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

黄草是一种双子叶（阔叶）植物，利用高效的C4光合途径。C4植物具有特殊的叶片解剖结构，由两种光合细胞组成，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，将二氧化碳集中在初级碳固定酶核酮糖1,5二磷酸羧化酶（Rubisco）附近的叶b细胞中。C4途径需要编码Rubisco酶的红细胞基因的细胞类型特异性表达，从而导致其在叶b细胞中的特异性定位。PI实验室先前的研究表明，C4红细胞基因表达模式在很大程度上是由转录后水平的调控决定的，包括对红细胞mRNA翻译和稳定性的控制。这个新项目将在转录后水平上研究介导红细胞mRNA特异性C4表达模式的分子过程。通过生物瞬时表达和转基因C4植物，研究细胞类型特异性红细胞mRNA的定位、功能和利用。为了实现这些目标，已经制备了包含FbRbcS mRNA （FbRbcS1）的特定区域的表达构建体，该区域与绿色荧光蛋白（GFP）报告基因相连。这些构建体将用于识别和表征FbRbcS1转录物中发生的顺式作用调控区域。本研究已经确定fbrbcs15 ‘和3’非翻译区（UTRs）本身赋予了强大的bs特异性GFP蛋白和mRNA的积累，这为转录稳定性调控是bs特异性基因表达的主要决定因素提供了强有力的证据。基于这些新发现，该项目将扩展到分离与FbRbcS1 mRNA特定区域相互作用以介导转录后C4表达模式的调节蛋白。C4植物在光合作用中将大气中的二氧化碳有效地同化为生物上有用的分子，特别是在高温条件和边缘干旱环境下。这项研究将为C4植物独特的光合基因表达模式和增强的固碳能力的遗传过程提供令人兴奋的新信息。了解这种特殊光合途径的分子基础将有助于了解这些植物如何能够在高温和水分胁迫的条件下茁壮成长，这可能严重限制许多利用更常见和不太专门的C3途径的作物植物的光合生产力。该研究将为提高重要农艺作物的光合效率和对边缘生境的适应性提供新的见解。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C4的一些特性设计到农业上重要的C3作物物种中，产生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.