2002 Annual Report 1.What major problem or issue is being resolved and how are you resolving it? The problem is that desirable multi-gene complexes present in one crop (or wild species) can't be transferred to other crops because genetic crossing between different species is difficult or impossible. Our overall objective is to understand pollen development so that it can be manipulated to improve agriculture. Pollen development and pollen-pistil interactions are complex processes, involving not only structural proteins, but also proteins that influence other proteins through signal recognition and signal transduction. Our specific objectives are to identify proteins that affect the early stages of pollen tube growth, and to elucidate the roles of pollen-specific receptor kinases and other signaling molecules in successful pollen germination. Although double fertilization was discovered 1898 we still know nothing about the proteins that mediate gamete recognition and fusion in plants. 2.How serious is the problem? Why does it matter? Developing the capability to regulate pollen development and pollen-pistil interactions through genetic engineering represents a high-priority goal for agricultural biotechnology. By regulating these processes precisely, genetic engineering may enable the successful hybridization of distantly related crops, production of microspore-derived haploid plants (which facilitate plant breeding) in a wider variety of crops, enhancement of the ability of pollen to survive and function under high temperatures, or the development of novel pollen sterility systems for efficient production of hybrid crops. 3.How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? 301 -- 30% Plant, Microbial & Insect Genetic Resources, Genomics and Genetic Improvement & 302 -- 70% Plant Biological and Molecular Processes. This project is related to 301 because we are identifying the roles of new genes and that goal falls under plant germplasm development. This project is related to 302 because manipulating gene expression will improve plant biological molecular processes. Research focuses on tomato, maize and the model plant Arabidopsis because: (1) Tomato is the most important vegetable crop in the US. The tomato genome and those of other Solanaceous crops (e.g., potato, pepper) share similar genetic repertoires so elucidating pollen development and pollen-pistil interactions in tomato may greatly facilitate research with other major crops; (2) Maize is the most important grain crop in the US. The availability of a cDNA library from maize sperm makes maize ideal for the study of gamete interactions in plants; (3) The genome sequence of Arabidopsis is complete and this therefore offers an invaluable resource for identifying gene homologs in other species. Furthermore, research to date suggests that, at a molecular level, the regulatory mechanisms for pollen development and pollen-pistil interactions may be particularly uniform across a very broad range of plant species, so that results of this research may be even more widely applicable than their similar genetic repertoires may indicate. If we identify a gene in tomato or maize and demonstrate that modification of its expression would be useful in other crop plants (e.g. for hybrid seed production, for haploid plant generation), it will be quite easy to obtain the homologous gene from the other crop plant and to generate transgenic plants for modified expression. Conversely, identification of mutations in the model plant Arabidopsis will facilitate the isolation of homologous genes from the crop species. Knowledge of gene control regions, such as promoters, may help direct the expression of a variety of pollen and pistil traits, whereas knowledge of the coding regions may be used to directly alter specific pollen developmental phases or pollen-pistil interactions. In the course of this work novel promoters will be isolated and characterized. These promoters may provide substitutes for proprietary materials for commercializing publicly developed transgenic plants. 4.What was your most significant accomplishment this past year? A. We postulate that pollen receptor kinases help mediate pollen tube growth and our goal is to better understand signaling between pollen and pistil during pollen tube growth. In the course of our work, the McCormick lab at the PGEC in Albany, CA, isolated and characterized novel promoters. We identified ligand candidates and downstream protein partners for pollen receptor kinases and confirmed interactions for several of these proteins. Manipulation of these signaling pathways might help expand interspecific hybrids for crop improvement. B. We are attempting to gain further understanding of signaling between pollen and pistil during pollen tube growth. To further our research, the McCormick lab at the PGEC in Albany, CA, has secured funding from NSF for "Functional Analyses of Plant Gamete Gene Expression." The data in the grant submission was generated as a result of a headquarter's funded postdoctoral fellowship (class of 2000). Determination of the genes expressed in sperm cells will be useful for the regulation of pollen fertility and hybrid production. C. Significant Accomplishments/Activities that Support Special Target Populations: NONE. D. None. 5.Describe your major accomplishments over the life of the project, including their predicted or actual impact? 1) Isolation of mutations that disrupt pollen development or germination. Raring-to-go prematurely germinates within the anther. A molecular understanding of raring-to-go and other novel mutants (polka dot pollen, giftwrapped pollen and emotionally fragile pollen) may provide novel insights into the earliest steps in pollen germination, which is a crucial and highly regulated step at the beginning of pollen-pistil interactions. 2) Development of receptor-like kinase constructs and of antibodies raised against the extracellular domains of the pollen kinases are critical tools that will be necessary for determining the functions of signal transduction during pollen- pistil interactions. 3) Demonstration that yeast two hybrid screening can be successfully used to identify proteins that interact extracellularly (i.e. receptors and their ligands) has encouraged other research groups to use this approach for their specific research projects. 4) Initiated analysis of sperm-egg interactions, to identify the sperm-surface proteins that recognize the egg and central cell. Project will provide gamete- specific promoters to manipulate gamete gene expression and perhaps thereby expand interspecies hybrids for crop improvement. It will improve annotations of sequenced plant genomes. 6.What do you expect to accomplish, year by year, over the next 3 years? . 1)Complete biochemical characterization of the kinase interaction clones that we have identified by yeast two-hybrid library screening. Reconstruct signaling pathway in other cells, to facilitate analyses. Test candidate proteins for biological relevance during pollen-pistil interactions, using mutational analyses and the development of bioassays for pollen tube growth chemotaxis. . 2)Complete cloning and cell biological characterization of the raring-to-go, giftwrapped pollen, polka dot pollen and emotionally fragile pollen, mutations in Arabidopsis that initiate pollen tube growth precociously, inside the anther. Perform suppressor and enhancer screens to identify other components of these pathways. . 3)Characterize sperm and embryo sac genes. Develop assays to test for interactions between egg and sperm that are mediated by surface-localized molecules on the two cell types. 7.What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? I provided pollen-specific promoter constructs to many academic and government labs. I provided cDNA libraries to several academic labs. Transfer of technology developed from our research may in some cases require acceptance of transgenic plants. 8.List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) McCormick, S. 2002. Sporophyte and gametophyte. Encyclopedia of Life Science, Web-based, MacMillan Publishers. Tsiantis, M., Hay, A., Ori, N., Kaur, H., Henderson, I., Holtan, H., McCormick, S. and Hake, S. (2002). Developmental signals regulating plant form. In Developmental Genetics and Plant Evolution. Editors: Cronk, Q.C.B., Bateman, R.M and Hawkins, J.A. Taylor and Francis, London. Eckardt, N.A., Cho, H-T., Perrin, R. M. Willmann, M.R. Plant Biology 2001(meeting report, mentions our pollen kinase project - presented in mini-symposium at the annual meeting of Plant Biologists). Plant Cell 13: 2165-2173. Review Publications Magnard, J-M., Yang, M., Chen, Y-C. S., Leary, M. and McCormick, S. 2001. The Arabidopsis thaliana gene Tardy Asynchronous Meiosis (TAM) is required for the normal pace and synchrony of cell division during male meiosis. Plant Physiol. 127: 1157-1166. KIM, H., COTTER, R., JOHNSON, S., SENDA, M., DODDS, P., KULIKAUSKAS, R., TANG, W., EZCURRA, I., HERZMARK, P., MCCORMICK, S.M. NEW POLLEN-SPECIFIC RECEPTOR KINASES IDENTIFIED IN TOMATO, MAIZE AND ARABIDOPSIS: THE TOMATO KINASES SHOW OVERLAPPING BUT DISTINCT LOCALIZATOIN PATTERNS ON POLLEN TUBES. PLANT MOLECULAR BIOLOGY. 2002. 33:12:1641-1650. Yang, M. and McCormick, S. 2002. The Arabidopsis thaliana MEI1 gene likely encodes a protein with BRCT domains. Sexual Plant Reprod. 14: 355-357. Published online March 15, 2002 (DOI 10.1007/s00497-002-0129-5).