Progress 03/17/06 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is to develop the ability to regulate pollen development and pollen-pistil interactions. Using yeast two hybrid screens, as well as genomic and proteomic tools, we have identified candidate proteins that play roles during these processes, and have developed tools (such as cell-specific promoters) that can facilitate functional studies of these proteins. The specific objectives of this project plan are: 1. To elucidate the molecular interactions that occur during pollen development, pollen-pistil interactions, and sperm-egg interactions, using both model (e.g., Arabidopsis) and crop (e.g., tomato, corn) species. 2. To define the mechanisms underlying hybridization barriers between species and determine if manipulating gene expression levels of key genes can overcome such barriers. Approach (from AD-416) 1) Use yeast 2-hybrid screens and biochemical techniques to characterize proteins expressed in female tissues and/or in pollen that are required for signaling through receptor kinases. Use similar techniques to identify sperm- and egg-expressed proteins that might mediate egg-sperm interactions. Develop sperm- and egg-specific promoter elements for gamete-specific gene manipulations. Use comparative genomics to identify protein domains important for specificity. Use gene disruptions to test interactions genetically. 2) Use pollination assays and microscopic imaging to determine where hybridization barriers occur between selected plant species pairs. Identify target genes that might act at these steps. Generate gene disruptions for selected candidates and assay for pollen tube growth/fertilization phenotypes. Introduce DNA constructs into transgenic plants. Use pollination assays to test for expanded hybridization potential in plants that overexpress or repress the expression of selected target genes. REPLACES 5335-21000-020-00D (4/06). Discovered that the correct regulation of gene expression of a novel gene pair in Arabidopsis is required for double fertilization. Demonstrated that a molecule called STIL is responsible for dephosphorylation of pollen receptor kinases, and that STIL also promotes pollen tube growth in vitro. Developed conditions for applying endomembrane and cytoskeleton inhibitors and markers to pollen tubes of Arabidopsis and used these methods to demonstrate that vesicle trafficking and actin dynamics are coordinately required for proper pollen tube growth. Accomplishments 01 Double fertilization is required for normal seed development. In the genome of the model plant, Arabidopsis thaliana, a gene named Kokopelli (KPL) overlaps, in an inverse direction, with a gene named ARIADNE14. Th role of KPL is to prevent ARI14 from being expressed in sperm; when ARI1 is expressed in sperm, there are defects in seed formation. KPL accomplishes this regulation because when each gene is expressed, their overlapping region forms a double-stranded RNA, and this double-stranded RNA (a so-called natural antisense small interfering RNA, or nat-siRNA) down-regulates the expression of ARI14 specifically in sperm cells. This finding by ARS researchers in Albany, CA, was the first demonstration th a cis-nat-siRNA plays a role in a developmental process, plant fertilization. This research provides a basic understanding of how plant produce seeds.
Impacts
(N/A)
Publications
- Zhang, Y., Mccormick, S.M. 2009. The Regulation of Vesicle Trafficking by Small GTPases and Phospholipids during Pollen Tube Growth. Sexual Plant Reproduction. 497, DOI 10.1007/s00497-009-0118-z.
- Zhang, Y., Mccormick, S.M. 2009. AGCVIII Kinases: at the crossroads of cellular signaling. Trends in Plant Science. 14(12):689-695.
- Guan, Y., Boavida, L.C., Mccormick, S.M. 2010. Gametogenesis. Encyclopedia for Life Sciences. Online 1/15/2010. DOI: 10.1002/9780470015902.a0002037. pub2.
- Ron, M., Alandete-Saez, M., Williams, L.C., Fletcher, J.C., Mccormick, E.J. 2010. Proper regulation of a sperm-specific cis-nat-siRNA is essential for double fertilization in Arabidopsis. Genes and Development. 24(10):24: 1010-1021.
- Zhang, Y., He, J., Lee, D., Mccormick, S.M. 2010. Interdependence of endomembrane trafficking and actin dynamics during polarized growth of Arabidopsis pollen tubes. Plant Physiology. 152(4):2200.
- Muschietti, J., Mccormick, S.M. 2010. Abscisic acid (ABA) receptors: light at the end of the tunnel. Faculty of 1000 Biology. 2:15 (doi: 10.3410/B2- 15).
- Wengier, D., Mazzeklla, M., Salem, T., Mccormick, S.M., Muschietti, J. 2010. STIL, a peculiar molecule from styles, specifically dephosphorylates the pollen receptor kinase LePRK2 and stimulates pollen tube growth in vitro. Biomed Central (BMC) Plant Biology. Online 1471-2229/10/33.
- Boavida, L.C., Mccormick, S.M. 2010. A22316 Gametophyte and sporophyte (version 2.0). Encyclopedia of Life Sciences. DOI: 10.1002/9780470015902. a0002037.pub2
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is to develop the ability to regulate pollen development and pollen-pistil interactions. Using yeast two hybrid screens, as well as genomic and proteomic tools, we have identified candidate proteins that play roles during these processes, and have developed tools (such as cell-specific promoters) that can facilitate functional studies of these proteins. The specific objectives of this project plan are: 1. To elucidate the molecular interactions that occur during pollen development, pollen-pistil interactions, and sperm-egg interactions, using both model (e.g., Arabidopsis) and crop (e.g., tomato, corn) species. 2. To define the mechanisms underlying hybridization barriers between species and determine if manipulating gene expression levels of key genes can overcome such barriers. Approach (from AD-416) 1) Use yeast 2-hybrid screens and biochemical techniques to characterize proteins expressed in female tissues and/or in pollen that are required for signaling through receptor kinases. Use similar techniques to identify sperm- and egg-expressed proteins that might mediate egg-sperm interactions. Develop sperm- and egg-specific promoter elements for gamete-specific gene manipulations. Use comparative genomics to identify protein domains important for specificity. Use gene disruptions to test interactions genetically. 2) Use pollination assays and microscopic imaging to determine where hybridization barriers occur between selected plant species pairs. Identify target genes that might act at these steps. Generate gene disruptions for selected candidates and assay for pollen tube growth/fertilization phenotypes. Introduce DNA constructs into transgenic plants. Use pollination assays to test for expanded hybridization potential in plants that overexpress or repress the expression of selected target genes. Significant Activities that Support Special Target Populations Showed that mutants which carry truncated versions of a protein disulfide isomerase have delayed embryo sac maturation and disrupted pollen tube guidance, whereas gene knockouts in this gene had no effect. Demonstrated that mutations in two different kinase genes cause pollen tubes to twist and turn, indicating that these kinases are important for normal polar tip growth; the mutant pollen tubes were also less competitive in reaching ovules. Determined which phase of pollen development is disrupted in over 50 different mutants, and identified novel mutants that affect the latest stages of the fertilization process.
Impacts
(N/A)
Publications
- Wang, H., Boavida, L.C., Ron, M., McCormick, S.M. 2008. Truncation of a Protein Disulfide Isomerase, PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance in Arabidopsis thaliana. The Plant Cell, 20:3300-3311.
- Zhang, D., Wengier, D., Shuai, B., Gui, C., Muschietti, J., McCormick, S.M. , Tang, W. 2008. The Pollen Receptor Kinase LePRK2 Mediates Growth- Promoting Signals and Positively Regulates Pollen Germination and Tube Growth. Plant Physiology, 148:1368-1379.
- Zhang, Y., He, J., McCormick, S.M. 2009. Two Arabidopsis AGC kinases are critical for the polarized growth of pollen tubes. Plant Journal. 58(3) 474:484.
- Boavida, L.C., Shuai, B., Yu, H., Pagnussat, G., Sundaresan, V., McCormick, S.M. 2009. A Collection of Ds Insertional Mutants Affecting Male Gametophyte Development and Function in Arabidopsis thaliana. Genetics. 181:1369-1385.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is to develop the ability to regulate pollen development and pollen-pistil interactions. Using yeast two hybrid screens, as well as genomic and proteomic tools, we have identified candidate proteins that play roles during these processes, and have developed tools (such as cell-specific promoters) that can facilitate functional studies of these proteins. The specific objectives of this project plan are: 1. To elucidate the molecular interactions that occur during pollen development, pollen-pistil interactions, and sperm-egg interactions, using both model (e.g., Arabidopsis) and crop (e.g., tomato, corn) species. 2. To define the mechanisms underlying hybridization barriers between species and determine if manipulating gene expression levels of key genes can overcome such barriers. Approach (from AD-416) 1) Use yeast 2-hybrid screens and biochemical techniques to characterize proteins expressed in female tissues and/or in pollen that are required for signaling through receptor kinases. Use similar techniques to identify sperm- and egg-expressed proteins that might mediate egg-sperm interactions. Develop sperm- and egg-specific promoter elements for gamete-specific gene manipulations. Use comparative genomics to identify protein domains important for specificity. Use gene disruptions to test interactions genetically. 2) Use pollination assays and microscopic imaging to determine where hybridization barriers occur between selected plant species pairs. Identify target genes that might act at these steps. Generate gene disruptions for selected candidates and assay for pollen tube growth/fertilization phenotypes. Introduce DNA constructs into transgenic plants. Use pollination assays to test for expanded hybridization potential in plants that overexpress or repress the expression of selected target genes. REPLACES 5335-21000-020-00D (4/06). Significant Activities that Support Special Target Populations Characterized 67 mutants with defects in male transmission and showed that they corresponded to genes affecting diverse functions and that the mutants had problems at various stages of pollen development and pollen tube growth. Showed that a protein from the female tissue stimulated pollen tube growth. Showed that a pollen receptor kinase was important for pollen tube growth; plants in which the expression of this gene was decreased had less pollen germination, shorter pollen tubes, and the pollen was less capable of responding to signals from the female. Showed that GEX3, a sperm- and egg-expressed protein was important for pollen tube guidance. Used a fluorescent protein tag and a sperm-specific promoter to isolate large quantities of plant sperm cells, then determined the gene expression pattern for sperm cells. Sperm cells express almost 6000 genes of the ~22,000 genes in the genome. This relates to NP 301, Component 2 and NP302, Component 2.
Impacts
(N/A)
Publications
- Boavida, L.C., McCormick, S.M. 2007. Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant Journal. 52(3):570-582.
- Alandete-Saez, M., Ron, M., McCormick, S.M. 2008. GEX3, Expressed in the Male Gametophyte and in the Egg Cell of Arabidopsis thaliana, Is Essential for Micropylar Pollen Tube Guidance and Plays a Role during Early Embryogenesis. Molecular Plant. 1(4):586-598.
- Borges, F., Gomes, G., Gardner, R., Moreno, N., McCormick, S.M., Feijo, J., Becker, J. 2008. Comparative Transcriptomics of Arabidopsis thaliana Sperm Cells. Plant Physiology. 10.1104/pp.108.125229.
- Zhang, Y., McCormick, S.M. 2007. A distinct mechanism regulating a pollen- specific guanine nucleotide exchange factor for the small GTPase Rop in Arabidopsis thaliana. Proceedings of the National Academy of Sciences. 104(47):18830-18835.
- Jasinski, S., Tattersall, A., Piazza, P., Hay, A., Martinez-Garcia, J.F., Schmitz, G., Theres, K., McCormick, S.M., Tsiantis, M. 2008. PROCERA encodes a DELLA protein that mediates control of dissected leaf form in tomato. Plant Journal. "Postprint"; doi: 10.1111/j.1365-313X.2008.03628.x
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) The long-term goal of this research is to develop the ability to regulate pollen development and pollen-pistil interactions. Using yeast two hybrid screens, as well as genomic and proteomic tools, we have identified candidate proteins that play roles during these processes, and have developed tools (such as cell-specific promoters) that can facilitate functional studies of these proteins. The specific objectives of this project plan are: 1. To elucidate the molecular interactions that occur during pollen development, pollen-pistil interactions, and sperm-egg interactions, using both model (e.g., Arabidopsis) and crop (e.g., tomato, corn) species. 2. To define the mechanisms underlying hybridization barriers between species and determine if manipulating gene expression levels of key genes can overcome such barriers. Approach (from AD-416) 1) Use yeast 2-hybrid screens and biochemical techniques to characterize proteins expressed in female tissues and/or in pollen that are required for signaling through receptor kinases. Use similar techniques to identify sperm- and egg-expressed proteins that might mediate egg-sperm interactions. Develop sperm- and egg-specific promoter elements for gamete-specific gene manipulations. Use comparative genomics to identify protein domains important for specificity. Use gene disruptions to test interactions genetically. 2) Use pollination assays and microscopic imaging to determine where hybridization barriers occur between selected plant species pairs. Identify target genes that might act at these steps. Generate gene disruptions for selected candidates and assay for pollen tube growth/fertilization phenotypes. Introduce DNA constructs into transgenic plants. Use pollination assays to test for expanded hybridization potential in plants that overexpress or repress the expression of selected target genes. REPLACES 5335-21000-020-00D (4/06). BSL 1; 7/1/05. Accomplishments Development of a robust pollen germination assay for Arabidopsis thaliana. Pollen germination in the model plant Arabidopsis thaliana was problematic, limiting the use of this model for studying genes that are required for pollen function. Scientists in the PGEC, Albany, CA found that an optimized medium and precisely controlled incubation temperatures were critical to improving the germination to ~90%, a level similar to that of widely studied plants such as tomato and tobacco. Characterization of mutants that affect pollen germination can now be easily done in this model plant. This research is relevant to Component 1, Functional Utilization of the Plant Genome: Translating Plant Genomics into Crop Improvement, and Problem Statement 1A, Advancing from Model Plants to Crop Plants, of the NP 302 Action Plan. Technology Transfer Number of New CRADAS and MTAS: 1 Number of Active CRADAS and MTAS: 1 Number of Web Sites managed: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 8
Impacts
(N/A)
Publications
- Mccormick, S.M. 2007. Seed to Seed, The Secret Life of Plants. Bioessays, 29(5):504-505.
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