Progress 03/01/11 to 08/31/13
Outputs
Target Audience: Laboratory workshop on plant biology, two mornings, summer 2013 - participants were nine 8th grade girls from socially disadvantaged groups in Holyoke, MA. Research seminars: Olomouc University, Olomouc, Czech Republic – November 2011 University of Toronto, Scarborough Campus, Canada – March 2012 Invited Meeting/Symposium Talks: Perspectives on Modern Plant Physiology Symposium: Frankfurt Germany, August 2011. 10th Anniversary Symposium of The Gregor Mendel Plant Research Institute (GMI): Vienna, Austria, November 2011. Tropical Vegetation and Rising Temperatures: Functional basis of ecological response. Smithsonian Tropical Research Institute. Panama City, Republic of Panama. May 2012. SPOT-IN: EU Framework Program on Pollen thermotolerance and crop fertility. Keynote Lecture: Frankfurt, Germany, January 2013 2013 International Symposium on Agricultural Biotechnology: Emerging topics in Plant Stress Biology. Academica Sinica, Taipei, Taiwan. May 2013. Changes/Problems: Research was slowed during this time period because of the relocation of the PIs lab to the University of Massachusetts. The lab was not functional until the summer of 2011 and new personnel were recruited to the project and had to be trained from ground zero. The project is now up and running and results from this award have been used to submit a proposal to the NSF for further baic research. What opportunities for training and professional development has the project provided? Postdoctoral Training: Dr. Shengbao Xu: March 2011-Jan 2013; Dr. Damian Guerra: April 2013 to present. Undergraduate training: Olena Gross, Michelle Rousseau, Nathen Bopp, Brett Higgins. Research Technician training: Angela Blais, Stephanie Craig. How have the results been disseminated to communities of interest? Publications, seminar and invited symposium and meeting lectures. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
In total this project has used careful analysis of GSNOR mutant phenotypes, bioinformatics of plant GSNOR genes, localization of GSNOR in Arabidopsis thaliana, and microarray analysis of a GSNOR null mutant to gain insights into the function and regulation of this critical enzyme in nitric oxide homeostasis. Arabidopsis thaliana homozygous for GSNOR loss-of-function alleles exhibit increased shoot branching stem, decreased trichome branching, reduced root growth and limited formation of lateral roots. We have carefully quantified these phenotypes in the background of two different Arabidopsis accessions and determined that they are complemented by the wild-type gene in one accession (Columbia). In addition, the mutants show reduced fertility. Reduced fertility is due in part to reduced stamen length, but even hand pollination does not achieve full seed set. Further analysis showed that a significant percentage of the GSNOR null mutant ovules are defective, with potentially incomplete formation of the gametophyte, as well as defective integuments. These phenotypes are being characterized further. Bioinformatics and structural modeling show that plant GSNORs likely localize to the cytosol, contain conserved, solvent-accessible cysteines, and tend to be encoded by a single gene. We hypothesize that three solvent exposed Cys residues may be targets of modification for regulation of GSNOR. We have determined that mutation of two of these residues to Cys does not significantly alter activity of the enzyme, and are proceeding to introduce these mutants into plants to determine if they alter plant phenotype in vivo. Complementation of the GSNOR null mutant with GFP-tagged GSNOR under control of the native promoter quantitatively rescued the morphological and fertility defects, indicating the fusion protein is a valid marker for localization of GSNOR. GSNOR-GFP showed fluorescence throughout Arabidopsis seedlings, consistent with ubiquitous expression of the protein, but with especially high fluorescence in the root tip, apical meristem and flowers. At the cellular level we observed cytosolic and nuclear fluorescence, with exclusion from the nucleolus. GSNOR with a C-terminal FLAG tag was also introduced into plants and shown to rescue the mutant phenotype. These plants are now ready to assess the presence of GSNOR-interacting proteins. Microarray analysis identified 98 up- and 117 downregulated genes (≥2-fold; p ≤ 0.01) in a GSNOR null mutant compared to wild type. Six members of the plant specific, ROXY glutaredoxins and three BHLH transcription factors involved in iron homeostasis were strongly upregulated, supporting a role for GSNOR in redox and iron metabolism. We suggest that these ROXY proteins are upregulated to reverse protein modifications that occur due to the high levels of NO and NO derived species in in the mutant. Antibodies against ROXY-specific peptides have been produced and we will continue to investigate the potential importance of these proteins. We have also obtained plants expressing redox-regulated GFP which we are crossing into the GSNOR null plants in order to better examine changes in the redox poise of GSNOR mutants. It was also observed that one third of downregulated genes are linked to pathogen resistance, providing further basis for the reported pathogen sensitivity of GSNOR null mutants. Together, these findings indicate GSNOR regulates multiple developmental and metabolic programs in plants and offer insight into putative routes of post-translational GSNOR regulation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Xu, S., D. Guerra, U. Lee, E. Vierling. S-Nitrosoglutathione reductases are low-copy number, cysteine-rich proteins in plants that control multiple developmental and defense responses in Arabidopsis. Front. Plant Sci., 4:Article 430, pp. 1-13 (2013). doi: 10.3389/fpls.2013.00430
- Type:
Other
Status:
Published
Year Published:
2013
Citation:
PDB ID: 4L0Q Crystal Structure of S-nitrosoglutathione Reductase From Arabidopsis Thaliana, C370a/c373a Double Mutant.
PDB ID: 4JJI Crystal Structure of S-nitrosoglutathione Reductase From Arabidopsis Thalina, Complex With Nad+
PDB ID: 4GL4 Crystal Structure of Oxidized S-nitrosoglutathione Reductase From Arabidopsis Thalina, Complex With Nadh
PDB ID: 3UKO Crystal Structure of S-nitrosoglutathione Reductase From Arabidopsis Thaliana, Complex With Nadh
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Progress 02/29/12 to 02/27/13
Outputs
Target Audience: Target audience is primarily scientists. Presented Research Seminar of project data at University of Toronto, Scarborough, Canada, March 2012. Presented project data in a Keynote Lecture to members of an EU Framework program on Pollen thermotolerance and crop fertility in Frankfurt, Germany, Jan 2013. Changes/Problems: Due to technical difficulties in characterizing redox modifications in theGSNOR mutant, we have not pursued this avenue further. We shifted our focus larger to understanding the fertility defect and the defect in lateral root formation. What opportunities for training and professional development has the project provided? Research Training was provided for postdoctoral research associate Dr. Shengbao Xu during this entire time period. Lucie Kubienová – March 2012. Visiting PhD student from the Czech Republic. Helped genotype transgenics and assay Cys mutants of GSNOR. Undergraduate Olena Gross worked with Dr. Xu for the summer and one semester to receive training in plant genetics and PCR. Undergraduate Michelle Russeau aided in analysis of GSNOR seed properties. UndergraduateNathen Bopp received training in basic lab maintenance tasks, but did not perofmr research specific to this project. Laboratory technician Angela Blais received training in plant genetics and performed PCR analysis. How have the results been disseminated to communities of interest? Presented Research Seminar of project data at University of Toronto, Scarborough, Canada, March 2012. Presented project data in a Keynote Lecture to members of an EU Framework program on Pollen thermotolerance and crop fertility in Frankfurt, Germany, Jan 2013. What do you plan to do during the next reporting period to accomplish the goals? Continue analysis of transgenic plants to localize GSNOR. Complete crosses to introduce ovule markers into the GSNOR mutant. Test activities of GSNOR in which Cys residues have been substituted with Ser or His, for possible introduction into plants. Deposit crystal coordinates in the protein data bank. Publish existing data.
Impacts
What was accomplished under these goals?
We overcame the low seed production of the homozygous GSNOR mutant so that we now have significant seed stocks for experimentation. We were also successful in directly transforming the homozygous GSNOR mutant and have created transgenic plants carrying a GSNOR-GFP translational fusion for localization studies of the enzyme, and plants carrying a flag-tagged enzyme to determine if the protein binds to any potential regulatory proteins in vivo. We have made the most progress on detailed phenotypic analysis of the GSNOR mutant, providing direct genetic evidence that multiple plant processes are impacted by NO homeostasis. Multibranching of shoots and reduced lateral root formation indicate perturbed hormone responses, potentially responses to auxin. This is being explored with transgenic plants expressing the auxin reporter DR5::Gus. Examination of the fertility defect shows that pollen develops normally, but ovule development is partially defective, preventing normal seed set. To investigate the basis of the fertility defect, the mutant was crossed with Gus and GFP marker lines expressing the marker in different cell types of the gametophyte. Microarray analysis reveals 270 differentially expressed genes (>2 fold, P<0.01) between hot5-2 and wild type, with a significant enrichment in genes related to redox regulation, stress responses, calcium signaling and Fe metabolism. Perturbation of redox metabolism is evidenced by increased levels of mRNA encoding six cytosolic glutaredoxins with an SCCMS active site. We hypothesize these proteins are involved in metabolism of glutathiolated proteins. We have not yet been able to detect alterations in NO or GSH modifications of proteins in hot5 mutants, although we predict this should be the case because the plants accumulate high levels of NO.
Publications
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Progress 03/01/11 to 02/28/12
Outputs
OUTPUTS: It is now possible to obtain sufficient seed from the homozygous S-nitrosoglutathione reductase (GSNOR) mutant so that diverse experiments can be more rapidly completed. The null mutant has been transformed, and homozygous lines carrying a GSNOR-GFP fusion protein are now available to localize the protein in the plant. GSNOR tagged with a FLAG affinity tag have also been generated in the null background in order to determine if interacting proteins can be identified. Yeast two hybrid analysis was completed with the GSNOR protein. Detailed phenotyping of the GSNOR null mutant was performed, examining growth in the light and dark, and extensive analysis of the reduced fertility defect was completed, including observations of defects in ovule development. Crosses are in progress to determine the interaction of GSNOR with nitrogen metabolism. Based on microarray data, specific genes that are elevated in the absence of GSNOR, are being suppressed with microRNAs in order to determine if they have related phenotypes to the mutant. These transgenic plants are setting seed. To examine changes in auxin distribution in the mutant, the DR5:GUS reporter has been introduced. To test the importance of Cys residues in GSNOR, all residues have been replaced with either Ser or His. A GSNOR inhibitor was recently defined and has been obtained to test how it may phenocopy the mutant and be useful for isolation of additional mutants in this pathway. A new population of mutagenized GSNOR seed is being produced for suppressor screening. These results have been communicated at scientific meetings, including the Annual Meeting of the Society of Plant Biologists, and at the NIFA meeting for project PIs. PARTICIPANTS: The PI has directed the project, trained students, prepared presentation materials and presented work at meetings. A postdoctoral fellow, Dr.Shengbao Xu worked full time on the project and presented initial data at the annual meeting of the American Society of Plant Biology. He completed analysis of previously obtained microarray data, has generated extensive transgenic material for further analysis and performed detailed studies of the mutant phenotype. A research technician, Angela Blais, has provided basic support for all aspects of the project. A student the laboratory of Marek Petrivalsky from the Czech Republic, spent one month in the laboratory learning about transgenic plants and working with recombinant GSNOR protein. The project is supported by interactions with the U of Arizona: R. Palanivelu (fertilization), Dept of Plant Sciences; W. Montfort (GSNOR structure) Dept of Chem & Biochem; and Dr. Marek Petrivalsky from the Czech Republic (GSNOR in tomato). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Progress was slowed during the past reporting period because the PI moved to a new institution and all new personnel had to be recruited and trained, and a new lab established.
Impacts
Nitric oxide (NO) is an endogenously produced radical gas that affects diverse physiological responses in plants, including stomatal movement, flowering and pathogen responses. Results are demonstrating how a key enzyme in nitric oxide metabolism, S-nitrosoglutathione (GSNO) reductase (GSNOR), functions to regulate nitric oxide homeostasis in plants to impact these diverse processes Microarray analysis reveals 270 differentially expressed genes (>2 fold, P<0.01) between the GSNOR mutant and wild type, with a significant enrichment in genes related to redox regulation, stress responses, calcium signaling and amino acid metabolism, as well as protein synthesis/degradation in the chloroplast (P <0.01). Perturbation of redox metabolism is evidenced by increased levels of mRNA encoding six cytosolic glutaredoxins with an SCCMS active site. These proteins are hypothesized to be involved in metabolism of glutathiolated proteins. Multi-branching and other phenotypes also indicate nitric oxide is linked to auxin signaling. Examination of the fertility defect shows that pollen develops normally, but ovule development is partially defective, preventing normal seed set. Further biochemical and genetic analysis of these phenotypes in the mutants should provide a wealth of information about the importance of nitric oxide in multiple plant processes.
Publications
- Shengbao Xu, Ung Lee and Elizabeth Vierling. (2011) Mutation of S-nitrosoglutathione reductase perturbs redox and pathogen response transcript levels and impacts fertility. ASPB Plant Biology 2011, Minneapolis, MN. http://abstracts.aspb.org/
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