The rbcL RNA s1-binding domain protein (RLSB) in C3 and C4 plants; Functional characterization, phylogeny, and analyses of its role in C4 evolution
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The discovery by our laboratory of the rbc L RNA S1-binding domain protein (RLSB) as an important regulatory factor controlling the expression of the chloroplast rbc L gene (encoding LSU of Rubisco) represents a significant step toward understanding photosynthetic gene expression in both C 3 and C 4 plants. The topics covered in this dissertation include characterization of RLSB binding specificity and the effects of altering its expression on the regulation of rbc L. Research presented here comprehensively establishes for the first time the importance of this S1-mRNA binding domain protein in the regulation of Rubisco, a key enzyme responsible for photosynthetic carbon assimilation in all plants. Phylogenetic analyses shows the presence of RLSB like sequences in all terrestrial plant taxa examined, and in an algal group ( Charophyta ), closely linked to early land plant evolution. This widespread distribution and high level of conservation provide strong evidence that RLSB originated just prior to the terrestrial adaptation of plants, and has been retained throughout all terrestrial plants. Results presented here also explore the possible role of this protein in the evolution of C 4 photosynthesis. This dissertation is divided into five chapters: Chapter 1, the Introduction , reviews general concepts of photosynthesis and processes associated with the regulation of gene photosynthetic gene expression. This chapter covers anatomical and biochemical features involved in this complicated multistep biochemical process. Information regarding the major pathways of photosynthesis, C 3 , C 4 and CAM, and the environmental conditions under which these occur are discussed, as well as anatomical and biochemical features associated with each pathway. The regulation of photosynthetic gene expression and known factors associated with this regulation are reviewed, with a primary focus on genes encoding the large and small subunits of Rubisco. This chapter introduces RLSB, the protein which is the focus of my studies, comparing it with other known Rubisco regulatory factors. The final section presents the overall goals of this study. Chapter 2 is titled “A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C 4 plants” is a published article in the journal BMC Plant Biology. I am a co-author and listed as an equal contribution author based on my extensive contributions to this study. My specific contributions to the manuscript are listed at the beginning of the chapter. This chapter covers the discovery and initial functional characterization of RLSB. Experiments, findings, and conclusions presented in this chapter have established RLSB as an important positive regulator of rbc L gene expression. More specifically, the chapter presents data about the co-localization of RLSB with LSU in C 3 and C 4 plants, and demonstrates it selective binding to rbc L mRNA. It details how rbc L expression is affected in the loss-of-function RLSB mutants in the C 3 dicot Arabidopsis and the C 4 monocot Maize. The data presented here are mostly from biochemical and molecular studies that reveal the close association between RLSB binding and post-transcriptional control of rbc L gene expression. Chapter 3, is titled “Evolution of RLSB, a nuclear-encoded S1 domain RNA binding protein associated with post-transcriptional regulation of plastid-encoded rbcL mRNA in vascular plants”. This chapter goes a step further and explores the evolution of this important regulatory RNA binding protein through analysis of phylogeny, conservation, synteny, and gene copy number. The findings presented in this chapter indicate that RLSB and its surrounding genomic region are highly conserved in all land plants, suggesting a critical function that has been retained throughout plant evolution. It is also present in Charophyta, an algal group closely associated with the early evolution of land plants, indicating an ancient plant-specific origin for this protein. The chapter also analyzes the variation in the RLSB gene copy number in some dicot and monocot species, and explores the significance of these variations. A range of phylogenetic tools were used to achieve the data and arrive at the conclusions presented this chapter. This RLSB evolution project was conceived and initiated by me, with the current study also involving collaborations with colleagues who are experts in the fields of molecular evolution and phylogenetics. My specific contributions are listed at the beginning of the chapter. Chapter 4 is titled, “C 3 to C 4 evolution in Flaveria species: A possible role for RLSB, a nuclear-encoded RNA binding protein associated with post-transcriptional rbc L gene expression in chloroplasts”. This is a comparative analysis of RLSB and Rubisco LSU co-localization in different species of genus Flaveria that possess C 3 , C 3 -C 4 , C 4 -like, or full C 4 type photosynthesis. The goal of this analysis was to determine if RLSB, as an rbc L regulatory factor, might have had a role in the evolution of bundle sheath cell-specificity that characterize Rubisco gene expression in C 4 plants. Additional analysis of RLSB and Rubisco LSU co-localization in photosynthetic and non-photosynthetic tissues of C 3 and C 4 Flaveria species, together with their co-regulated synthesis and accumulation in response to light, provide further evidence for the tight relationship between these proteins. The work discussed in this chapter was done by myself, working with a Masters Student, Erin Agar, who worked under my supervision and direction. My specific contributions are discussed at the beginning of the chapter. Chapter 5 of this dissertation discusses the significance and implications of RLSB as regulatory factor associated with the production of Rubisco. The primary conclusions from the studies work presented in the chapters 2, 3 and 4 are summarized. This chapter also suggests future directions that might be pursued to further the understanding RLSBs role as a regulatory factor and the possible mechanisms through which it regulates the production of Rubisco.