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

黄顶菊是一种利用高效C4光合途径的双子叶（阔叶）植物。 C4植物具有特殊的叶片解剖结构，由两种光合细胞组成：维管束鞘细胞（bs）和叶肉细胞（mp）。 这些细胞提供了一个“CO2泵”的框架，该泵将CO2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bs细胞中。 C4途径需要编码Rubisco酶的RbcS基因的细胞类型特异性表达，导致其在叶bs细胞中的特异性定位。 PI实验室先前的研究表明，C4 RbcS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RbcS mRNA翻译和稳定性的控制。 这个新的项目将调查介导的RbcS mRNA在转录后水平的专门的C4表达模式的分子过程。 细胞类型特异性RbcS mRNA的定位，功能和利用将使用基因枪瞬时表达和转基因C4植物进行研究。 为了实现这些目标，已经制备了表达构建体，其含有与绿色荧光蛋白（GFP）报告基因连接的FbRbcS mRNA（FbRbcS 1）的限定区域。 这些构建体将用于鉴定和表征FbRbcS 1转录物内发生的顺式作用调控区。 这项研究已经确定，FbRbcS 1 5'和3'非翻译区（UTR）本身赋予GFP蛋白以及mRNA的强烈的bs特异性积累，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，本研究将进一步分离与FbRbcS 1 mRNA特定区域相互作用的调控蛋白，从而调控转录后C4的表达模式。C4植物在光合作用中能有效地将大气CO2同化为生物学上有用的分子，特别是在高温和边缘干旱环境下。 这项研究将提供令人兴奋的新信息，负责独特的光合基因表达模式和C4植物物种的固碳能力增强的遗传过程。了解这种专门的光合作用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业的C3途径的作物的光合生产力。 该研究将为提高重要农作物的光合效率和边缘生境适应性提供新的见解。 如果能够在C4植物中阐明负责高水平、细胞特异性基因表达模式的机制，那么最终有可能将一些C4特征工程化到农业上重要的C3作物物种中，产生具有改善的CO2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.