Progress 09/01/08 to 08/31/11
Outputs
Target Audience: Presentations: Adaptation Potential in Plants: Gregor Mendel Institute, Vienna Austria, March 2009. Gordon Conference: Stress Proteins in Growth, Development and Disease, Andover, NH. July, 2009. Keystone Conference: Plant Abiotic Stress Tolerance Mechanisms, Water and Global Agriculture, CO, Jan 2011. Perspectives on Modern Plant Physiology Symposium: Frankfurt Germany, August 2011. Changes/Problems: The PI spent 22 months of the grant period as a Program Officer at the NSF. This slowed progress on the proposed project. The PI moved from The University of Arizona to the University of Massachusetts in January 2011. This required a complete change of personnel, which also slowed progress on the award. This report is meant to reflect work completed at the University of Arizona. A separate report was submitted for the remaining time on the grant through the University of Massachusetts. What opportunities for training and professional development has the project provided? Undergraduates have presented work from this project in university science poster sessions. Products: Antibodies have been generated to detect S-nitroglutathione reductase and are available to the community of plant scientists. The high resolution structure of S-nitroglutathione reductase from a higher plant (Arabidopsis) has been determined. The coordinates of this structure have been deposited in the public database. A new collaboration was been initiated with MarekPetrivalsky from the Czech Republic. Dr. Petrivalsky studies S-nitroglutathione reductase and pathogen infection in tomato. He obtained a grant from the Czech government for collaborative research and will be sending a student to the US and will also visit my laboratory. A research technician, Deborah Raynes, has generated all of the recombinant DNA vectors necessary for the project. A postdoctoral Fellow, Dr. Ung Lee performed all of the research involved in the Plant Cell publication. How have the results been disseminated to communities of interest? Seminars and publications. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Analysis of the S-nitrosoglutathione reductase mutant is providing new insights into nitric oxide metabolism and its impact onother processes in plants. Data from microarray analysis of gene transcript levels in the S-nitrosoglutathione reductase mutantlinks activity of this enzyme to glutathione metabolism. The analysis shows that of 129 genes that are significantly up-regulated (more than two-fold), six of the highest up-regulated genes encode glutaredoxins, small enzymes that are oxidizedby substrates and non-enzymatically reduced by glutathione. Glutaredoxins are proposed to be involved in glutathiolation anddeglutathiolation, which would be perturbed in plants with elevated S-ntirosoglutathione, which is the case for the S-nitrosoglutathione mutants. Another of the most highly upregulated genes is asparagine synthetase, critical to nitrogen metabolism. Interestingly, nitrate reductase transcript levels were found to be upregulated, but the nitrate reductase protein and activity were significantly decreased in the mutant. Because nitric oxide is also produced from nitrate, the absence of S-nitrosglutathione may be expected to perturb nitrogen metabolism. Analysis of the fertility defect in S-nitrosoglutathione mutants indicates that there is both a male and female defect. Vectors have been produced to tag GSNOR with GFP for localizing expression in plants. In addition, GSNOR has been tagged with a FLAG epitope for use in isolation of GSNOR associated proteins.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2008
Citation:
Lee, U., C.Wie*, B. O. Fernandez, M. Feelisch, E. Vierling. Modulation of nitrosative stress by S-nitrosoglutathione reductase is critical for thermotolerance and plant growth. Plant Cell 20: 786-802, (2008).
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Activities: Genetic material has been generated to investigate the floral defect in S-nitrosoglutathione reductase mutants. Microarray analysis has been completed to describe the changes in gene regulation in the mutant compared to wild type. Vectors have been generated to produce mutant recombinant proteins for investigation of the role of conserved cysteine residues in protein activity. To further understand regulation, we have also identified a potential suppressor mutation of a weak allele of S-nitroglutathione reductase. The mutant has a pale green phenotype that appears to co-segregate with the suppressor phenotype. A mapping population has been generated for this suppressor. Events: Undergraduate students have presented work from this project in university science poster sessions. Products: Antibodies have been generated to detect S-nitroglutathione reductase and are available to the community of plant scientists. The high resolution structure of S-nitroglutathione reductase from a higher plant (Arabidopsis) has been determined. The coordinates of this structure are available on request, and will be deposited publically after publication. A new collaboration has been initiated with Marek Petrivalsky from the Czech Republic. Dr. Petrivalsky studies S-nitroglutathione reductase and pathogen infection in tomato. He obtained a grant from the Czech government for collaborative research and will be sending a student to the US and will also visit my laboratory. Dessemination: Work on this project was presented at an international symposium: Biotechnology for Better Crops, Energy and Health, at the Academica Sinica in Taipei, Taiwan,2008. Work was also presented in a conference entitled Adaptation Potential in Plants held at the Gregor Mendel Institute, Vienna Austria, March 2009. PARTICIPANTS: The PI has directed the project, trained students, prepared presentation materials and presented work at international meeting. A research technician, Deborah Raynes, has generated all of the recombinant DNA vectors necessary for the project. A postdoctoral fellow, Dr.Ung Lee performed all research involved in the Plant Cell publication. He completed microarray analysis of the GSNOR mutant compared to WT, and performed tests of the response of the GSNOR mutant to GSH and antioxidants. In addition he completed genetic analysis of a suppressor mutation which is currently available for map-based cloning. Collaborators at the University of Arizona:R. Palanivelu (fertilization), Dept of Plant Sciences; W. Montfort (GSNOR structure) Dept of Chem & Biochem; K. Miranda (NO chemistry), Dept of Chem & Biochem; Dept of Plant Sciences. Other collaborators: J. Greenberg (pathogen responses), U of Chicago. Dr. Marek Petrivalsky from the Czech Republic (GSNOR in tomato). The project has trained three undergraduate students in plant genetics and/or protein biochemistry: Amr Badran, Tarik Ozumerzifon and Shiqi Li. TARGET AUDIENCES: The project has trained three undergraduate students in plant genetics and/or protein biochemistry: Amr Badran, Tarik Ozumerzifon and Shiqi Li.These students performed laboratory research and presented their work formally in university events. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Analysis of the S-nitrosoglutathione reductase mutant is providing new insights into nitric oxide metabolism and its impact on other processes in plants. Data from microarray analysis of gene transcript levels in the S-nitrosoglutathione reductase mutant links activity of this enzyme to glutathione metabolism. The analysis shows that of 129 genes that are significantly up-regulated (more than two-fold), six of the highest up-regulated genes encode glutaredoxins, small enzymes that are oxidized by substrates and non-enzymatically reduced by glutathione. Glutaredoxins are proposed to be involved in glutathiolation and deglutathiolation, which would be perturbed in plants with elevated S-ntirosoglutathione, which is the case for the S-nitrosoglutathione mutants. Another of the most highly upregulated genes is asparagine synthetase, critical to nitrogen metabolism. Interestingly, nitrate reductase transcript levels were found to be upregulated, but the nitrate reductase protein and activity were significantly decreased in the mutant. Because nitric oxide is also produced from nitrate, the absence of S-nitrosglutathione may be expected to perturb nitrogen metabolism. Analysis of the fertility defect in S-nitrosoglutathione mutants indicates that there is both a male and female defect.
Publications
- Lee, U., C.Wie, B. O. Fernandez, M. Feelisch, E. Vierling. Modulation of nitrosative stress by S-nitrosoglutathione reductase is critical for thermotolerance and plant growth. Plant Cell 20: 786-802, (2008).
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