Flaveria bidentis is a dicotyledonous (broadleaf) plant that utilizes the highly efficient C4 photosynthetic pathway. C4 plants possess a specialized leaf anatomy consisting of two photosynthetic cell types, the bundle sheath cells (bs) and mesophyll (mp) cells. These cells provide the framework for a "CO2 pump" that concentrates CO2 in leaf bs cells in the vicinity of the primary carbon fixation enzyme, ribulose 1,5 bisphosphate carboxylase (Rubisco). The C4 pathway requires cell type-specific expression of RbcS genes that encode the Rubisco enzyme, leading to its specific localization in leaf bs cells. Previous research from the PI's laboratory has shown that C4 RbcS gene expression patterns are determined in large part by regulation at post-transcriptional levels, including control of RbcS mRNA translation and stability. This new project will investigate molecular processes that mediate the specialized C4 expression patterns of RbcS mRNA at post-transcriptional levels. Cell type-specific RbcS mRNA localization, function, and utilization will be investigated using biolistic transient expression and transgenic C4 plants. To accomplish these goals, expression constructs have been prepared that contain defined regions of an FbRbcS mRNA (FbRbcS1), linked to a green fluorescent protein (GFP) reporter gene. These constructs will be used to identify and characterize cis-acting regulatory regions that occur within the FbRbcS1 transcript. This research has already determined that the FbRbcS1 5' and 3' untranslated regions (UTRs) in themselves confer strong bs-specific accumulation of GFP protein as well as mRNA, providing strong evidence that regulation of transcript stability is a major determinant of bs-specific gene expression. Based on these new findings, this project will be expanded to isolate regulatory proteins that interact with specific regions of FbRbcS1 mRNA to mediate post-transcriptional C4 expression patterns. C4 plant species are very efficient in the photosynthetic assimilation of atmospheric CO2 into biologically useful molecules, especially under conditions of high temperatures and in marginal arid environments. This study will provide exciting new information about genetic processes responsible for the unique photosynthetic gene expression patterns and the enhanced carbon-fixation capabilities of C4 plant species. Understanding the molecular basis of this specialized photosynthetic pathway will provide insights into how such plants are able to thrive under conditions of high temperature and water stress, which can severely limit photosynthetic productivity in many crop plants that utilize the more common and less specialized C3 pathway. This research will provide new insights for improving photosynthetic efficiency and adaptability to marginal habitats for agronomically important crop species. If mechanisms responsible for high-level, cell-specific gene expression patterns can be elucidated in C4 plants, then ultimately it may be possible to engineer some C4 characteristics into agriculturally-important C3 crop species, producing artificial C3-C4 intermediates with improved CO2 assimilation. In addition this project will provide education, training, and career development for new scientists in the fields of molecular biology, plant science, and photosynthesis.
