Progress 05/01/03 to 04/30/09
Outputs
OUTPUTS: Several important accomplishments have been made during the course of this project: 1. Identification of two nuclear genes, MSH1 and RECA3, that control mitochondrial genome recombination in Arabidopsis. 2. Demonstration of mitochondrial recombination in five additional crop species by RNAi suppression of MSH1. 3. Complete sequencing and assembly of the Arabidopsis mitochondrial genomes of Col-0, Ler, and a mitochondrial mutant form msh1. 4. Identification of all mitochondrial intermediate repeated sequences in Arabidopsis that undergo asymmetric recombination under conditions of relaxed MSH1 gene expression. 5. Characterization of global gene expression changes associated with mitochondrial genome rearrangement activity. 6. Identification of novel stress response and developmental pathways altered in response to changes in mitochondrial status in Arabidopsis and five other crop species. PARTICIPANTS: Several graduate students were trained on this project: 1. Dr. Ricardo Abdelnoor, Ph.D. conferred 2006. Currently a Research Leader on the Soybean Project at EMBRAPA. 2. Dr. Ajay Sandhu, Ph.D. conferred 2008. Currently a Discovery Postdoctoral Researcher at Pioneer Hibred Intl. 3. Dr. Xuehui Feng, Ph.D. conferred 2008. Currently a postdoctoral trainee at Danforth Center, St. Louis. 4. Dr. Saleem Mohammed, Ph.D. conferred 2008. Currently a Research Scientist at Monsanto Corp. 5. Dr. Vikas Shedge, Ph.D. conferred 2009. Currently a Postdoctoral Research Associate at DuPont Co., Delaware. 6. Veronica Murray, M.S. conferred 2007. Currently a Research Technician at Dow, Indianapolis. 7. Bryan Munoz-Castillo, M.S. Conferred 2007. Currently an International Grants Specialist for Costa Rica. 8. Wilson de Paula, M.S. Conferred 2009. Currently pursuing Ph.D. training at University of London. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Our initial plan was to emphasize the mitochondrial proteome, but the resolution was found to be much greater with emphasis on the transcriptome. Consequently, most of our work was focused on cataloging gene expression changes at the transcript level.
Impacts
The following are novel insights gained from this project: 1. We have shown that plant mitochondrial genetic variation is controlled by the activity of particular, identifiable nuclear genes. 2. This mitochondrial genetic variation influences plant stress responses and developmental processes. 3. Developmental pathways influenced by mitochondrial status in plants appear to be conserved. We have shown similar effects of mitochondrial perturbation on development in Arabidopsis, sorghum, millet, soybean, tobacco and tomato. 4. Our results have implications for plant adaptive mechanisms. We have shown that MSH1 expression is responsive to environmental changes, implying that plants under stress have the capacity to induce mitochondrial genomic changes that might, in turn, influence developmental and stress responses in the plant.
Publications
- Abdelnoor, R.V., Yule, R., Elo, A., Christensen, A., Meyer-Gauen, G. and Mackenzie, S. 2003 Substoichiometric Shifting in the Plant Mitochondrial Genome is influenced by a Gene Homologous to MutS. Proc. Natl Acad. Sci. USA 100:5968-5973.
- Elo, A., Lyznik, A., Gonzalez, D.O., Kachman, S.D. and Mackenzie, S. 2003. Nuclear genes encoding mitochondrial proteins for DNA and RNA metabolism are clustered in the Arabidopsis genome. Plant Cell 15:1619-1631.
- Christensen AC, Lyznik A, Mohammed S, Elowsky CG, Elo A, Yule R, Mackenzie SA. 2005. Dual-domain, dual-targeting organellar protein presequences in Arabidopsis can use non-AUG start codons. Plant Cell10:2805-16.
- Mackenzie SA. 2005. Plant organellar protein targeting: a traffic plan still under construction. Trends Cell Biol. 10:548-54.
- Mackenzie, S.A, 2005. The mitochondrial genome of higher plants: A target for natural adaptation. . Diversity and Evolution of Plants, R. J. Henry, ed. CABI Publishers, Oxon, UK. Pp. 69-80.
- Mackenzie, S.A., 2005. The influence of mitochondrial genetics on crop breeding strategies. Plant Breeding Reviews Vol 25:115-138. John Wiley and Sons
- Abdelnoor, R.V., Christensen, A.C., Mohammed, S., Munoz-Castillo, B., Moriyama, H. and Mackenzie, S.A. 2006. Mitochondrial genome dynamics in plants and animals: Convergent gene fusions of a MutS homolog. J. Molec. Evol. 63(2):165-73.
- Sandhu, A.S., Abdelnoor, R.V. and Mackenzie, S.A. 2007. Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants. Proc Natl Acad Sci U S A. 104:1766-70.
- Shedge, V., Arrieta-Montiel, M., Christensen, A.C. and Mackenzie, S.A. 2007. Plant mitochondrial recombination surveillance requires novel RecA and MutS homologs. Plant Cell 19:1251-1264.
- Mackenzie, S.A. 2007. The unique biology of mitochondrial genome instability in plants, In: Plant Mitochondria (D. Logan, ed., Blackwell Publ.), Pp. 36-46.
- Wamboldt, Y., Mohammed, S., Elowsky, C., Wittgren, C., de Paula, W. and Mackenzie S. 2009. Alternative translation initiation leads to differentially targeted protein isoforms in Arabidopsis. Plant Cell 21:157-67
- Feng, X., Kaul, A., Dweikat, I. and Mackenzie, S. 2009. The mitochondrial genome of millet (Pennisetum americanum) undergoes evolutionary transition patterns that may be representative of higher plants. Theor. Appl Genet. 118:1361-70.
- Arrieta-Montiel MP, Shedge V, Davila J, Christensen AC, Mackenzie SA. 2009. Diversity of the Arabidopsis mitochondrial genome occurs via nuclear-controlled recombination activity. Genetics 183:1261-8.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Three major project outcomes have been obtained during this project to date. The first is the identification of a second component of the DNA recombination surveillance apparatus, RecA3, and the determination that both Msh1 and RecA3 participate in suppressing ectopic recombination in the mitochondrial genome of plants. The second is the manipulation of Msh1 expression in six plant species to genetically induce cytoplasmic male sterility. These species are tomato, soybean, tobacco, millet, sorghum and Arabidopsis. These observations are unprecedented. We have also observed the induction of several additional traits of potential agronomic value including heat tolerance and highly branced, nonflowering plant types. The third important discovery is that dual targeting of organellar proteins to the chloroplast and mitochondrion can utilize alternative translation initiation to control protein targeting behavior. All three of these observations are unprecedented. PARTICIPANTS: Graduate Students: Dr. Ajay Pal Sandhu, Ph.D. conferred 12/2008 Dr. Saleem Mohammed, Ph.D. conferred 12/2008 Dr. Xuehui Feng, Ph.D. conferred 12/2008 Mr. Vikas Shedge, Ph.D. candidate Ms. Veronica Murray, M.S. conferred 8/2008 Dr. Sandhu developed the CMS induction strategy and is now employed by Pioneer Hibred Intl. Dr. Saleem Mohammed carried out the bioinformatics work and is now emmployed by Monsanto Co. Dr. Feng carried out CMS induction work in Sorghum and millet and is now employed as a Postdoc at Danforth Center, St. Louis, MO. Ms. Murrray carried out molecular biology research and is now employed by Dow AgroSciences. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: We have opted to focus our work of modifications in mitochondrial genome structure and gene expression in response to alterations in expression of Msh1 and RecA3. Our original goal was to investigate these genes directly, and to implement a proteomics approach. Our rationale for changing strategy was the discovery of massive recombination induced by disruption of these two nuclear genes.
Impacts
We have now developed a stable cytoplasmic male sterile tomato line that we intend to pursue for development of seedless tomato varieties and for establishment of a hybrid seed production system in tomato. Both would have important agricultural implications for the crop and its production. We are also developing a highly branched sweet sorghum type for enhanced sugar production in a nonflowering growth habit. both types emerged from our studies of mitochondrial recombination.
Publications
- Sandhu, A.S., Abdelnoor, R.V. and Mackenzie, S.A. 2007. Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants. Proc Natl Acad Sci U S A. 104:1766-70.
- Wamboldt, Y., Mohammed, S., Elowsky, C., Wittgren, C., De Paula, W., Mackenzie, SA. 2009. Participation of Leaky Ribosome Scanning in Protein Dual Targeting by Alternative Translation Initiation in Higher Plants. Plant Cell, manuscript pending final acceptance.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Our laboratory has used Arabidopsis to investigate nuclear genes controlling mitochondrial genome processes. In the past year, we have made the following progress with this strategy: 1. We have now extrapolated our findings in Arabidopsis to tobacco, soybean, tomato, millet and sorghum. In all cases, we have successfully induced cytoplasmic male sterility to the crop by RNAi suppression of a nuclear gene in each species that represents the cognate to MSH1. MSH1 is a gene discovered in Arabidopsis that controls mitochondrial genome recombination behavior. 2. We have carried out work to investigate the relationship of MSH1 in Arabidopsis to two other nuclear genes, RECA3 and OSB1, and we have shown that these genes all participate in the control of illegitimate recombination of the mitochondrial genome. 3. We have shown that illegitimate recombination activity in Arabidopsis or tomato results in several distinct mitochondrial genome configurations (mitotypes)that undergo
sorting. These various mitotypes confer distinct physiological effects on the plant ranging from male sterility, leaf variegation, and plant stunting, to enhanced reactive oxygen species, lowered ATP, and, most remarkably, enhanced heat tolerance.
PARTICIPANTS: Vikas Shedge, to complete Ph.D. degree in December, 2008 Ricardo Abdelnoor, completed Ph.D. degree in 2006. Now a Research Leader at EMBRAPA in Brazil. Ajay Pal S. Sandhu, to complete Ph.D. degree in August, 2008. Veronica Murray, completed M.S. degree in December, 2007. Now employed at Dow AgroSciences. Saleem Mohammed, to complete Ph.D. degree in December, 2008. Bryan Munoz-Castillo, completed M.S. degree in December, 2007.
TARGET AUDIENCES: We have filed a patent on the process for transgenic induction of CMS in crop plants. We hope to make this available to industries for facilitation of hybrid breeding and containment of transgenes in crops. We have filed a disclosure on the induction of plant thermotolerance with mitochondrial genome rearrangement. We hope to pursue the underlying basis of this thermotolerance trait and to make this information available for the development of thermotolerant crop plants.
PROJECT MODIFICATIONS: We have emphasized transcriptome and mutation analysis in Arabidopsis rather than the originally proposed proteomic approaches because of the relative ease of the experimentation and the sensitivity of microarray analysis over current proteomic approaches.
Impacts
The most significant impacts of our work to date come in the demonstration that we can now induce cytoplasmic male sterility in a non-transgenic form that can be deployed. This demonstration has been made in five distinct agricultural crops: soybean, tomato, millet, sorghum and tobacco. The second signficant impact is that we have now shown that modification of the mitochondrial genome can also greatly enhance plant thermotolerance. Both traits are of significant agricultural importance and are difficult to attain through conventional breeding alone.
Publications
- Shedge, V., Arrieta-Montiel, M., Christensen, A.C. and Mackenzie, S.A. 2007. Plant mitochondrial recombination surveillance requires novel RecA and MutS homologs. Plant Cell 19:1251-1264.
- Arrieta-Montiel, M.P., Sandhu, A.P.S., Shedge, V.,Xu Y.-Z., Mohammed, S., Christensen,A.C., Schwarzlaunder, M. and Mackenzie, S.A. 2008. Profound effects of mitochondrial genome instability on plant cellular physiology and development. Plant Cell, submitted.
- Mackenzie, S.A. 2007. The unique biology of mitochondrial genome instability in plants, In: Plant Mitochondria (D. Logan, ed., Blackwell Publ.), Pp. 36-46.
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Progress 10/01/05 to 09/30/06
Outputs
The Mackenzie laboratory has successfully identified several nuclear genes that influence mitochondrial genome stability. Two of these, Msh1 and RecA3 are involved in controlling (suppressing) illegitimate, asymmetric recombination within the mitochondrial genome. We have shown a direct link between this illegitimate recombination activity and the induction of cytoplasmic male sterility, and we are now investigating the relationship of mitochondrial genome dynamics to the pollination process or source-sink relationships within the plant. We are also assessing the ecological cost to the plant of male sterility induction.
Impacts
Currently our focus has been to extend our analysis of CMS induction from tomato and tobacco to soybean and corn, where potential for economic impact may be greater for the hybrid seed industry.
Publications
- Sandhu, A., Abdelnoor, R. Mackenzie, S. 2007. Transgenic induction of mitochondrial rearrangements for cytoplasmic male sterility in crop plants. Proc. Natl. Acad. Sci. USA, in press.
- Shedge, V., Arrieta-Montiel, M., Christensen, A.C. and Mackenzie, S.A., 2007 Plant Mitochondrial Recombination Surveillance Requires Unusual RecA and MutS Homologs, Plant Cell, revised, pending acceptance.
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Progress 10/01/04 to 09/30/05
Outputs
Progress in 2005 was made in four primary areas: 1. Molecular characterization of the nuclear gene MSH1 that influences mitochondrial genome stability.This study involves characterization of the MSH1 gene product and its functional domains. One of these domains, Domain VI, encodes a YIG-GIY homing endonuclease motif. Only one other example of a MutS homolog in association with a homing endonuclease activity has been observed. This second example exists in corals. This work is detailed in a Journal of Molecular Evolution manuscript that is in press. 2. Identification and characterization of a RecA-homologous locus that influence mitochondrial genome stability. We have characterized an example of reversible mitochondrial DNA rearrangement, investigated its developmental implications, and shown that disruption of mitochondrial DNA maintenance influences plant cell cycle. This manuscript is in preparation. 3. Transgenic manipulation of mitochondrial genome stability for
induction of cytoplasmic male sterility. We have shown that RNAi modulation of MSH1 gene expression in tobacco and tomato results in reproducible mitochondrial rearrangements and a phenotype of cytoplasmic male sterility. This manuscript is in preparation. 4. Organellar protein targeting: We have shown that dual targeting of proteins to mitochondrial and plastids can utilize non-AUG translation initiation and likely internal ribosome entry sites. These observations have implications for the evolution of endosymbiosis and the modulation of translation during plant development. These data were published this year in Plant Cell and Trends in Cell Biology.
Impacts
The demonstration of transgenic induction of cytoplasmic male sterility in crop plants is significant for the vegetable and agronomic crop seed industry and hybrid seed production systems. The observation of an association between mitochondrial DNA maintenance and cell cycle control in plants, if further substantiated, is significant to our understanding of nuclear-mitochondrial interactions and cell division check points in plants.
Publications
- Abdelnoor, R.V., Christensen, A.C., Mohammed, S., Munoz-Castillo, B., Moriyama, H. and Mackenzie, S.A. 2006. Mitochondrial genome dynamics in plants and animals: Convergent gene fusions of a MutS homolog. J. Molec. Evol., in press.
- Mackenzie, S.A, 2005. The mitochondrial genome of higher plants: A target for natural adaptation. . Diversity and Evolution of Plants, R. J. Henry, ed. CABI Publishers, Oxon, UK. Pp. 69-80.
- Mackenzie, S.A., 2005. The influence of mitochondrial genetics on crop breeding strategies. Plant Breeding Reviews Vol 25:115-138. John Wiley and Sons Christensen AC, Lyznik A, Mohammed S, Elowsky CG, Elo A, Yule R, Mackenzie SA. 2005. Dual-domain, dual-targeting organellar protein presequences in Arabidopsis can use non-AUG start codons. Plant Cell10:2805-16.
- Mackenzie SA. 2005. Plant organellar protein targeting: a traffic plan still under construction. Trends Cell Biol. 10:548-54.
- Vallejos, C.E., Astua-Monge, G., Jones, V., Plyler, T.R., Sakiyama, N.S., and Mackenzie, S.A. 2005. Genetic and molecular characterization of the I locus of Phaseolus vulgaris . Genetics, in press.
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Progress 10/01/03 to 09/30/04
Outputs
Three important advances have been made in the past year: 1. Characterization of organellar protein targeting for plant genetic components of DNA maintenance. We have found evidence that dual targeting of proteins to mitochondria and plastids can involve non-AUG start codons and upstream sequences that have been incorporated to the gene to influence targeting of the encoded protein. A manuscript detailing these results has been submitted. 2. A nuclear gene, MSH1, was identified last year that influences mitochondrial genome configuration. We have further characterized the gene and found it to represent a form of convergent evolution with a similar form found in corals, a family of animals. A manuscript detailing these findings is in preparation. 3. A second nuclear gene, RecA3, has been identified that operates together with MSH1 to influence mitochondrial genome configuration. This gene is a component of mitochondrial DNA recombination, providing evidence of an
association of the recombination apparatus in mitochondrial DNA replication functions. Detailed study of RecA3 action is underway. In addition, we are pursuing additional components of mitochondrial DNA maintenance using analysis of protein-protein interactions, analysis of protein functional domains, and transgenic RNAi gene suppression methods.
Impacts
We anticipate that the identification of the MSH1 locus in various plant species will permit us to develop a strategy for the transgenic induction of cytoplasmic male sterility mutations by directing mitochondrial nonhomologous recombination. The characterization of the dual domain targeting presequence structure will likely impact our ability to predict targeting capacity of proteins by available prediction programs.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
We have made significant progress in three primary areas. 1. Cloning of genes involved in mitochondrial DNA maintenance. We have cloned and characterized the MSH1 locus involved in mitochondrial nonhomologous recombination, and have shown that it is a highly conserved gene that is unique to plants. We are currently investigating the function of the MSH1 gene product, and its relationship to other genes involved in mitochondrial recombination such as RecA. 2. Cloning and characterization of gene involved in mitochondrial DNA metabolism. We have conducted tandem affinity purification tagging with the RecA and SSB mitochondrial proteins, and are currently in the process of identifying the products retrieved from this procedure. 3. Characterization of organellar protein targeting. We have identified two features of dual targeting presequences that are important. Dual targeting presequences have two definable domains, one for plastid and one for mitochondrial. These
dual-domain, dual-targeting presequences may initiate translation at non-AUG start codons. These two features will now allow the refinement of protein prediction programs that will permit the more accurate prediction of protein targeting features.
Impacts
We anticipate that the identification of the MSH1 locus in various plant species will permit us to develop a strategy for the transgenic induction of cytoplasmic male sterility mutations by directing mitochondrial nonhomologous recombination. The characterization of the dual domain targeting presequence structure will likely impact our ability to predict targeting capacity of proteins by available prediction programs.
Publications
- Abdelnoor, R.V., Yule, R., Elo, A., Christensen, A., Meyer-Gauen, G. and Mackenzie, S. 2003 Substoichiometric Shifting in the Plant Mitochondrial Genome is influenced by a Gene Homologous to MutS. Proc. Natl Acad. Sci. USA 100(10):5968-5973.
- Elo, A., Lyznik, A., Gonzalez, D.O., Kachman, S.D. and Mackenzie, S. 2003. Nuclear genes encoding mitochondrial proteins for DNA and RNA metabolism are clustered in the Arabidopsis genome. Plant Cell 15: 1619-1631.
- Mackenzie, S. 2004. The influence of mitochondrial genetics on crop breeding strategies. Plant Breeding Reviews, in press.
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