Post-transcriptional control of C4 RbcS Gene Expression in Flaveria bidentis

黄花是一种利用高效C4光合作用途径的双子叶(阔叶)植物。C4植物具有特殊的叶片解剖结构，由两种光合作用细胞组成，即束鞘细胞(Bs)和叶肉细胞(MP)。这些细胞为“二氧化碳泵”提供了框架，该泵将二氧化碳集中在主要的碳固定酶--核酮糖-1，5-二磷酸羧基酶(Rubisco)附近的叶BS细胞中。C4途径需要编码Rubisco酶的RBCs基因在细胞类型上的特定表达，导致其在叶BS细胞中的特异性定位。PI实验室之前的研究表明，C4RBC基因的表达模式在很大程度上是由转录后水平的调控决定的，包括控制RBC mRNA的翻译和稳定性。这一新项目将在转录后水平上研究调节红细胞C4基因表达模式的分子过程。将利用生物瞬时表达和转基因C4植物来研究特定细胞类型的红细胞mRNA的定位、功能和利用。为了实现这些目标，已经准备了包含FbRbcS mRNA(FbRbcS1)的定义区域的表达构建体，该区域与绿色荧光蛋白(GFP)报告基因相连。这些结构将用于识别和表征FbRbcS1转录本中出现的顺式作用调节区。这项研究已经确定FbRbcS15‘和3’非翻译区(UTRs)本身具有很强的bs特异性GFP蛋白和mRNA的积累，这提供了强有力的证据，表明转录稳定性的调节是bs特异性基因表达的主要决定因素。基于这些新发现，该项目将扩大到分离与FbRbcS1 mRNA特定区域相互作用的调控蛋白，以介导转录后C4表达模式。C4植物在将大气中的二氧化碳光合作用同化为生物有用分子方面非常有效，特别是在高温和边缘干旱环境下。这项研究将提供有关C4植物独特的光合作用基因表达模式和增强的固碳能力的遗传过程的令人兴奋的新信息。了解这种特殊的光合作用途径的分子基础将有助于深入了解这些植物如何能够在高温和水分胁迫条件下茁壮成长，这可能严重限制许多利用更常见和较不专门的C3途径的作物的光合作用生产力。这项研究将为提高光合作用效率和提高对边缘生境的适应能力提供新的见解。如果能够在C4植物中阐明高水平、细胞特异性基因表达模式的机制，那么最终有可能将某些C4特性改造成对农业具有重要意义的C3作物物种，生产出具有改善二氧化碳同化作用的人工C3-C4中间体。此外，该项目还将为分子生物学、植物科学和光合作用领域的新科学家提供教育、培训和职业发展。

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.