Final Report Summary - PRONITROARAB (NO-dependent protein translocation and S-nitrosylation of nuclear proteins <br/>in Arabidopsis thaliana)
Nitric oxide (NO) is a small, highly reactive, membrane-permeable molecule, which has turned out to be an important biological messenger in animals and plants [1,2]. In the last few years, many studies have described NO as both a cytotoxic and a cytoprotecting regulator involved in different physiological processes in plants. It has been implicated in disease resistance, stomata closure, seed germination, iron homeostasis, different development processes, and in response to abiotic stresses such as drought, salinity, and high temperature [3,4]. One of the most important regulatory mechanisms of NO is S-nitrosylation - the covalent attachment of NO to cysteine residues. In the first objective we studied the S-nitrosylation of Arabidopsis nuclear proteins after pathogen infection. To generate a map of nuclear proteins, which are candidates for S-nitrosylation, Arabidopsis cell cultures were infected with avirulent Pseudomonas syringae. Afterwards, nuclei were enriched and proteins were extracted and treated with S-nitrosoglutathione as an NO donor. After that, the samples were subjected to the biotin switch assay and biotin-labelled proteins were purified using affinity chromatography. Identification of purified proteins was done by mass spectrometry. In this way, a map of proteins which are potential candidates for S-nitrosylation was generated. In total 195 candidate proteins were identified which 57% (111 proteins) of them were described as nuclear proteins according to the Cell eFP Browser database (http://bar.utoronto.ca/cell_efp/cgi-bin/cell_efp.cgi(s’ouvre dans une nouvelle fenêtre)). This work provides a new collection of S-nitrosylated nuclear proteins in Arabidopsis after pathogen infection. The majority of the S-nitrosylated nuclear proteins were involved in protein synthesis, degradation, targeting, folding, and amino acid activation, RNA metabolism, and stress response, which indicates that S-nitrosylated nuclear proteins are involved in a variety of functions. In addition, most of the identified candidates are novel targets for S-nitrosylation indicating organelle specific regulation of the nuclear proteins by S-nitrosylation (Figure 1 in attached file). Manuscript is in preparation.
In recent years, many experimental methods based on the biotin switch assay have been developed for identification of S-nitrosylated proteins. However, these methods have several limitations, such as side reactions in multi-step procedures, detection of low abundant proteins, instability of proteins, or inaccuracy of instruments. Computational methods could overcome such problems. The analyses can be done very easy with a complex protein dataset available in databases, independent of protein abundance and instability and complex chemical reaction. Using the GPS-SNO 1.0 software, a recently developed S-nitrosylation site prediction program to identify candidate proteins for S-nitrosylation in the whole Arabidopsis proteome (27416 proteins). 31907 S-nitrosylated sites were predicted in 16610 candidate proteins using the medium threshold. Potential target proteins were detected in all cellular compartments and ranged from 86% to 37% of the total protein content per compartment. More than 70% of S-nitrosylated candidates were identified in “chloroplast”, “CUL4-RING ubiquitin ligase complex”, and “membrane” compartments. Moreover, the highest 10% high-confident S-nitrosylation sites were extracted for further study. 3190 sites were predicted in 3005 target proteins. These candidates were detected also in all compartments and ranged from 17% to 5% of the total protein content per compartment. These targets were enriched in “chloroplast”, “intracellular”, and “plasmodesmata”. The high number of identified candidates compared to the total number of proteins reflects the importance of redox-signalling in the particular compartments. In general, the currently available algorithm seems to be useful tool to characterize the S-nitrosylome. However, still need to be improved to be accurate in the identification of S-nitrosylation sites. Interestingly, computational prediction of candidates for S-nitrosylation from Arabidopsis can offer a starting point for experimental verification and further studies of S-nitrosylation in plants. Probably the combination of both computational predictions and experimental verifications could be a good approach to get insight into the molecular mechanisms and the regulatory function of S-nitrosylation in plants. Manuscript has been submitted to Plos One (resubmission process).
The bZIP transcription factor TGA1 and the regulatory protein NPR1 together are playing a crucial role in systemic acquired resistance during the inducible plant defense response. Both proteins are regulated in a redox-dependent manner, and it has been reported already the enhanced DNA-binding activity of TGA1 in presence of S-nitrosoglutathione. However, neither the molecular mechanisms nor the structural alterations responsible for these effects are unknown. It is very important to identify the 3-dimensional structure of NPR1 and TGA1 to could clarify the exact mechanism of how nitric oxide regulates NPR1-TGA1 activity in plant defense. This could be very useful for improving plant resistance in the future. The purified recombinant proteins showed an adequate purity grade, a high amount of both proteins will be produced for crystallization. Scaling-up procedure is in progress. (Figure 2 in attached file).
The project was published at the institute of Biochemical Plant Pathology web-site. Scientific students were contacted me showing their interest to do the PhD thesis in this project. Moreover, my results were presented in Fascination of Plants Day, the event was open to general public where they showed the interest to my data. The Project was also discussed with the students at the Technical University of Munich. Additionally, I was invited by Dr. Marek Petřivalský from the University of Olomouc to give a seminar. The visit was published in web-site of the University. The audience showed the interest to the presentation. Afterwards, I had a workshop in the Department of Biochemistry at the same University. I presented an overview of methods and protocols related to nitric oxide research in plants. I had a discussion with the members and PhD students of the department. Moreover, the project will be presented as oral communication in the 5th Plant NO Club Meeting which takes part on 24th of July in Munich, Germany.
Project website address:
http://www.helmholtz-muenchen.de/biop/research-groups-units/redox-signalling-and-antimicrobial-peptides/no-dependent-protein-translocation/index.html(s’ouvre dans une nouvelle fenêtre)
http://www.helmholtz-muenchen.de/biop/research-groups-units/redox-signalling-and-antimicrobial-peptides/analysis-of-no-dependent-structural-alternations-of-npr1-and-tga1/index.html(s’ouvre dans une nouvelle fenêtre)
Scientist in charge:
Dr. Christian Lindermayr
lindermayr@helmholtz-muenchen.de
Tel: +498931872285
Fax: +498931873383
Marie Curie Fellow:
Mounira Chaki
mchaki@hotmail.com, mounira@ujaen.es
Tel: +34667699202
References
1. Stamler JS, Singel DJ, Loscalzo J (1992) Biochemistry of nitric oxide and its redox-activated forms. Science 258: 1898-1902.
2. Wendehenne D, Pugin A, Klessig DF, Durner J (2001) Nitric oxide: comparative synthesis and signaling in animal and plant cells. Trends Plant Sci 6: 177-183.
3. Delledonne M, Xia Y, Dixon RA, Lamb C (1998) Nitric oxide functions as a signal in plant disease resistance. Nature 394: 585-588.
4. Durner J, Wendehenne D, Klessig DF (1998) Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose. Proc Natl Acad Sci U S A 95: 10328-10333.
In recent years, many experimental methods based on the biotin switch assay have been developed for identification of S-nitrosylated proteins. However, these methods have several limitations, such as side reactions in multi-step procedures, detection of low abundant proteins, instability of proteins, or inaccuracy of instruments. Computational methods could overcome such problems. The analyses can be done very easy with a complex protein dataset available in databases, independent of protein abundance and instability and complex chemical reaction. Using the GPS-SNO 1.0 software, a recently developed S-nitrosylation site prediction program to identify candidate proteins for S-nitrosylation in the whole Arabidopsis proteome (27416 proteins). 31907 S-nitrosylated sites were predicted in 16610 candidate proteins using the medium threshold. Potential target proteins were detected in all cellular compartments and ranged from 86% to 37% of the total protein content per compartment. More than 70% of S-nitrosylated candidates were identified in “chloroplast”, “CUL4-RING ubiquitin ligase complex”, and “membrane” compartments. Moreover, the highest 10% high-confident S-nitrosylation sites were extracted for further study. 3190 sites were predicted in 3005 target proteins. These candidates were detected also in all compartments and ranged from 17% to 5% of the total protein content per compartment. These targets were enriched in “chloroplast”, “intracellular”, and “plasmodesmata”. The high number of identified candidates compared to the total number of proteins reflects the importance of redox-signalling in the particular compartments. In general, the currently available algorithm seems to be useful tool to characterize the S-nitrosylome. However, still need to be improved to be accurate in the identification of S-nitrosylation sites. Interestingly, computational prediction of candidates for S-nitrosylation from Arabidopsis can offer a starting point for experimental verification and further studies of S-nitrosylation in plants. Probably the combination of both computational predictions and experimental verifications could be a good approach to get insight into the molecular mechanisms and the regulatory function of S-nitrosylation in plants. Manuscript has been submitted to Plos One (resubmission process).
The bZIP transcription factor TGA1 and the regulatory protein NPR1 together are playing a crucial role in systemic acquired resistance during the inducible plant defense response. Both proteins are regulated in a redox-dependent manner, and it has been reported already the enhanced DNA-binding activity of TGA1 in presence of S-nitrosoglutathione. However, neither the molecular mechanisms nor the structural alterations responsible for these effects are unknown. It is very important to identify the 3-dimensional structure of NPR1 and TGA1 to could clarify the exact mechanism of how nitric oxide regulates NPR1-TGA1 activity in plant defense. This could be very useful for improving plant resistance in the future. The purified recombinant proteins showed an adequate purity grade, a high amount of both proteins will be produced for crystallization. Scaling-up procedure is in progress. (Figure 2 in attached file).
The project was published at the institute of Biochemical Plant Pathology web-site. Scientific students were contacted me showing their interest to do the PhD thesis in this project. Moreover, my results were presented in Fascination of Plants Day, the event was open to general public where they showed the interest to my data. The Project was also discussed with the students at the Technical University of Munich. Additionally, I was invited by Dr. Marek Petřivalský from the University of Olomouc to give a seminar. The visit was published in web-site of the University. The audience showed the interest to the presentation. Afterwards, I had a workshop in the Department of Biochemistry at the same University. I presented an overview of methods and protocols related to nitric oxide research in plants. I had a discussion with the members and PhD students of the department. Moreover, the project will be presented as oral communication in the 5th Plant NO Club Meeting which takes part on 24th of July in Munich, Germany.
Project website address:
http://www.helmholtz-muenchen.de/biop/research-groups-units/redox-signalling-and-antimicrobial-peptides/no-dependent-protein-translocation/index.html(s’ouvre dans une nouvelle fenêtre)
http://www.helmholtz-muenchen.de/biop/research-groups-units/redox-signalling-and-antimicrobial-peptides/analysis-of-no-dependent-structural-alternations-of-npr1-and-tga1/index.html(s’ouvre dans une nouvelle fenêtre)
Scientist in charge:
Dr. Christian Lindermayr
lindermayr@helmholtz-muenchen.de
Tel: +498931872285
Fax: +498931873383
Marie Curie Fellow:
Mounira Chaki
mchaki@hotmail.com, mounira@ujaen.es
Tel: +34667699202
References
1. Stamler JS, Singel DJ, Loscalzo J (1992) Biochemistry of nitric oxide and its redox-activated forms. Science 258: 1898-1902.
2. Wendehenne D, Pugin A, Klessig DF, Durner J (2001) Nitric oxide: comparative synthesis and signaling in animal and plant cells. Trends Plant Sci 6: 177-183.
3. Delledonne M, Xia Y, Dixon RA, Lamb C (1998) Nitric oxide functions as a signal in plant disease resistance. Nature 394: 585-588.
4. Durner J, Wendehenne D, Klessig DF (1998) Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose. Proc Natl Acad Sci U S A 95: 10328-10333.