymbiotic plant hemoglobins in stress related responses

 

The first plant hemoglobins that were discovered were leghemoglobins which are highly expressed in the root nodules of legumes. Nonsymbiotic hemoglobins possess distinct nucleotide sequences and biochemical properties compared to them. Whereas leghemoglobins are nowadays well known both structurally and functionally, the function and mechanism of action of nonsymbiotic hemoglobins is still unclear. The main role of nonsymbiotic hemoglobins was attributed to NO scavenging and thus increasing plant survival during hypoxia. The current project aims to track the mechanism which governs nonsymbiotic hemoglobins involvement in plant stress tolerance using Arabidopsis thaliana and Medicago truncatula as plant models. This will involve transgenic lines of A. thaliana and M. truncatula and will lead to the identification of the nonsymbiotic hemoglobins’ physiological roles in plant-pathogen interaction and in tolerance of other abiotic stress factors. Additionally, in vitro biochemical characterization of two previously unexplored truncated nonsymbiotic hemoglobins from M. truncatula will be performed. The outcome of the current project is expected to lead to solid explanations and predictions on the enzymatic activities of the nonsymbiotic hemoglobins in plants. In the longer term, it is foreseen that the results provide novel occasions for rational crop plans design to improve plant stress tolerance.

 

Objectives (achieved and under progress):

 

ü  In vitro biochemical characterization of two previously unexplored truncated nsHbs from Medicago. This will imply isolation, purification, spectroscopic investigations (UV-vis, EPR, FTIR, CD) upon their deoxy, oxy and met forms and their interaction with putative interaction small molecules (O2, CO, CN-, N3-, NO, NO2, H2O2, S22-, SO32- - including kinetics taking advantage of stopped-flow, freeze quench and/or flash photolysis techniques), ligand binding affinities (O2, NO, CO).

ü  In vitro characterization of their putative enzymatic activities (enzymatic parameters, kinetics, inhibitory study) in comparison with the activities of the three nsHbs from Arabidopsis.

ü  Obtaining and characterization of stable transgenic nsHb overexpressing lines of Arabidopsis thaliana and transient root transformed Medicago truncatula plants and obtaining of nsHbs knock-out mutants (by screening of Tnt1 insertion lines).

·       Identification and, if identified, characterization of a metHb reductase as a putative in vivo partner in nsHbs enzymatic activities.

·       Identification of nsHbs physiological roles in S. sclerotiorum – Arabidopsis and Medicago interaction.

·       Identification of nsHbs physiological roles in tolerance of abiotic stress factors in Arabidopsis and Medicago.

Impact:

The outcome of the current project is expected to be visible in:

· Explaining and making predictions on the catalytic activities of the nsHbs in plants that are currently unclear or unexplored.

· Identifying of new physiological roles in different pathways involving nsHbs

· Explaining, at least in part, the interaction of the devastating phytopathogenic S. sclerotiorum with its hosts.

· In the longer term, it is foreseen that the results provide novel occasions for rational crop plans to improve plant stress tolerance.

· A welcome opportunity to train a passionate group of young researchers (PhD students, postdocs) and undergraduate students.

 

Results and benefits:

1.     Articles:

·        Mioto Paulo Tamaso, Ruiz Marta Rodríguez, Augustin C. Mot, Corpas Francisco J., Freschi Luciano, Mercier Helenice, Alternative fluorimetric-based method to detect and compare total S-nitrosothiols in plants, Nitric oxide – Biology and Chemistry, accepted, http://www.sciencedirect.com/science/article/pii/S1089860316302221

·       Augustin C. Mot, Erik Prell, Maria Klecker, Christin Naumann, Frederik Faden, Bernhard Westermann, Nico Dissmeyer, Real-time detection of N-end rule-mediated ubiquitination via fluorescently labeled substrate probes, New Phytologist, accepted

http://onlinelibrary.wiley.com/doi/10.1111/nph.14497/full

·       Miclea Patricia, Bischin Cristina-Maria, Naumova-Letia Galaba, Augustin C. Mot*, Dorneanu Sorin, Dorina Podar, Nico Dissmeyer, Silaghi-Dumitrescu Radu, Autoxidation and redox state controlling of class 1, 2 and 3 hemoglobins from Arabidopsis thaliana, in preparation

 

2.      Conferences:

·       Augustin C. Mot, Cristina Bischin, Galaba Naumova Leția, Grigore Damian, Nicoleta Dina, Nicolae Leopold, Radu Silaghi-Dumitrescua, Spectroscopic characterization of nonsymbiotic plant hemoglobins using UV-vis, Raman and EPR techniques, National Conference of Biophysics, June 2016, Cluj-Napoca, Romania;

·       Galaba Naumova Leția, Cristina Bischin, Augustin C. Mot, Grigore Damian, Nicolae Leopold, Nico Dissmeyer, Radu Silaghi-Dumitrescu, Mechanistic study of the reaction between nitrite and three distinct nsHbs from Arabidopsis thaliana by means of UV-Vis, Raman and EPR spectroscopy, 6th Plant Nitric Oxide International Meeting, Septembrie 2016, Granada, Spain

·       Nicoleta E. Dina, Alia Colniță, Cristina Mircea, Augustin C. Moț, Nicolae Leopold, Marcel Parvu, Radu Silaghi-Dumitrescu, Resonant Raman investigation of the ROS generated stress interference with the carotenogenesis of R.  mucilaginosa, 33rd European Congress on Molecular Spectroscopy (EUCMOS 2016), August 2016, Szeged, Hungary

·       Cristina Bischin, Patricia Miclea, Galaba Naumova Leția, Augustin Moț, Grigore Damian, Radu Silaghi-Dumitrescu, Alternative path for controlling the oxidations state and oxidative stress of class 1, 2 and 3 from Arabidopsis thaliana, EuroBIC, August 2016, Budapesta, Hungary

·       Galaba Naumova Leția, Augustin C. Mot, Cristina Bischin, Grigore Damian, Nico Dissmeyer, Radu Silaghi-Dumitrescu, In vitro evaluation of peroxidase activity in phytoglobins - a comparative study, XIX International Conference on Oxygen Binding and Sensing Proteins, September 2016, Hamburg, Germany

 

 

 

3.     Purchased equipment:

·       Plant Growth Chamber GC-300TL/ 300TLH, value: 44200 lei

Lab Companion Model GC-300TL/ 300TLH 300 Liter Plant Growth Chamber

 

 

·       Fully equipped Biorad Electrophoresis and Western blotting system, value: 10116 lei

Mini-PROTEAN Tetra Cell and PowerPac Universal Power Supply with FastCast Acrylamide Kit

 

4.     Students involved:

·       1 Dissertation thesis, June 2016, student: Miclea Patricia

·       3 Bachelor students for their diploma research project, under progress