The Role of Copper and Transcriptional Regulatory Networks Governing Copper Homeostasis in Pollen Fertility in A. thaliana.

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The global demand for high-yielding crops is increasing due to current population growth, a trend that is forcing the utilization of marginal lands for agricultural purposes. For instance, limited copper availability in the soil causes copper deficiency in crop plants, a condition that leads to plant infertility and consequently low yields; however, total crop failure can be expected under acute copper deficiencies. Although a great deal is known about how plants acquire copper from the soil, and how it is mobilized within the plant, not much is understood about how copper affects plant fertility. This project will use interdisciplinary approaches to provide fundamental insights on the role copper plays in pollen development and fertility in the model plant Arabidopsis thaliana. More specifically, this project will also help to explain how two proteins, SPL7 and the newly discovered CITF1, coordinate copper uptake and delivery to the reproductive organs of A. thaliana, and how they orchestrate pollen development and fertility. This new knowledge and understanding will facilitate the development of crops with better grain yields in soils currently under cultivation or marginal soils. Research opportunities will be offered to undergraduate and graduate students with a special effort to attract women and underrepresented minorities. A multi-media web module "Mineral nutrition: from plants to humans" will be developed with the intent to increase the awareness of high school students and K-12 teachers about the role of micronutrients in plant growth and development as it pertains to food safety and human well-being. Although copper is recognized as an essential micronutrient for plant fertility, very little is known about copper delivery to reproductive organs, or about transcriptional regulatory networks that coordinate copper homeostasis and plant reproduction. In Arabidopsis thaliana, SPL7 is the only transcription factor known to function in copper homeostasis. This project is based on the recent discovery of CITF1 in the PI's lab. CITF1 is a member of the bHLH family of TFs that enables A. thaliana to survive copper deficiency. Preliminary evidence indicates that CITF1 interacts with the SPL7-pathway. Disrupting this interaction causes seedling lethality and pollen sterility. The sites of copper action in anthers and pollen, and the role played by SPL7- CITF1 in pollen development will be characterized through the analysis of mutant lines grown under different copper levels. Synchrotron X-ray fluorescent microscopy will be used to image (2D & 3D) copper distribution during pollen development. In-air scanning electron microscopy and histological analyses will be used to characterize copper-level effects on the anatomy and morphology of control and mutant lines. Targeted chromatin-immunoprecipitation and functional genetic assays will identify direct upstream regulators of CITF1. Global expression profiling coupled with computational co-expression network analyses will reveal likely downstream copper-responsive targets of SPL7 and CITF1, the hierarchy of regulatory components and their relationship to copper uptake, and organ partitioning and delivery. The long-term goals are to understand the regulatory and signaling networks underlying copper homeostasis, and to apply this knowledge to produce cultivars adapted to soils with low copper availability.