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:
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).
ü Identification and, if
identified, characterization of a metHb reductase as
a putative in vivo partner in nsHbs
enzymatic activities.
ü Identification of nsHbs
physiological roles in S. sclerotiorum –
Arabidopsis and Medicago interaction.
ü 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:
1.
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, 68, 2017, 7-13
http://www.sciencedirect.com/science/article/pii/S1089860316302221
2.
Naumova-Letia Galaba, Mot Augustin-Catalin, Probing
reducing power for ferryl phytoglobins of several
phenolic compounds using their kinetic profiles assisted by chemometric methods,
STUDIA UBB CHEMIA, LXII, 2, Tom I, 2017,
9-66;
http://chem.ubbcluj.ro/~studiachemia/issues/chemia2017_2/tom1/05Letia_Mot_49_66.pdf
3.
Mot
Augustin-Catalin, Parvu
Marcel, Parvu Alina, Rosca-Casian
Oana, Dina Nicoleta, Leopold Nicolae, Silaghi-Dumitrescu Radu, Mircea Cristina, Reversible naftifine-induced carotenoid
depigmentation in Rhodotorula mucilaginosa
(A. Jörg.) F.C. Harrison causing onychomycosis, Scientific
Reports, 2017, 11;7(1):11125
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593942/
4.
Christin
Naumann, Augustin C. Mot, Nico Dissmeyer, Generation
of artificial N-end rule substrate proteins in
vivo and in vitro, Plant Proteostatis series, Methods in Molecular Biology,
ed. Clifton, N.J., Volume: 1450, 201, 2017,
pag. 55-83;
https://link.springer.com/protocol/10.1007%2F978-1-4939-3759-2_6
5. 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, 2018, 217(2):613-624
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763331/
6.
Miclea Patricia, Bischin
Cristina-Maria, Naumova-Letia Galaba,
Augustin C. Mot, Dorneanu Sorin, Dorina
Podar, Nico Dissmeyer, Silaghi-Dumitrescu Radu, Redox control and autoxidation of class 1, 2 and 3 phytoglobins from
Arabidopsis thaliana, Scientific Reports, 2018, 13714
https://www.nature.com/articles/s41598-018-31922-4
7.
Daniela
Benedec, Ilioara Oniga, Daniela Hanganu, Ana Maria
Gheldiu, Cristina Pușcaș,
Radu Silaghi-Dumitrescu, Mihaela Duma, Brîndușa Tiperciuc, Rodica Vârban, Laurian Vlase, Sources for developing new medicinal
products: biochemical investigations on alcoholic extracts obtained from aerial
parts of some Romanian Amaryllidaceae species, BMC
Complementary and Alternative Medicine, 2018; 18: 226.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063020/
8.
Anca
D Farcas, Augustin C Mot, Cezara
Zagrean-Tuza, Vlad Toma, Claudia Cimpoiu,
Anamaria Hosu, Marcel Parvu,
Ioana Roman, Radu Silaghi-Dumitrescu,
Chemo-mapping and biochemical-modulatory
and antioxidant/prooxidant effect of Galium verum extract during acute restraint and dark stress in
female rats, PLOS One, 2018
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200022
9.
Augustin
C Mot, Cristina Puscas, Sorin Aurel
Dorneanu, Radu Silaghi-Dumitrescu,
EPR detection of sulfanyl radical during sulfhemoglobin formation–Influence of catalase, Free
Radical Biology and Medicine, 2019, 110-115.
https://www.sciencedirect.com/science/article/pii/S0891584919302631
2. Conferences (Oral presentations
or posters):
1. 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;
2. 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
3. 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
4. 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
5. 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
6. Cezara Zăgrean-Tuza,
Nico Dissmeyer, Augustin C. Moț,
Structural and mechanistic characterization of key enzymes in Arabidopsis
thaliana, N-term 2017 - Proteostasis via the N-terminus Conference, Halle (Saale),
Germany
7. Cezara Zăgrean-Tuza,
Augustin C. Moț, Structural and mechanistic
characterization of key enzymes in Arabidopsis
thaliana, International Conference Students for Students, 2017, Cluj-Napoca
8. Cezara Zăgrean-Tuza,
Augustin C. Moț, Structural and mechanistic
characterization of a new enzyme from Arabidopsis thaliana, 11th biennial
International Conference on Processes in Isotopes and Molecules, 2017, Cluj-Napoca
9. Cezara Zăgrean-Tuza,
Augustin C. Moț, On
finding the three hidden rings and their involvement in plant protein
degradation, International Conference of Young Researchers in Sciences, 2017, Cluj-Napoca
10. Anca D. Farcaș,
Luiza Buimagă-Iarinca, Augustin C. Moț, Vlad Al. Toma1, Silvia Neamțu,
Marcel Pârvu, Structure - biological activity
relationship of estrogenic flavonoids from Plantago
sempervirens, 19th IUPAB and 11th EBSA Congress, 2017, Edinburgh, Scotland
11. Augustin C. Mot, Galaba
Naumova Leția, Cezara Zăgrean-Tuza, Kim Hebelstrup,
Costel Sarbu, Profiling
non-symbiotic plant hemoglobins as regulators in
oxidative and nitrosative stress: a chemometric approach,
Euroanalysis,
2017, Stockholm, Sweden
3. Purchased equipment:
· Plant Growth Chamber GC-300TL/
300TLH, value: 44200 lei
· Fully equipped Biorad
Electrophoresis and Western blotting system, value: 10116 lei
· Eppendorf microcentrifuge: 8740 lei
· Retsch Mixer Mill MM 400: 31200 lei
4. Students involved:
· 1 Dissertation thesis, June 2016
· 5 Bachelor students for their
diploma research project