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United States Department of Agriculture

Agricultural Research Service

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Location: Plant Gene Expression Center

2013 Annual Report

1a. 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. [NP 301, C 4, PS 4A] 2. To define the mechanisms underlying hybridization barriers between species and determine if manipulating gene expression levels of key genes can overcome such barriers. [NP 301, C 4, PS 4A]

1b. 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. REPLACING 5335-21000-030-00D (9/10).

3. Progress Report:
This is the final report for project 5335-21000-036-00D terminating in February 2013, which has been replaced by Project No. 5335-21000-037-00D. Progress was made on both of the project Objectives. Progress on this project focuses on addressing Problem 3A – the need for fundamental knowledge of plant biological and molecular processes. Under Objective 1, we made significant advances in elucidating the molecular interactions that occur during pollen development, pollen-pistil interactions, and sperm-egg interactions. Towards accomplishing this, we used biochemical techniques such as pulldowns and imaging techniques such as bimolecular fluorescence complementation to analyze the protein interactions of pollen receptor kinases and other pollen proteins important for pollen tube growth. Progress was made on the objective of identifying candidate genes from the RNA-Seq dataset and functionally characterizing them. Specifically, we characterized mutations in a pollen-specific transcription factor and showed that this transcription factor was important for the 2nd mitosis during pollen development, namely, the mitosis that gives rise to the two sperm cells. Plants that are mutant in this transcription factor have reduced seed set, implicating this transcription factor in controlling processes required for correct double fertilization. Additionally we characterized a family of proteins called tetraspanins. In animals, a tetraspanin is required for sperm-egg fusion. We showed that some plant tetraspanins are localized to the sperm-sperm interface in pollen grains, implicating these proteins in interactions with the two fusion partners for sperm,i.e., the egg and the central cell.

4. Accomplishments
1. Callose plug deposition in pollen tubes correlates with pollen tube growth. Efficient pollen tube growth is critical for reproductive success. This work therefore addresses the key biological question of how pollen tube growth is mechanistically controlled. ARS scientists in the Plant Gene Expression Center in Albany, California, used 14 ecotypes of Arabidopsis (ecotypes are plants collected from diverse geographical locations) and 7 different species of tomato for a comparative study of pollen tube growth parameters. Pollen tubes produce periodic callose plugs during tube growth; callose plugs are depositions of a sugar molecule. We found that callose plug deposition in pollen tubes is correlated with pollen tube growth, and showed that the patterns of deposition varies in different ecotypes of Arabidopsis and in different species of tomato. These results lay the groundwork for genetic mapping of the genes underlying these differences thus providing tools for breeders.

2. Directional pollen tube growth towards female cues relies on a protein called Cobra-like 10 (COBL10). COBL10 is anchored at the plasma membrane at the pollen tube tip by a glycosylphosphatidylinositol (GPI) group, which is a small sugar lipid. Mutations in COBL10 caused gametophytic male sterility due to reduced pollen tube growth and these pollen tubes lacked directional sensing in the female transmitting tract. We further showed that cell wall deposition at the pollen tube tip was defective. Thus pollen tube localization of COBL10 at the apical plasma membrane is critical for its function and requires proper GPI processing and the C-terminal part of the protein. Because GPI-anchored proteins are widespread cell sensors in mammals, especially during egg-sperm communication, showing that COBL10 is critical for directional growth of pollen tubes suggests that GPI-anchored proteins also play critical roles in cell-cell communication in plant reproduction.

Review Publications
Qin, P., Ting, D., Shieh, A., Mccormick, S.M. 2012. Callose plug deposition patterns vary in pollen tubes of Arabidopsis thaliana ecotypes and in tomato species. Biomed Central (BMC) Plant Biology. 12:178.

Li, S., Ge, F., Xu, M., Zhao, X., Huang, G., Zhou, L., Wang, J., Kombrink, A., Mccormick, S.M., Zhang, X.S., Zhang, Y. 2013. Aridopsis COBRA-LIKE 10, a GPI-anchored protien, mediates directional growth of pollen tubes. Plant Journal. 74:486-497.

Last Modified: 07/25/2017
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