Location: Plant Gene Expression Center2012 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.
3. Progress Report:
Progress was made on the objective of elucidating the molecular mechanisms that occur during pollen development, pollen-pistil interactions and sperm-egg interactions. In collaboration with researchers at Univ. N. Carolina-Charlotte, we used deep sequencing technology to discover that Arabidopsis pollen expresses about 1000 more genes than previously known, that it expresses some genes that are not in the current genome annotation or that are incorrectly annotated, and that it exhibits alternative splicing. We then used the RNA-seq dataset to select candidate genes for functional analyses and thereby discovered: 1) that a pollen transcription factor is important for the cell divisions that give rise to sperm cells and 2) that a pollen-expressed protein phosphatase is localized in nuclear bodies in sperm cells. The speed of pollen tube growth can contribute to reproductive success. We 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 eventual genetic mapping of the genes underlying these differences. The work contributed to NP 301, Component 4A (Plant Biological and Molecular Processes; Functional Utilization of Plant Genomes – Translating Plant Genomics into Crop Improvement).
1. Identification and characterizaton of gamete expressed proteins. GEX1 is a plasma membrane protein first identified as a sperm-expressed protein (the name GEX stands for Gamete-expressed). ARS scientists at the Plant Gene Expression Center showed that GEX1 performs functions during both male and female gametophyte development and during early embryogenesis. Different parts of the GEX1 protein are important for these different roles; for example, the predicted GEX1 extracellular domain is sufficient and necessary for GEX1 function during the development of both gametophytes, and the predicted cytoplasmic domain is necessary for correct early embryogenesis of GEX1 in the zygote. The discovery shows that gamete-expressed proteins can have unexpected roles in later stages of plant development.
2. Lipid signaling in pollen. Autophagy is a pathway by which nutrient remobilization occurs, and it plays a key role in many physiological processes including pollen germination and tube growth. Arabidopsis thaliana PTEN, a pollen-specific protein and lipid dual phosphatase, regulates autophagy in pollen tubes by disrupting the dynamics of a key signaling lipid. PTEN is involved in tumor suppression in animals. ARS scientists at the PGEC found that overexpression of PTEN in pollen caused accumulation of autophagic bodies in pollen tubes and resulted in gametophytic male sterility. The novel role of PTEN during pollen development shows that signaling lipids are important for pollen tube growth. Manipulation of lipid signaling might be a new target to improve reproductive success.
Zoulias, N., Koeing, D., Hamidi, A., Mccormick, S.M., Kim, M. 2011. A role for PHANTASTICA in medio-lateral regulation of adaxial domain development on tomato and tobacco leaves. Annals Of Botany. 109:407-418, 2012.