2 Identification of Potential Biomineralization Linked Genes in Emiliania huxleyi via High-Throughput Sequencing with RT-PCR Verification By Chrystal Grace Schroepfer Research Thesis Submitted for the Masters Degree in Biological Sciences Department of Biological Science, College of Science and Mathematics California State University San Marcos November 2011 3 Table of Contents Table of Contents . 2 Acknowledgements . .. 3 Abstract . 4 Introduction . 5 Coccolithophorids . 5 Biomineralization and Coccolithogenesis . 7 Sequence Profiling . 10 Methods and Materials . 14 Strains and Growth Conditions . 14 Scanning Electron Microscopy . 15 2+ - Ca Titration Estimates of CaCO3 . 16 Measuring Photosynthesis Rates . 17 RNA Extraction . 18 RNA Gel Electrophoresis . 20 Experion RNA Electrophoresis . 20 RNA Sequencing . 21 Primer Design . 23 Real Time RT-PCR . 23 Annotation . 26 Results . 27 Cell Growth . 27 Scanning Electron Microscopy . 29 Calcium Titration . 31 Photosynthesis Rates . 34 Solexa Profiles . 35 RNA Extraction & Gel Electrophoresis . 38 Comparative Reverse Transcriptase Real-Time PCR . 43 Annotation . 52 Discussion . 61 References . 77 Appendices. 86 4 Acknowledgements I would like to thank my committee members Dr. Betsy Read, Dr. Matthew Escobar and Dr. Jose Mendoza for their guidance and support throughout the thesis completion process. I would like to thank my parents, John and Mary Schroepfer, my closest friends, Steve and Jeanne Bâby, Gearald Denny, Tom Bento and my Church Family at Bostonia Church of Christ for their love, encouragement and support over the years. To my brothers, David and Jason Schroepfer, thanks for all the laughs. Lastly, I would like to thank all my friends that I made from the laboratory: Estela Carrasco, James Fuller, Jessica Garza, Karina Gonzalez, Latha Kannan, Ray Liang, Tien Nguyen, Alyse Prichard, Analisa Sarno, Andrew Segina, Christina Vanderwerken and William Whalen for their help and all the fun times we shared together. Christina and William: Thank you for being there. Christina: Thank you for being my best friend. William: Thank you for guidance with the completion of this thesis paper. Andrew: Peasant day was a blast. Thank You!!!!! 5 ABSTRACT Emiliania huxleyi and Isochrysis galbana are two of the three living Isochrysidales. E. huxleyi produces distinguishing shells of calcium carbonate called coccoliths through the process of biomineralization, while I. galbana does not. The study of the genetic underpinnings for biomineralization in coccolithophorids is in its infancy. This research is aimed at moving such study forward by identifying genes involved in biomineralization using transcript profiling via Solexa high-throughput RNA sequencing (RNAseq). Transcriptome profiles were generated from cells grown in normal and calcium deplete conditions with and without a sodium bicarbonate spike. Cell counts and rates of calcium uptake and photosynthesis confirmed previous studies that calcification and photosynthesis are independent processes. Photosynthesis rates and Ca2+ uptake were monitored during cell growth and SEM was used to determine the effect of treatments on calcification and coccolith morphology. As previously demonstrated cells grown in the absence of calcium showed fragments of coccoliths while cells grown in the presence of calcium showed well formed and intact coccoliths. 126 differentially expressed genes were identified using a negative binomial distribution to compare transcript levels of E. huxleyi cells grown under calcifying conditions in 9 mM Ca2+ as compared to non-calcifying conditions in 0 mM Ca2+. Real time RT-PCR analysis was performed to independently validate the differential expression of 25 potential biomineralization genes, 20 of which were significantly up-regulated and 3 that were significantly down-regulated. Although many of the 126 genes identified as being differentially expressed lacked significant homology to known sequences, others made biological sense with regard to biomineralization and/or coccolithogenesis. These included the differential expression of C-type lectins, titin-like proteins, glycosyltransferases and several proteoglycans. As part of this thesis work, gene predictions for cyclophilins in E. huxleyi were also manually curated and uploaded on the JGI E huxleyi genome portal. Keywords: Emiliania huxleyi, Isochrysis galbana, coccolith, coccolithophorid, coccolithogenesis, biomineralization, scanning electron microscopy, high- throughput RNA sequencing, transcriptome profiling, real-time PCR, manual gene model annotation, cyclophilin. 6 INTRODUCTION Coccolithophorids Coccolithophorids are unicellular marine algae categorized in the Haptophyta division under the Prymnesiophyceae class (Edvardsen et al. 2000). Prymnesiophyceae contains four orders which are Isochrysidales, Coccosphaerales, Prymnesiales and Pavlovales. The order Isochrysidales consists of three living species: 1) the coccolith bearing Emiliania huxleyi and Gephyrocapsa oceanica, and 2) the non-coccolith bearing Isochrysis galbana. Coccolithophorids have a dimorphic life cycle producing cells which alternate between a haploid (or motile) and diploid (or nonmotile) phase. In the nonmotile phase E. huxleyi and G. oceanica, cells share common traits such as the presence of two flagellar apparatuses of equal or subequal size, small delicate organic scale layers external to the plasmalemma, the production of coccoliths, and a small or nonexistent haptonema (Edvardsen et al. 2000). E. huxleyi and G. oceanica have similar intracellular structures, life cycles and coccolith morphology. The similar morphology of the coccoliths suggests a close phylogenetic relationship, perhaps they are even cryptic species (Fujiwara et al. 1994). As sister species, E. huxleyi and G. oceanica are identical except for one nucleotide difference in the 18s rDNA sequence 7 (Edvardsen et al. 2000). Also they are identical in the nucleotide sequences of rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase) (Fujiwara et al. 1994). Similarly, the nonmotile cells of I. galbana have body scales similar to the cell coverings in E. huxleyi and G. oceanica, which also supports a close phylogenic relationship between species. E. huxleyi, G. oceanica and I. galbana are unique among the coccolithophorids because of their shared morphological and ultrastructural characteristics. By comparison, G. oceanica is a little bit larger then E. huxleyi, has structurally different coccoliths and is more sensitive to temperature changes. By similarity, they both form large blooms and are producers of calcium carbonate deposits in the world’s oceans (Winter and Siesser 1994; Rhodes. 1995). Even though the coccolith morphology is distinctively different in the two species, they have similar structures and presumably similar calcification mechanisms (Young et al. 1999). As a non-calcifying sister species I. galbana will serve as an ideal subject for comparison to unravel the molecular mechanisms of calcification in coccolithophorids. 8 Biomineralization and Coccolithogenesis Biomineralization is by definition the formation and deposition of inorganic solids in biological systems (Mann 2001). The most common inorganic solids are biominerals that are made from calcium carbonate, calcium phosphate or silica (Livingston et al. 2006). Forms of calcium carbonate include calcite, magnesium calcite or aragonite which are produced by organisms such as coccolithophorids, sea urchins and mollusks, respectivly. Coccolithophorids deposit calcium carbonate in the form of elaborate calcite shells called coccoliths that encapsulate the cell. Individual cells are typically surrounded by 10-50 coccoliths (Westbroek et al. 1984; Tyrrell and Taylor. 1996). The structure of the coccolith consists of a base plate and radial arrays of calcium carbonate crystalline segments that include a flat radially oriented lower element, a hammer shaped upper element, a central element and a medially directed element. Figure 1: Diagram of coccolith elements, from the International Nanoplankton Association, Natural history museum, London, England ©2010 9 While a variety of organisms are capable of carrying out biomineralization processes, there are two principal methods of mineral deposition, biologically-induced mineralization and biologically-mediated mineralization (Mann. 2005). In biologically-induced mineralization, minerals are deposited adventitiously along the surface of the cell, as a result of various metabolic activities of the cell. In contrast, biologically-mediated mineralization is a highly regulated process that occurs intracellularly and results in materials with specialized functions and structures. Coccolithophorids exhibit biologically-mediated mineralization, where mineralization occurs within a vesicle where matrix molecules nucleate and shape the crystalline structure. Coccoliths of E. huxleyi and G. oceanica are created within a specialized intracellular compartment called the coccolith vesicle (CV) derived from the Golgi apparatus (Mann 2001; Marsh 2003; Nguyen et al. 2005). Coccolithophorids convert inorganic carbon into organic and biomineralized 2+ - product whereby Ca + 2HCO3 CaCO3 + CO2 + H2O (Marsh 2003). The coccoliths are formed inside the CV in a process called coccolithogenesis, where the orientation, size and shape of the crystalline elements are carefully controlled. The CV is attached to a convoluted membrane system called the reticular body, forming the c.v - r.b complex, which is thought to be responsible for the