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Molecular evolutionary analysis of TALE homeobox in Viridiplantae Wang, Ming Hsiu 2015

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MOLECULAR EVOLUTIONARY ANALYSIS OF TALE HOMEOBOX IN VIRIDIPLANTAE by  Ming Hsiu Wang  B.Sc., The University of British Columbia, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2015  © Ming Hsiu Wang, 2015 ii  Abstract  The emergence of embryophytes from their charophyte-like ancestor is estimated to have occurred 476-432 MYA. During the adaptation to land, embryophytes evolved to have sporic meiosis; whereas charophyte algae undergo zygotic meiosis. The transition to land required the embryophytes to develop specialized tissues and a cuticle to survive drier terrestrial environments. This transition resulted in increasing elaboration of the body plan in the diploid phase, establishing the sporophyte. It is hypothesized that diversification of heterodimeric TALE homeobox genes in the ancestral charophyte algae may have acted as new types of master regulators to control diploid-specific developmental program, which initiated the development of novel sporophytic body plan. This study is focused on determining TALE homeobox genealogy by comparing genetic sequences and gene structure of TALE homeobox found in the transcriptomes of Picocystis salinarum (prasinophyte), Mougeotia sp. (charophyte). and Cosmocladium constrictum (charophyte). The interaction of TALE homeobox proteins from Picocystis salinarum was tested with a Y2H assay. Prior to this study, it was known that the diploid developmental program was regulated by KNOX and BELL classes of TALE homeobox genes in embryophytes and KNOX and GSP1 classes of TALE homeobox genes in Chlamydomonas reinhardtii (chlorophyte). Through phylogenetic analysis, I found that charophytes express KNOX, BELL and GSP1 classes, and P. salinarum expresses KNOX, GSP1, divergent TALE, and two red algal homologs of the TALE homeobox. Furthermore, comparison of intron location indicated that the BELL and GSP1 genes in the charophytes may be homologous. Intron comparisons and phylogenetic analysis of the KNOX genes indicate that KNOX II class from streptophyta and KNOX from chlorophyta share the greatest similarity, iii  whereas KNOX I class can be hypothesized to have emerged by gene duplication in the early charophyte ancestor. The Y2H assay of TALE homeobox from Picocystis salinarum shows that GSP1 and KNOX can interact, whereas the possibility of an interaction with the red algal homolog is inconclusive.  iv  Preface  The figures in chapter 1 are reproduced with permission (noted in caption), or designed by me. The research question in chapter 1 was produced by me with the guidance of supervisor Dr. Jae-Hyeok Lee and committee members Dr. Carl Douglas and Dr. Sean Graham.  The data in chapter 2 is credited to Dr. Sunjoo Joo, who produced the phylogeny, and supervised undergraduate student Gary Lui to collect TALE homeobox sequences in green algal transcriptomes.  The cloning of TALE homeobox from charophytes and prasinophytes in chapter 3 was initiated by Dr. Sunjoo Joo. I carried out the original cloning scheme proposed by Dr. Joo. I found the upstream Knox 1 and Pox domains for Mougeotia KNOX II and BELL genes, and designed the cloning procedure for these regions. The analysis of TALE homeobox gene structure was done by me. The Y2H experiment in chapter 4 was carried out by me. Dr. Lee supervised the cloning procedure for the Y2H constructs and the experimental set up for the Y2H assay.  v  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ............................................................................................................................. viiii List of Figures ............................................................................................................................... ix List of Abbreviations .....................................................................................................................x Acknowledgements ..................................................................................................................... xii Dedication ................................................................................................................................... xiii Chapter 1: TALE Homeobox in the Evolutionary History of Viridiplantae ...........................1 1.1 Motivation for the Study and Thesis Question ............................................................... 6 1.2 Objective of Study .......................................................................................................... 7 Chapter 2: Phylogenetic Analysis of TALE Homeobox in Viridiplantae .................................8 2.1 Introduction ..................................................................................................................... 8 2.2 Results ............................................................................................................................. 9 2.2.1 Three Classes of TALE Homeobox Found in Chlorophyta: KNOX, GSP1 and Divergent TALE ..................................................................................................................... 9 2.2.2 KNOX Class Is Resolved, But Non-KNOX Branches Are Not Well Resolved ......... 10 2.3 Discussion ..................................................................................................................... 14 2.3.1 The Ancestral State of TALE Homeobox in the Common Algal Ancestor of the Viridiplantae May Have Included Three Classes of TALE Homeobox ............................... 14 vi  2.3.2 Relationship Among BELL, GSP1 Homologs, and BELL-related Classes of TALE Homeobox in Green Algae Cannot Be Well Resolved By The Present Phylogenetic Analysis................................................................................................................................. 15 2.3.3 Mougeotia sp., Cosmocladium constrictum, and Picocystis salinarum Transcriptomes Express the Most TALE Homeobox Classes ........................................................................ 15 2.4 Materials and Methods .................................................................................................. 16 2.4.1 Collecting TALE Homeobox Genes for Phylogenetic Analysis ................................. 16 2.4.2 Building the Phylogeny................................................................................................ 16 Chapter 3: Gene Structure of TALE Homeobox in Mougeotia sp., Cosmocladium constrictum, and Picocystis salinarum ........................................................................................24 3.1 Introduction ................................................................................................................... 24 3.2 Results ........................................................................................................................... 25 3.2.1 Defining TALE Homeobox Gene Structure of Picocystis salinarum ......................... 25 3.2.2 Defining TALE Homeobox Gene Structures of Two Charophytes: Mougeotia sp. and Cosmocladium constrictum ................................................................................................... 26 3.2.3 Search for Upstream Domains for BELL Class TALE Homeobox in Mougeotia sp. . 33 3.2.4 Comparison of Intron Locations in TALE Homeobox. ............................................... 35 3.3 Discussion ..................................................................................................................... 38 3.3.1 Using Introns to Infer the Phylogenetic Relationship of TALE Homeobox in Viridiplantae ......................................................................................................................... 38 3.4 Materials and Methods .................................................................................................. 44 3.4.1 Cultures ........................................................................................................................ 44 3.4.2 Cloning Genomic Sequences of TALE Homeobox from the Transcriptome. ............. 44 vii  3.4.3 Defining Introns, Exons and Conserved Domains. ...................................................... 45 Chapter 4: Y2H Analysis of TALE Homeobox from Picocystis salinarum ............................47 4.1 Introduction ................................................................................................................... 47 4.2 Results ........................................................................................................................... 48 4.3 Discussion ..................................................................................................................... 49 4.4 Materials and Methods .................................................................................................. 50 4.4.1 Yeast Strain .................................................................................................................. 50 4.4.2 Y2H Constructs ............................................................................................................ 50 4.4.3 Yeast Competent Cells and Transformation ................................................................ 51 Chapter 5: Conclusion .................................................................................................................53 5.1 Major Findings and Contributions to TALE Homeobox Research .............................. 53 5.2 Future Directions .......................................................................................................... 55 References .....................................................................................................................................56  viii  List of Tables  Table 2.1 TALE homeobox sequences used for phylogenetic analysis in Chapter 2 .................. 18 Table 3.1 Primer pairs used to amplify TALE homeobox genes from genomic DNA of Mougeotia sp., Cosmocladium constrictum, and Picocystis salinarum. ....................................... 45 Table 4.1 Primer pairs used to amplify TALE homeobox genes from genomic DNA of Picocystis salinarum ..................................................................................................................... 51 Table 4.2 Y2H transformation solution........................................................................................ 52   ix  List of Figures  Figure 1.1 Overview of the Viridiplantae clade ............................................................................. 4 Figure 1.2 Life cycle of embryophytes (top) vs. algae (bottom). .................................................. 5 Figure 1.3 Genealogy of TALE homeobox ................................................................................... 6 Figure 2.1 Bayesian phylogeny of Homeodomain in chlorophytes and prasinophytes ............... 12 Figure 2.2 Phylogeny of TALE Homeodomain from prasinophytes, chlorophytes, charophytes, and embryophytes. ........................................................................................................................ 13 Figure 3.1 Sequence alignment of TALE homeobox gene structure in Picocystis salinarum .... 27 Figure 3.2 Sequence alignment of TALE homeobox gene structure in Mougeotia sp. ............... 29 Figure 3.3 Sequence alignment of TALE homeobox gene structure in Cosmocladium constrictum .................................................................................................................................... 32 Figure 3.4 Multiple sequence alignment of Pox domain from embryophytes, Mougeotia sp. and Penium exiguum. ........................................................................................................................... 35 Figure 3.5 Comparison of intron locations between KNOX class genes in embrophytes, charophytes, chlorophytes and prasinophytes............................................................................... 36 Figure 3.6 Comparison of intron locations between BELL and GSP1 class genes in embryophytes, charophytes, chlorophytes, and prasinophytes ..................................................... 38 Figure 3.7 Model of evolution for TALE homeobox classes in the Viridiplantae. ..................... 43 Figure 4.1 Yeast-2-hybrid assay of Picocystis salinarum TALE homeobox interactions with pGBKT7 fusion proteins (bait) and pGADT7 fusion proteins (prey)........................................... 49    x  List of Abbreviations  1KP 1000 plants project BBM Bold’s basal media BELL BEL1-like Homeobox BELL-related TALE homeobox homologous to BELL, but does not cluster with BELL class members on phylogeny CCAC Culture Collection of Algae at the University of Cologne DDO Double drop out media  (-leucine/-tryptophan) DMSO Dimethyl sulfoxide GSM1 Gamete-specific minus mating type molecule 1 GSP1 Gamete-specific plus mating type molecule 1 GSP1 homologs TALE homeobox homologous to GSP1 found in Chlamydomonas reinhardtii JGI Joint Genome Institute KNOX Knotted-like Homeobox LiAc Lithium acetate MAFFT Multiple Alignment using Fast Fourier Transform MYA Million years ago NCMA National Center for Marine Algae and Microbiota OD Optical density PCR Polymerase chain reaction QDO Quadruple drop out media (-leucine/-tryptophan/-adenine/-histidine) xi  TALE Three amino acid loop extension TE Tris and Ethylenediaminetetraacetic acid (EDTA) Waris-H + 3V Waris-H media with 3 times more vitamin than the original Waris-H media  WAG Whelan And Goldman w/v Concentration of solution expressed as weight/volume percentage v/v Concentration of solution expressed as volume/volume percentage Y2H Yeast two hybrid YPDA Yeast-peptone-dextrose-adenine media  xii  Acknowledgements  This research was funded by NSF (US National Science Foundation), and teaching assistantships from the Botany department at the University of British Columbia. I am grateful for everyone who contributed and supported my graduate research over the past two years.  I would like to thank Dr. Jae Hyeok Lee for giving me the opportunity to join his team in researching the evolution of TALE homeobox in green algae. I am grateful to have committee members, Dr. Carl Douglas and Dr. Sean Graham, for providing supportive and constructive feedback to improve my project. I owe my deepest gratitude to Dr. Sunjoo Joo, who patiently taught me all of the molecular biology techniques required to complete my project. I am also lucky to have great lab mates: Yuan Xiong, Jack Munz, Thamali Kariyawasam, and Evan Cronmiller, who provided me with constructive feedback at every lab meeting.  Finally, I would not have been able to complete my graduate education without the emotional and financial support from my family members: Leo, Yuchun, Peter, Maggie and Paris.    xiii  Dedication  This thesis is dedicated to my dad, whose optimism and big heart inspired me to pursue my dreams.1  Chapter 1: TALE Homeobox in the Evolutionary History of Viridiplantae  The Viridiplantae lineage is a monophyletic group consisting of chlorophyta and streptophyta (figure 1.1), which diverged at least 1 billion years ago (Lewis and McCourt, 2004). After the early divergence of the Viridiplantae, the chlorophyta lineage underwent multiple divergence events in the prasinophyte-like ancestors. A new mode of cell division arose during one such divergence event estimated 900-700 MYA. The use of a phycoplast to mediate cytokinesis during this time marked the emergence of the core chlorophyte lineage (Leliaert et al., 2012). The streptophyta clade contains embryophytes (land plants) and charophytes (fresh water green algae). The early charophytes initially colonized fresh water environments. The embryophytes emerged when their algal ancestor colonized dry land approximately 432-476 MYA (Leliaert et al., 2011). Migration to dry land required embryophytes to develop new survival strategies. Such adaptations included the development of cuticle and sporopollenin to protect against desiccation, and stomata for gas exchange. Ever since the migration, embryophytes continued to evolve specialized organs to adapt to terrestrial life. Such adaptations included the emergence of the vascular system, leaf, root, seed, and flower structures. The development of these specialized tissues required the elaboration of the ancestral body plan. Interestingly, specialized body plans evolved in the diploid phase of the life cycle, whereas the haploid phase has not shown elaboration throughout the embryophytes.  The interpolation theory (antithetic theory) and the homologous theory are two conflicting theories for the origin of the sporophyte, which is the three-dimensional diploid body of embryophytes. The homologous theory suggests the embryophytic diploid sporophyte is a 2  modified version of the haploid gametophyte, with both generations sharing homologous structures (reviewed in Blackwell, 2003). Under the more widely accepted interpolation theory, the sporophyte stage was inserted into the embryophytic life cycle (reviewed in Blackwell, 2003).The diploid zygote of embryophytes undergoes mitosis to form a mass of diploid cells known as the sporophyte. As more adaptations were developed to survive in the terrestrial environment, the body plans of embryophytes became increasingly diverse during the sporophytic stage (Langdale & Harrison, 2008). After many mitotic divisions, a sub population of the sporophytic cells will undergo meiosis to form haploid spores (Figure 1.2). By contrast, the diploid zygote of algae undergoes immediate meiosis to form haploid vegetative cells. The vegetative cells then undergo mitotic divisions to grow, and differentiate as gametes for sexual reproduction (Figure 1.2). Comparison of the two life cycles indicate that sporophytes emerged by delaying the time of meiosis from directly after zygote formation (zygotic meiosis) to a time after sporophyte formation (sporic meiosis). In charophytes, the diploid stage is unicellular (Graham et al., 2000). Thus, the algal ancestors did not have the capacity for specialized tissue differentiation in the diploid phase of their life cycle. It is hypothesized that the addition of mitosis prior to sporic meiosis created a multicellular diploid sporophytic stage that could escape the algal life cycle (Lee et al., 2008). The change in body plan during the transition from algae to embryophyte is postulated to have been induced by changes in the master regulators that control the diploid developmental program. Within the Viridiplantae, studies on Chlamydomonas reinhardtii and Physcomitrella patens have shown that the haploid-diploid transition and the diploid developmental program is regulated by a group of transcription factors belonging to the TALE homeobox superclass (Sakakibara, 2013; Lee et al., 2008). In addition to its role in the Viridiplantae, TALE homeobox 3  genes have been found to be involved in the haploid-diploid transition of all major eukaryotic lineages (Bürglin, 1997). This suggests that the TALE homeobox is an ancient group of genes that emerged near the base of eukaryotic evolution. Bürglin (1997) found that gene classes within the TALE homeobox diversified from a common ancestral gene. The diversification of existing TALE homeobox genes created new combinations of heterodimers, which had the potential to regulate novel developmental programs that could lead to diversification of novel body plans.  TALE homeobox superclass is a branch of the Homeobox family. Members from the Homeobox family share a highly conserved 60 amino acid Homeodomain (reviewed in Bürglin, 2011). However, members of the family that cluster into the TALE homeobox branch are characterized by the presence of three extra amino acids in the loop region between helix 1 and helix 2 of the protein (Bürglin, 1997). Generally, TALE homeobox proteins contain a C-terminal Homeodomain that allows specific binding to target DNA, and conserved domains outside of the Homeodomain that is used for heterodimerization interactions (Bellaoui et al., 2001; Chen et al., 2003; Muller et al., 2001; Smith et al., 2002). Phylogenetic analysis indicates that the TALE homeobox superclass can be further divided into eight classes (figure 1.3A) (Bürglin, 1997).  In embryophytes, members of the TALE homeobox superclass include the KNOX and BELL classes (figure 1.3A). Heterodimer interactions between members of embryophytic KNOX and BELL classes have been well documented (Bellaoui et al., 2001; Chen et al., 2003; Muller et al., 2001; Smith et al., 2002). Functional homologs of the embryophytic KNOX and BELL classes have been found in the green algae, Chlamydomonas reinhardtii (Lee et. al, 2008). Chlamydomonas reinhardtii has three genes that belong to the TALE homeobox superclass: GSM1, GSP1, and HDG1. Phylogenetic analysis indicated that GSM1 belongs to the KNOX 4  class, while GSP1 and HDG1 are relatives of the BELL class (figure 1.3B) (Lee et al., 2008). Of the three TALE homeobox proteins, GSM1 (a member of the KNOX class) and GSP1 (a relative of the BELL class) are able to heterodimerize with each other. This evidence suggests that a diversification event occurred in the KNOX and BELL classes during the emergence of embryophytes. The diversification allowed different combinations of heterodimerization to form, which may have been involved in recruiting genes necessary for sporic meiosis, allowing the elaboration of diploid body plans to occur. To address this hypothesis, this study is focused on the diversification of TALE homeobox proteins that have specific functions in regulating the haploid-diploid transition of the life cycle.    Figure 1.1 Overview of the Viridiplantae clade. Reproduced with permission from Leliaert et al., (2012) Copyright © 2012, Taylor and Francis Online. The Viridiplantae diverged into the chlorophyta and streptophyta lineages at least 1 billion years ago. The chlorophyta lineage originally contained prasinophyte-like ancestors, which underwent early divergence events. One such divergence event approximately 900-700 MYA gave rise to the core chlorophyte lineage. The early streptophyta lineage consisted of charophyte-like algal ancestors, which dominated freshwater environments. Approximately 476-432 MYA, charophyte-like algae began to colonize dry land, which gave rise to the embryophyte lineage. 5   Figure 1.2 Life cycle of embryophytes (top) vs. algae (bottom). In embryophytes, the diploid zygote undergoes mitosis to form the sporophyte. The meiotic event is delayed to a later time in the diploid stage of the embryophytic life cycle (sporic meiosis). In green algae, the diploid zygote undergoes meiosis immediately after zygote formation (zygotic meiosis). The mitosis event does not occur in the diploid stage of the green algal life cycle.  Algae Embryophyte 6  Figure 1.3 Genealogy of TALE homeobox. A) Reproduced with permission from Bürglin (1997), Copyright © 1997, Oxford University Press. TALE homeobox from embryophytes, fungi and animals can be divided into 8 classes. The KNOX and BELL classes (red boxes) are unique to embryophytes. B) Reproduced with permission from Lee et al. (2008) Copyright © 2008, Elsevier Inc. Embryophytic TALE homeobox clusters into KNOX (grey shaded box) and BELL (blue shaded box). GSM1 (black arrow) and is a TALE homeobox from Chlamydomonas reinhardtii that clusters into the KNOX class (grey shaded box). GSP1 (red arrow) is a TALE homeobox from Chlamydomonas reinhardtii that clusters into the GSP1 homologs cluster (pink shaded box).   1.1 Motivation for the Study and Thesis Question The genome sequences of green algal species revealed that the TALE homeobox proteins in C. reinhardtii are homologous to the KNOX and BELL classes of TALE homeobox proteins found in embryophytes (figure 1.3B) (Lee et al., 2008). However, TALE homeobox in the prasinophytes and charophytes have not been well studied. This thesis is focused on the genealogy of TALE homeobox genes in the prasinophyte and charophytes. The outcomes of this 7  study have provided better understanding of the ancestral state of TALE homeobox in the Viridiplantae and evolutionary events that contributed to the emergence of embryophytes.  The thesis addressed the changes that occurred in the genetic sequence and the heterodimerization interactions of TALE homeobox in the Viridiplantae.   1.2 Objective of Study Objective 1: Determine the phylogeny of TALE homeobox genes in prasinophytes and charophytes Objective 2: Determine the gene structure of TALE homeobox genes identified in charophytes and prasinophytes  Objective 3: Determine the interaction network of TALE homeobox proteins in prasinophytes using the yeast-2-hybrid system 8  Chapter 2: Phylogenetic Analysis of TALE Homeobox in Viridiplantae  2.1 Introduction The first objective of this study is to determine the phylogeny of TALE homeobox genes in prasinophytes, and charophytes. Generating a phylogeny will determine the classes of TALE homeobox that are found in green algal transcriptome libraries provided by the Worden laboratory and the 1KP project (available at http://camera.calit2.net/mmetsp/ and https://www.bioinfodata.org/Blast4OneKP/), and the prasinophyte genomes at JGI (http://genome.jgi.doe.gov/). The purpose of classifying TALE homeobox in prasinophytes and charophytes is to extrapolate the ancestral state of TALE homeobox in the Viridiplantae.  Prior to this study, Lee et al. (2008) built a phylogeny using limited genetic TALE homeobox information from the chlorophyta. The study found that chlorophyta contained two classes of TALE homeobox, an algal version of KNOX class and a GSP1 homologs class (Lee et al., 2008). Lee et.al’s (2008) phylogeny indicated that the KNOX class genes from embryophytes and chlorophyta formed a cluster supported by 61% bootstrap value. Embryophytic KNOX class proteins have been reported to heterodimerize with a BELL class protein to increase the affinity to the target DNA (Bellaoui et al., 2001; Chen et al., 2003; Muller et al., 2001; Sharma et al., 2014; Smith et al., 2002). As KNOX class members were found in chlorophyta, it is reasonable to believe there are BELL class TALE homeobox genes in green algae. However, it was found that the BELL class genes from embryophytes formed a cluster with 49% bootstrap support and the algal GSP1 homologs formed a separate cluster with 48% bootstrap support (Lee et al., 2008). Lee et al.(2008) found that chlorophytes and embryophytes both use two classes of TALE homeobox to regulate diploid developmental programs. However, 9  embryophytes use KNOX and BELL classes, whereas chlorophytes use KNOX and a potentially novel GSP1 homologs class. The phylogenetic tree presented by Lee et al. (2008) was limited to only the chlorophycean TALE homeobox. Charophytes, the closest living algal group to embryophytes, was omitted from the analysis because genomic information was unavailable at the time. The limitation to information may have resulted in the low bootstrap support for the BELL and GSP1 homologs class. Given the observed bootstrap support, we cannot conclude that green algae have a novel GSP1 homologs class of TALE homeobox. Is the GSP1 homologs cluster found by Lee et al. (2008) a newly discovered true class of TALE homeobox or a separated branch that reflects lack of sampling in prasinophytes and charophytes? To answer this question, we aimed to create a new phylogeny which will include the previously available TALE homeobox information from the chlorophyta, as well as TALE homeobox information from unpublished and published transcriptomes of charophytes and prasinophytes. The sequences for the phylogenetic tree were collected with the help of Dr. Sunjoo Joo and undergraduate student, Gary Lui. Dr. Joo put together the alignment of the TALE homeobox genes to generate the phylogenetic trees for figure 2.1 and 2.2.   2.2  Results 2.2.1 Three Classes of TALE Homeobox Found in Chlorophyta: KNOX, GSP1 and Divergent TALE The TALE homeobox sequences of prasinophytes show that there are three classes of TALE homeobox present: KNOX, GSP1 homologs (previously called BELL-related1 by Lee et al., 2008), or divergent TALE homeobox (figure 2.1). The divergent TALE homeobox genes do not show homology to sequences from species that are not part of the same family. Within each 10  species of prasinophytes examined, either the KNOX class, GSP1 homologs class, or no TALE homeobox was found in the transcriptomes (figure 2.1). Picocystis salinarum was an exception to this trend because five TALE homeobox sequences were identified in the transcriptome. Each sequence was found to cluster into separate clades (figure 2.1, orange triangles). Two of the P. salinarum TALE homeobox belong to the KNOX and prasinophyte GSP1 homologs clades, two sequences show homology to TALE homeobox from the red alga Cyanidioschyzon merolae, and the last one is a divergent TALE homeobox (figure 2.1).  To ensure that the red algae-like TALE homeobox genes were not caused by contamination of the culture used to prepare the transcriptome, these sequences were amplified from genomic DNA extracted from a single cell colony of P. salinarum CCMP1897. All five TALE homeobox sequences could be amplified from the genomic DNA, confirming that these TALE homeobox sequences are encoded by the P. salinarum genome.  2.2.2 KNOX Class Is Resolved, But Non-KNOX Branches Are Not Well Resolved The comparison of TALE homeobox from chlorophyta and streptophyta shows a division between the KNOX and non-KNOX classes (BELL, BELL-related, GSP1 homologs) at the base of the phylogeny, with 61% bootstrap support (figure 2.2). The KNOX class genes from charophytes (red branches) and embryophytes (green branches) cluster together with 57% bootstrap support (figure 2.2). Within the KNOX class cluster, the KNOX I and KNOX II classes are found with 43% and 99% bootstrap support respectively (figure 2.2). Bifurcation of the KNOX class is not seen in the algal KNOX branch containing sequences from chlorophyta (figure 2.2).  11   The non-KNOX branch includes BELL, BELL-like, and GSP1 homologs classes. Similar to the previous study by Lee et al.(2008), the BELL and GSP1 homologs classes form two separate clusters with low bootstrap support. The BELL cluster has 53% bootstrap support, and contains sequences from charophytes (light blue branches) and embryophytes (green branches) (figure 2.2). All charophyte sequences that cluster with embryophyte sequences in the BELL class branch belong to species from the Zygnematales order. Within the BELL-class cluster, there is a branch with 48% bootstrap support that suggests that the TALE homeobox from Mougeotia sp., Mesotaenium caldariorum, and Cylindrocystis sp. (blue) have the closest relationship to the BELL class genes from embryophytes (green) (figure 2.2). Mougeotia sp. and Cosmocladium constrictum are of interest for further analysis because the TALE homeobox classes found in their transcriptomes possibly represent an intermediate state to the TALE homeobox configuration found in embryophytes and chlorophyta. TALE homeobox from Mougeotia sp. falls into the KNOX class I, KNOX class II, BELL, and GSP1 homologs clusters (figure 2.2 blue triangles). Cosmocladium constrictum sequences fall into the KNOX class I, KNOX class II, and GSP1 homologs clusters (figure 2.2, pink triangles).   12   Figure 2.1 Bayesian phylogeny of Homeodomain in chlorophytes and prasinophytes. KNOX, GSP1 homologs and divergent TALE sequences are colored in green, blue and red respectively. Posterior probabilities from MrBayes (above) and bootstrap values from PhyML (below) are indicated on the branches. KNOX cluster (green branches), GSP1 homologs (blue branches), and divergent TALE (red branches) are highlighted on the tree. Orange triangles show five TALE homeobox sequences in Picocystis salinarum.  13   Figure 2.2 Phylogeny of TALE Homeodomain from prasinophytes, chlorophytes, charophytes, and embryophytes. Bootstrap values are indicated above branches. Charophyte KNOX I (red) and KNOX II classes (pink), embryophyte KNOX I and KNOX II classes (green), charophyte GSP1 homologs class (light blue), charophyte BELL and BELL-like classes (dark blue) are highlighted on the tree. Blue triangles denote Mougeotia sp., and pink triangles denote Cosmocladium constrictum. 14  2.3 Discussion Similar to the results of the previous phylogeny by Lee et al. (2008), the present phylogeny found that TALE homeobox genes from chlorophyte, prasinophyte and charophyte algae can be divided into two major branches, KNOX and non-KNOX (figure 2.1 and figure 2.2). Within the non-KNOX group, the BELL, GSP1 homologs and the previously undiscovered BELL-like clusters have low bootstrap support (figure 2.2).   2.3.1 The Ancestral State of TALE Homeobox in the Common Algal Ancestor of the Viridiplantae May Have Included Three Classes of TALE Homeobox The phylogenetic tree indicates that there are three classes of TALE homeobox found in the chlorophyta (figure 2.1). An exception to this trend is the prasinophyte, P. salinarum, where five TALE homeobox sequences were found in the transcriptome. Two of these sequences showed homology to C. merolae (figure 2.1). It is possible that these genes are present due to horizontal gene transfer from red algae.  The KNOX and GSP1 homologs class genes are also likely to have existed in the common ancestor of Viridiplantae. The phylogeny showed that KNOX, and GSP1 homologs are found in streptophyta and chlorophyta. The BELL class is only found in the streptophyta, suggesting that this class emerged after the Viridiplantae had diverged into the chlorophyta and streptophyta.    15  2.3.2 Relationships Among BELL, GSP1 homologs, and BELL-related classes of TALE Homeobox in Green Algae Cannot Be Well Resolved By The Present Phylogenetic Analysis The non-KNOX branch of the phylogeny (figure 2.2) contains three clusters of interest: BELL, BELL-related and GSP1 homologs. A previous phylogeny by Lee et al. (2008) showed that the BELL and GSP1 homologs clusters do not have good bootstrap support, thus we cannot conclude that the GSP1 homolog cluster is a novel class of TALE homeobox unique to green algae. The phylogeny in figure 2.2 included TALE homeobox from more algal species than the previous phylogeny from Lee et al. (2008). However, the branches of the BELL and GSP1 homologs still were not well resolved. In addition, the phylogeny shows charophytes have another group of TALE homeobox, the BELL-related cluster. This group was not previously found by Lee et al. (2008) because charophyte transcriptomes were not available at the time. The three clusters of non-KNOX TALE homeobox may be difficult to resolve on the phylogeny due to the limited number of characters in the alignment. As the phylogenetic analysis cannot clearly discriminate the relationships amongst GSP1 homologs, BELL-related and BELL clusters, intron locations will be used to provide more insight into the evolutionary history of the three clusters in chapter 3.   2.3.3 Mougeotia sp., Cosmocladium constrictum, and Picocystis salinarum Transcriptomes Express the Most TALE Homeobox Classes Based on the phylogenetic trees in figure 2.1 and 2.2, three species were found to express multiple TALE homeobox belonging to separate clusters. Picocystis salinarum expresses TALE homeobox from KNOX, GSP1 homologs, as well as two red algal homologs (figure 2.1). The classes of TALE homeobox found in P. salinarum are similar to classes that are hypothesized to 16  be found in the common ancestor of Viridiplantae (chapter 2.3.1). Therefore, the TALE homeobox from P. salinarum will be further characterized in chapters 3 and 4.  Mougeotia sp. expresses genes in the KNOX I, KNOX II, BELL, and GSP1 homologs classes (figure 2.2). Cosmocladium constrictum expresses genes in the KNOX I, KNOX II, and GSP1 homologs classes (figure 2.2). The classes of TALE homeobox found in these two charophytes are an intermediate combination between the chlorophytes and embryophytes. Thus, the TALE homeobox from Mougeotia sp. and C. constrictum will also be further analyzed.   2.4 Materials and Methods 2.4.1 Collecting TALE Homeobox Genes for Phylogenetic Analysis The TALE homeobox sequences were collected by searching through published prasinophyte genomes (available at http://genome.jgi.doe.gov/), and green algal transcriptomes from the Worden laboratory and 1KP project (available at http://camera.calit2.net/mmetsp/ and https://www.bioinfodata.org/Blast4OneKP/). The conserved 60 amino acid homeobox Pfam sequence was used as a query. The resulting transcripts that were found are summarized in table 2.1.   2.4.2 Building the Phylogeny Characterized sequences belonging to separate classes of TALE homeobox were collected from either Arabidopsis thaliana or Physcomitrella patens. These sequences were included in the alignment to mark the clusters that are KNOX I, KNOX II, or BELL class. Chlamydomonas reinhardtii and Ostreococcus sequences were used to mark the GSP1 homologs and algal KNOX clusters. The conserved Homeodomain of 75 genes were aligned using MAFFT 17  in figure 2.1 and 135 genes were aligned in figure 2.2. The alignment was manually adjusted to ensure the known conserved motifs within the Homeodomain were aligned. The phylogeny was generated using MrBayes algorithm equipped with the Geneious 7.1 software, using a WAG amino acid substitution model. Posterior probabilities were calculated from 10000 Bayesian trees. The phylogeny was estimated by the maximum likelihood method using the PhyML 3.0 program, using a WAG amino acid substitution model. The phylogeny was rooted between the KNOX and non-KNOX branches.18  Table 2.1 TALE homeobox sequences used for phylogenetic analysis in Chapter 2. Transcripts were collected from genomes at JGI (http://genome.jgi.doe.gov/), transcriptomes from the Worden lab (http://camera.calit2.net/mmetsp/), and transcriptomes from 1KP (https://www.bioinfodata.org/Blast4OneKP/).  Transcript name Species Family Source ID Lineage AaKNOX1 Chlamydomonas reinhardtii  Polyphysaceae Genbank AAD51632.1  Chlorophyte AtBEL1 Arabidopsis thaliana Camelineae Genbank NP_198957.1 Embryophyte AtBP Arabidopsis thaliana Camelineae Genbank CAB81151.1 Embryophyte ATH1 Arabidopsis thaliana Camelineae Genbank NP_195024.1 Embryophyte AtKNAT3 Arabidopsis thaliana Camelineae Genbank NP_197904.1 Embryophyte AtSTM Physcomitrella patens Camelineae Genbank NP_176426.1 Embryophyte AZZW-2008127p Chlorokybus atmophyticus Klebsormidiaceae 1KP AZZW-2008127p Charophyte AZZW-4310 Chlorokybus atmophyticus Klebsormidiaceae 1KP AZZW-4310 Charophyte Bathy06g01510 Bathycoccus prasinos Mamiellaceae 1KP Bathy06g01510 Prasinophyte Bathy08G03820 Bathycoccus prasinos Mamiellaceae 1KP Bathy08G03820 Prasinophyte Bathy10g00740 Monomastix opisthostigma Mamiellaceae 1KP Bathy10g00740 Prasinophyte BFIK 2029965 Entransia fimbriata Klebsormidiaceae 1KP BFIK 2029965 Charophyte BFIK-2024268 Entransia fimbriata Klebsormidiaceae 1KP BFIK-2024268 Charophyte BHBK-19923 Cosmarium tinctum Desmidiaceae 1KP BHBK-19923 Charophyte BHBK-2043117 Cosmarium tinctum Desmidiaceae 1KP BHBK-2043117 Charophyte BHBK-38400 Cosmarium tinctum Desmidiaceae 1KP BHBK-38400 Charophyte BHBK-7914 Cosmarium tinctum Desmidiaceae 1KP BHBK-7914 Charophyte BTFM-2049640  Micromonas pusilla Monomasticaceae 1KP BTFM-2049640 Prasinophyte CCMP1545 Micromonas sp. Mamiellaceae JGI 60177 Prasinophyte CCMP1545 Ostreococcus lucimarinus Mamiellaceae JGI 53806 Prasinophyte CCMP1545_53806  Micromonas pusilla Mamiellaceae  JGI 53806 Prasinophyte CCMP1545_60177  Micromonas pusilla Mamiellaceae  JGI 60177 Prasinophyte CCMP1545_69285 Tetraselmis cordiformis Mamiellaceae  JGI 69285 Prasinophyte 19  ChiHDG Arabidopsis thaliana Chlamydomonadaceae Genbank AAW82031 Chlorophyte ChrGSM1 Chlamydomonas reinhardtii Chlamydomonadaceae Genbank ABJ15867.1 Chlorophyte ChrGSP1 Chlamydomonas reinhardtii Chlamydomonadaceae Genbank AAD23383.3 Chlorophyte ChrHDG1 Uronema sp. Chlamydomonadaceae Genbank ABY60735.1 Chlorophyte CMH049C Cyanidioschyzon merolae strain 10D Cyanidiaceae Genbank BAM79748.1 Rhodophyta CMR153C Cyanidioschyzon merolae strain 10D Cyanidiaceae Genbank BAM82406.1 Rhodophyta CMR176C  Cyanidiaceae Genbank BAM82421.1 Rhodophyta DFDS-2037218 Desmidium aptogonum Desmidiaceae 1KP DFDS-2037218 Charophyte DUMA-2002154 Tetraselmis striata Chlorodendraceae 1KP DUMA-2002154 Prasinophyte FFGR-55042 Netrium digitus Mesotaeniaceae 1KP FFGR-55042 Charophyte FQLP-3749 Klebsormidium subtile Klebsormidiaceae 1KP FQLP-3749 Charophyte GBGT-2097171 Xanthidium antilopaeum Desmidiaceae 1KP GBGT-2097171 Charophyte GBGT-2105682 Xanthidium antilopaeum Desmidiaceae 1KP GBGT-2105682 Charophyte GGWH-20590 Onychonema laeve Desmidiaceae 1KP GGWH-20590 Charophyte GYRP-2181145 Euastrum affine Desmidiaceae 1KP GYRP-2181145 Charophyte GYRP-2197240 Euastrum affine Desmidiaceae 1KP GYRP-2197240 Charophyte HAOX-2020577 Spirogyra sp. Zygnemataceae 1KP HAOX-2020577 Charophyte HHXJ-2042504 Tetraselmis striata Chlorodendraceae 1KP HHXJ-2042504 Prasinophyte HHXJ-2042517 Tetraselmis chui Chlorodendraceae 1KP HHXJ-2042517 Prasinophyte HKZW-2006257 Mesotaenium caldariorum Mesotaeniaceae 1KP HKZW-2006257 Charophyte HKZW-2011495 Mesotaenium caldariorum Mesotaeniaceae 1KP HKZW-2011495 Charophyte HKZW-2045628 Mesotaenium caldariorum Mesotaeniaceae 1KP HKZW-2045628 Charophyte HKZW-2046084 Mesotaenium caldariorum Mesotaeniaceae 1KP HKZW-2046084 Charophyte HVNO-2016205 Tetraselmis chui Chlorodendraceae 1KP HVNO-2016205 Prasinophyte HVNO-2063673 Prasinoderma coloniale Chlorodendraceae 1KP HVNO-2063673 Prasinophyte HYHN-2013671 Prasinoderma coloniale Prasinococcaceae 1KP HYHN-2013671 Prasinophyte ISGT-2038335 Pedinomonas tuberculata Chaetophoraceae 1KP ISGT-2038335 Chlorophyte 20  KMNX-0003056 Nucleotaenium eifelense Mesotaeniaceae 1KP KMNX-0003056 Charophyte KMNX-2032586 Nucleotaenium eifelense Mesotaeniaceae 1KP KMNX-2032586 Charophyte MMETSP0033_2-Dolte064008 Crustomastix stigmata Mamiellaceae MMETSP MMETSP0033_2-Dolte064008 Prasinophyte MMETSP0803_2 Tetraselmis astigmatica Prasinophyceae MMETSP MMETSP0803_2 Prasinophyte MMETSP0804 Picocystis salinarum Prasinophyceae MMETSP MMETSP0804 Prasinophyte MMETSP0806_2 Dolichomastix tenuilepis Pycnococcaceae MMETSP MMETSP0806_2 Prasinophyte MMETSP0807_2 Picsa0325 Picocystis salinarum unknown MMETSP MMETSP0807_2 Picsa0325 Prasinophyte MMETSP0807_2 Picsa03466 Picocystis salinarum unknown MMETSP MMETSP0807_2 Picsa03466 Prasinophyte MMETSP0807_2 Picsa4387 Picocystis salinarum unknown MMETSP MMETSP0807_2 Picsa4387 Prasinophyte MMETSP0807_2-Picsa02499 Picocystis salinarum unknown MMETSP MMETSP0807_2-Picsa02499 Prasinophyte MMETSP0807_2-Picsa04995 Prasinococcus capsulatus unknown MMETSP MMETSP0807_2-Picsa04995 Prasinophyte MMETSP0941 Pyramimonas amylifera Pyncnococcaceae MMETSP MMETSP0941 Prasinophyte MMETSP1081 Pyram029342 Pyramimonas amylifera Pyramimonadaceae MMETSP MMETSP1081 Pyram029342 Prasinophyte MMETSP1081 Pyram035111 Pycnococcus sp. Pyramimonadaceae MMETSP MMETSP1081 Pyram035111 Prasinophyte MMETSP1081 Pyram049838 Pyramimonas amylifera Pyramimonadaceae MMETSP MMETSP1081 Pyram049838 Prasinophyte MMETSP1082 Micromonas sp. Mamiellaceae MMETSP NEPCC29.03919 Prasinophyte MMETSP1084 Micromonas pusilla Mamiellaceae MMETSP RCC472a.021003 Prasinophyte MMETSP1084 Cyanidioschyzon merolae strain 10D Mamiellaceae MMETSP RCC472a.0361 Prasinophyte MMETSP1085 Micromonas sp. Pyncnococcaceae MMETSP MMETSP1085 Prasinophyte MMETSP1086 Bathycoccus prasinos Glaucosphaeraceae MMETSP MMETSP1086 Glaucophyta MMETSP1089 Glow014496 Cyanoptyche gloeocystis Glaucocystaceae MMETSP MMETSP1089 Glow014496 Glaucophyta MMETSP1089 Glow103052 Gloeochaete wittrockiana Glaucocystaceae MMETSP MMETSP1089 Glow103052 Glaucophyta MMETSP1390 Ostreococcus tauri Mamiellaceae MMETSP MMETSP1390 Prasinophyte MvTALE Mesostigma viride Mesostigmataceae 1KP KYIO Charophyte NNHQ-2768 Spirotaenia minuta Mesotaeniaceae 1KP NNHQ-2768 Charophyte Ol_27999 Micromonas sp. Prasinophyceae JGI 27999 Prasinophyte Ol_33640 Micromonas pusilla Prasinophyceae JGI 33640 Prasinophyte 21  Ol_5313 Ostreococcus lucimarinus Prasinophyceae JGI 5313 Prasinophyte Ot_10780 Ostreococcus tauri Prasinophyceae JGI 10780 Prasinophyte Ot_12440 HD Ostreococcus tauri Prasinophyceae JGI 12440 Prasinophyte Ot_14130 Micromonas pusilla Prasinophyceae JGI 14130 Prasinophyte PpMKN13 Physcomitrella patens Funariaceae Genbank XP_001772723 Embryophyte PpMKN4 Gloeochaete wittrockiana Funariaceae Genbank XP_001781477.1 Embryophyte PUAN-20127 Dunaliella salina Pedinomonadaceae 1KP PUAN-20127 Chlorophyte QPDY-2006061 Coleochaete irregularis Coleochaetaceae 1KP QPDY-2006061 Charophyte QWFV-89532 Bambusina borreri Desmidiaceae 1KP QWFV-89532 Charophyte RCC299_102835 Micromonas pusilla Mamiellaceae JGI 102835 Prasinophyte RCC299_62285 Micromonas pusilla Mamiellaceae JGI 62285 Prasinophyte RCC299_64147 Micromonas pusilla Mamiellaceae JGI 64147 Prasinophyte RCC809_14799 Micromonas pusilla Mamiellaceae JGI 14799 Prasinophyte RCC809_18825 Picocystis salinarum Mamiellaceae JGI 18825 Prasinophyte RCC809_56605 Ostreococcus lucimarinus Mamiellaceae JGI 56605 Prasinophyte RHVC-18141 Neochlorosarcina sp. Mamiellaceae JGI RHVC-18141 Chlorophyte RPGL-2010848 Cylindrocystis brebissonii Mesotaeniaceae 1KP RPGL-2010848 Charophyte RPGL-2092257 Cylindrocystis brebissonii Mesotaeniaceae 1KP RPGL-2092257 Charophyte RPGL-2106649 Cylindrocystis brebissonii Mesotaeniaceae 1KP RPGL-2106649 Charophyte RPQV-2021281 Phymatodocis nordstedtiana Desmidiaceae 1KP RPQV-2021281 Charophyte RPRU-2010600 Staurodesmus omearii Desmidiaceae 1KP RPRU-2010600 Charophyte RPRU-34654 Staurodesmus omearii Desmidiaceae 1KP RPRU-34654 Charophyte RQFE-2954 Cosmocladium cf. constrictum Desmidiaceae 1KP RQFE-2954 Charophyte RQFE-39816 Cosmocladium cf. constrictum Desmidiaceae 1KP RQFE-39816 Charophyte RQFE-41411 Cosmocladium cf. constrictum Desmidiaceae 1KP RQFE-41411 Charophyte SNOX 2020439 Planotaenium ohtanii Mesotaeniaceae 1KP SNOX 2020439 Charophyte SNOX-30615 Planotaenium ohtanii Mesotaeniaceae 1KP SNOX-30615 Charophyte 22  SNOX-8735 Planotaenium ohtanii Mesotaeniaceae 1KP SNOX-8735 Charophyte spiroTALE Spirogyra sp. Zygnemataceae 1KP HAOX Charophyte STKJ-0034964 Cosmarium ochthodes Desmidiaceae 1KP STKJ-0034964 Charophyte STKJ-0109435 Cosmarium ochthodes Desmidiaceae 1KP STKJ-0109435 Charophyte STKJ-0111060partial Cosmarium ochthodes Desmidiaceae 1KP STKJ-0111060partial Charophyte TALE-Mesostigma Mesostigma viride Mesostigmataceae 1KP KYIO Charophyte TALE-Spirogyra Spirogyra sp. Zygnemataceae 1KP HAOX  Charophyte TGNL-2003026 Pyramimonas parkeae Chlorophyta incertae sedis 1KP TGNL-2003026 Prasinophyte TNAW-2000934 Pyramimonas parkeae Pyramimonadaceae 1KP TNAW-2000934 Prasinophyte TNAW-2002934 Pyramimonas parkeae Pyramimonadaceae 1KP TNAW-2002934 Prasinophyte TNAW-2068641 Pyramimonas parkeae Pyramimonadaceae 1KP TNAW-2068641 Prasinophyte TNAW-2078050 coccoid-prasinophyte Pyramimonadaceae 1KP TNAW-2078050 Prasinophyte TPHT-2004315 Spirotaenia sp. Mesotaeniaceae 1KP TPHT-2004315 Charophyte TPHT-2010979 Spirotaenia sp. Mesotaeniaceae 1KP TPHT-2010979 Charophyte TPHT-2033101 Spirotaenia sp. Mesotaeniaceae 1KP TPHT-2033101 Charophyte USIX-2728 Volvox cateri Chlorosarcinaceae 1KP USIX-2728 Chlorophyte VAZE-2004482 Cylindrocystis sp. Mesotaeniaceae 1KP VAZE-2004482 Charophyte VAZE-2007516 Cylindrocystis sp. Mesotaeniaceae 1KP VAZE-2007516 Charophyte VAZE-2042997 Cylindrocystis sp. Mesotaeniaceae 1KP VAZE-2042997 Charophyte VcHDG1 Dolichomastix tenuilepis Volvocaceae JGI 127454 Chlorophyte VQBJ-2043250 Coleochaete scutata Coleochaetaceae 1KP VQBJ-2043250 Charophyte WCQU-2047117 Staurodesmus convergens Desmidiaceae 1KP WCQU-2047117 Charophyte WCQU-2056543 Staurodesmus convergens Desmidiaceae 1KP WCQU-2056543 Charophyte WCQU-2056944 Staurodesmus convergens Desmidiaceae 1KP WCQU-2056944 Charophyte WDCW-2001839 Mesotaenium endlicherianum Mesotaeniaceae 1KP WDCW-2001839 Charophyte WDCW-2003591 Mesotaenium endlicherianum Mesotaeniaceae 1KP WDCW-2003591 Charophyte WDGV-2056101 Cosmarium subtumidum Desmidiaceae 1KP WDGV-2056101 Charophyte 23  WSJO-2006096 Mesotaenium braunii Mesotaeniaceae 1KP WSJO-2006096 Charophtye WSJO-2041695 Mesotaenium braunii Mesotaeniaceae 1KP WSJO-2041695 Charophtye XJGM-2023451 Prasinococcus capsulatus Prasinococcaceae 1KP XJGM-2023451 Prasinophyte XMCL-2015979 Micromonas pusilla Prasinococcaceae 1KP XMCL-2015979 Prasinophyte XOAL-2001289 Chlamydomonas incerta Dolichomastix tenuilepis 1KP XOAL-2001289 Chlorophyte XRTZ-2004971 Roya obtusa Mesotaeniaceae 1KP XRTZ-2004971 Charophyte YOXI-2007594 Cylindrocystis brebissonii Mesotaeniaceae 1KP YOXI-2007594 Charophyte YOXI-2009126 Cylindrocystis brebissonii Mesotaeniaceae 1KP YOXI-2009126 Charophyte YOXI-2024772 Cylindrocystis brebissonii Mesotaeniaceae 1KP YOXI-2024772 Charophyte YOXI-2056088 Cylindrocystis brebissonii Mesotaeniaceae 1KP YOXI-2056088 Charophyte ZRMT-0006388 Mougeotia sp.  Zygnemataceae 1KP ZRMT-0006388 Charophyte ZRMT-0029219 Mougeotia sp.  Zygnemataceae 1KP ZRMT-0029219 Charophyte ZRMT-0030532 Mougeotia sp.  Zygnemataceae 1KP ZRMT-0030532 Charophyte ZRMT-0037661 Mougeotia sp.  Zygnemataceae 1KP ZRMT-0037661 Charophyte     24  Chapter 3: Gene Structure of TALE Homeobox in Mougeotia sp., Cosmocladium constrictum, and Picocystis salinarum   3.1 Introduction The second objective of this study is to determine the gene structure of TALE homeobox identified in charophytes and prasinophytes. The purpose of analyzing the gene structure of TALE homeobox is to use intron locations as a marker to infer the phylogenetic relationships of TALE homeobox classes found in the phylogenetic tree in Chapter 2. The previous chapter showed that the phylogeny of TALE homeobox cannot conclude whether BELL, GSP1 homologs and BELL-related branches are divergent clusters of the same lineage or originated from separate lineages (figure 2.2). One of the difficulties inherent to Homeodomain-based phylogenetic analysis is that the 63 amino acid long Homeodomain sequences may not have enough information to trace events that are estimated to have occurred 1.5 billion years ago. This problem is exacerbated in less conserved clades, such as clusters from the non-KNOX branch of TALE homeobox in the Viridiplantae. In this chapter, the location of introns, exons and conserved domains of TALE homeobox found in Mougeotia sp., Cosmocladium constrictum, and Picocystis salinarum were characterized. With the gene structures determined, a comparison of intron locations within each conserved domain can give more insight into the evolutionary relationships between the TALE homeobox classes of Viridiplantae. Bürglin (1997) had previously used intron locations as a marker to infer the phylogenetic relationship between TALE homeobox classes from embryophytes and animals. Likewise, intron locations can infer the phylogenetic relationships 25  among TALE homeobox classes found in the Viridiplantae. Information on intron locations can also give more hints as to whether BELL and GSP1 homologs classes have a common origin. If BELL and GSP1 homologs classes are found to share common intron locations that are unique only to these two classes, it is likely for the two classes to have common origin. The charophyte Mougeotia sp. was selected for analysis because the classes of TALE homeobox found in the transcriptome shows a combination of classes found in embryophytes and classes from chlorophyta. Cosmocladium constrictum transcriptome expresses KNOX I, KNOX II and GSP1 homolog classes, but BELL class is not found. Since the genome is not available, it cannot be concluded whether C. constrictum encodes the BELL class gene or not. The TALE homeobox genes of P. salinarum were analyzed because five classes of TALE were found, and all five sequences could be amplified from genomic DNA.   3.2 Results 3.2.1 Defining TALE Homeobox Gene Structure of Picocystis salinarum  The PicoGSM1 gene (in algal KNOX class) contains the Knox 1, Knox 2, Elk domains and Homeodomain (figure 3.1A). The PicoGSP1 gene contains only the Homeodomain (figure 3.1B). Comparison between the cDNA and genomic DNA indicates that introns do not exist in the coding region of the GSM1 or GSP1 gene. The Pico4684 gene is a homolog of red algal TALE homeobox. The coding region contains the Homeodomain and one intron outside of the Homeodomain (figure 3.1C).    26  3.2.2 Defining TALE Homeobox Gene Structures of Two Charophytes: Mougeotia sp. and Cosmocladium constrictum  In Mougeotia sp., both KNOX I and KNOX II class genes contain Knox 1, Knox 2, Elk domains and Homeodomain (figure 3.2A and 3.2B). In the KNOX II class gene, the Knox 1 domain is on a separate contig (assembled transcript) from the Homeodomain containing contig. The region in between the two contigs was amplified by PCR and sequenced. The sequenced region between the two contigs cannot be matched to any reads in the Mougeotia sp. transcriptome library. Thus, the locations of exons and introns in the region between the Knox 1 and Knox 2 domains cannot be determined. The contigs containing the gene from BELL and GSP1 homologs classes in Mougeotia sp. contain only the Homeodomain (figure 3.2C and 3.2D). The search for upstream domains in the BELL class gene is described in chapter 2.3.2. The search identified a potential Pox domain upstream of the Mougeotia BELL class gene. However, the region between the contig containing the Pox domain and the contig containing the Homeodomain could not be amplified by PCR. Thus, the intron and exon locations of the full length BELL class gene in Mougeotia sp. could not be identified.     In C. constrictum, the KNOX I and KNOX II class genes contain Elk and Homeodomain (figure 3.3A and 3.3B). The Knox 1 and Knox 2 domains were not found. The GSP1 gene contains only the Homeodomain (figure 3.3 C).    27  A) PicoGSM (algal KNOX)   B) PicoGSP1 (GSP1 homolog)   28  C) Pico4684 (Red algal homolog)   Figure 3.1 Sequence alignment of TALE homeobox gene structure in Picocystis salinarum. Intron locations are indicated by the gap in the cDNA of sequence. Knox 1 domain (dark blue), Knox 2 domain (light blue), Elk domain (green) and Homeodomain (yellow) are labeled for A) PicoGSM1 (algal KNOX) B) PicoGSP1 C) Pico4684 (Red algal homolog)    29  A) Mougeotia KNOX I  30  B) Mougeotia KNOX II     31  Fig 3.2 C) Mougeotia BELL   D) Mougeotia GSP1   Figure 3.2 Sequence alignment of TALE homeobox gene structure in Mougeotia sp. Intron locations are indicated by the gap in the cDNA of sequence. Knox 1 domain (dark blue), Knox 2 domain (light blue), Elk domain (green) and Homeodomain (yellow) are labeled for A) KNOX I, B) KNOX II, C) BELL D) GSP1 32  A) Cosmocladium KNOX I  B) Cosmocladium KNOX II  33  C) Cosmocladium GSP1  Figure 3.3 Sequence alignment of TALE homeobox gene structure in Cosmocladium constrictum. Intron locations are indicated by the gap in the cDNA of sequence. Elk domain (green) and Homeodomain (yellow) are labeled for. A) KNOX I, B) KNOX II, C) GSP1  3.2.3 Search for Upstream Domains for BELL Class TALE Homeobox in Mougeotia sp. The contig containing the Mougeotia BELL class gene is truncated, as it only contains the Homeodomain (figure 3.2C). Since embryophyte BELL class genes contain a Pox domain upstream of the Homeodomain, it is possible this upstream domain also exists in the BELL class gene of Mougeotia sp. However, the transcriptome of Mougeotia sp. does not reveal any linkage between the Homeodomain and Pox domain. To search for the Pox domain in the transcriptome of Mougeotia sp., a multiple sequence alignment of the embryophyte Pox domain was used to query against 47 available charophyte transcriptomes. The purpose of searching multiple transcriptomes was two-fold: 1) the transcriptome of Mougeotia sp. may not express the Pox domain, but this does not conclude the 34  Pox domain does not exist in other charophytes; 2) the Pox domain of Mougeotia sp. may have low similarity to embryophyte Pox. If potential Pox domains are found in a subset of charophyte species, these sequences can be queried against the charophyte transcriptomes to search for a charophyte version of the Pox domain. The results of the query show that transcripts ZRMT-27890 (Mougeotia sp.) and YQST-10176 (Penium exiguum) have similarities with the embryophyte Pox domain query (figure 3.4, sequences 6 and 7). The Cosmarium broomei transcriptome had 11 reads with significant similarity to the embryophyte Pox query. The high sequence similarity may be caused by contamination during the preparation of the Cosmarium broomei transcriptome library. Thus, the significant hits from Cosmarium broomei were not further analyzed. Conserved residues between the Pox domain of embryophytes, Mougeotia sp., and Penium exiguum include F43, G48, Y56, T57, S65, F68, and R69.    To avoid the possibility that the embryophyte Pox domain is too diverged from charophyte sequences to permit retrieval, all 47 charophyte transcriptomes were searched again using the alignment of the Mougeotia and Penium exiguum Pox domain as query. This search also did not result in any hits with significant E-value scores.  The Pox domain found in Mougeotia sp. is on a separate contig from the Homeodomain. Since the genome is unavailable, the region in between the domains is of unknown size. A primer was designed to amplify genomic DNA that would span both transcripts. However, the genomic DNA of the full length BELL gene could not be amplified in Mougeotia sp.   35   Figure 3.4: Multiple sequence alignment of Pox domain from embryophytes, Mougeotia sp. and Penium exiguum. Sequences from embryophytes (pink side bar) and charophytes (red side bar) were aligned. Scaffold 27890 from Mougeotia sp. is shown in sequence 6 and scaffold 10176 from Penium exiguum is shown in sequence 7.     3.2.4 Comparison of Intron Locations in TALE Homeobox  Two introns were conserved in all KNOX genes of Viridiplantae: one in the Homeodomain and the other in the Knox 2 domain (figure 3.5). However, these introns were not found in the Prasinophytes P. salinarum, Micromonas and Ostreococcus (figure 3.5 sequences 12-15). The conserved intron in the Knox 1 domain is shared between streptophyta KNOX I class and chlorophyta algal KNOX (figure 3.5, sequences 1-4 and 16). The intron in the Elk domain is unique to the streptophyta KNOX II class and chlorophyta algal KNOX (figure 3.5, sequences 5-11, 18, and 19).  Within the BELL and GSP1 homologs classes, the Homeodomain shows one conserved intron across the Viridiplantae between amino acids labeled 60 and 61 in figure 3.6. The prasinophyte P. salinarum does not have this intron (figure 3.6, sequence 10). The intron between amino acid 40 and 41 of the Homeodomain in figure 3.6 is shared between the genes from charophyte BELL and GSP1 homologs classes, and the embryophyte BELL class (figure 3.6, sequences 1-8).    36     37    Figure 3.5 Comparison of intron locations between KNOX class genes in embryophytes, charophytes, chlorophytes and prasinophytes. The conserved domains of the KNOX class gene are shown in the schematic diagram at the top. The multiple sequence alignment of each conserved domain is shown below. The consensus sequence for each domain alignment is shown at the top with the Knox 1 (dark blue), Knox 2 (light blue), Elk (green) and Homeodomain (yellow) highlighted by the bar below the consensus. The red box below each sequence indicates intron locations in embryophyte KNOX I (orange side bar) and KNOX II (green side bar) classes, charophyte KNOX I and KNOX II (red side bar) classes, and chlorophyta algal KNOX (blue side bar).   38   Figure 3.6 Comparison of intron locations between BELL and GSP1 class genes in embryophytes, charophytes, chlorophytes, and prasinophytes. The consensus sequence is shown at the top of the multiple sequence alignment, with the Homeodomain highlighted by the yellow bar below the consensus sequence. The red box below each sequence indicates intron locations in embryophyte BELL class (pink side bar), charophyte BELL (red side bar) GSP1 (purple side bar) classes, and chlorophyta GSP1 (blue side bar).  3.3 Discussion 3.3.1 Using Introns to Infer the Phylogenetic Relationship of TALE Homeobox in Viridiplantae Three introns were found to be shared across the Viridiplantae, except for prasinophytes (figure 3.5: Knox 2 domain and KNOX class Homeodomain; figure 3.6: BELL/GSP1 Homeodomain). The ancestral state of TALE homeobox may have contained conserved introns, and the Prasinophyte lineage lost the conserved introns over time.  39  Previous literature indicates two introns are conserved in the KNOX class genes of embryophytes. Bürglin (1997) found the intron in the Homeodomain is unique to KNOX class genes of embryophytes when comparing TALE homeobox intron locations between embryophytes and animals. The present intron comparison found that this intron is not only conserved in the KNOX class genes of embryophytes, but also conserved in the KNOX class genes from algal lineages (figure 3.5). Kerstetter et al.(1994) found the intron in the Elk domain of maize KNOX class genes was unique to the KNOX II class genes, but lacking in the KNOX I class genes. The present intron comparison between KNOX class genes found that this intron is not only conserved in the Elk domain in embryophytes, but also found in charophyte KNOX II, and chlorophyta algal KNOX (figure 3.5).  Bürglin (1997) found one intron conserved between the Homeodomain of embryophyte BELL class and the Homeodomain of four classes of vertebrate TALE homeobox. Similar to Bürglin (1997), the intron comparisons found that this intron is conserved in all embryophyte BELL class genes examined (figure 3.6, between residues 60 and 61). In addition, the GSP1 homologs from the green algal lineages also share this intron location. Similar to the KNOX scenario, prasinophytes may have lost conserved introns over time. The shared intron locations suggest that the GSP1 homologs and BELL classes shared common ancestry. The second conserved intron in the Homeodomain of GSP1 and BELL is found between residues 40 and 41. This intron was previously reported to be conserved only in embryophyte BELL class genes when compared to animal TALE homeobox genes (Bürglin, 1997). Intron comparison in figure 3.6 shows that this intron is also found in the charophyte GSP1 homologs and BELL classes, but does not exist in the chlorophyta GSP1 homologs class (figure 3.6).   40  Figure 3.7 shows an evolution model of the TALE homeobox classes in the Viridiplantae. The intron comparison suggests that the ancestral KNOX gene likely contained introns at Knox1, Knox 2, Elk domains and Homeodomain. After the divergence of chlorophyta and streptophyta, the ancestral KNOX in both lineages retained the introns. The two classes of streptophyta KNOX may have emerged by a duplication event in the ancestral charophytes. After duplication, the KNOX I class gene lost the intron at the Elk domain and retained the remaining ancestral introns. By contrast, the KNOX II class gene lost the intron at the Knox 1 domain and retained the remaining ancestral introns. The chlorophyta KNOX retained all of the ancestral introns. This scenario would have resulted in two classes of KNOX in the Streptophyta, and an algal KNOX in Chlorophytes.  At present, two models of evolution are proposed for the evolution of the GSP1 and BELL classes (figure 3.7). Analysis of intron locations in TALE homeobox of red algal species will indicate whether the gain of intron (figure 3.7, left) or lost of intron (figure 3.7, right) model of evolution is more probable for the BELL and GSP1 classes in the Viridiplantae. In the gain of intron model (figure 3.7, left), the ancestral state of the GSP1 and BELL classes may have been an ancestral GSP1-like gene with one conserved intron in the Homeodomain that is indicated between residues 60 and 61 in figure 3.6. After divergence, both chlorophyta and streptophyta retained the ancient intron between residues 60 and 61 of the Homeodomain. The chlorophyta GSP1 class retained the ancestral intron over time. However, the ancestral GSP1-like gene in the charophytes gained an additional intron between residues 40 and 41 in the Homeodomain, followed by a gene duplication event to create the BELL class gene. Both GSP1 and BELL classes were retained in the charophytes, but the GSP1 class was lost in the embryophytes. In the lost of  intron model (figure 3.7 right), the ancestral state of GSP1 and BELL classes was an 41  ancestral GSP1-like gene with two conserved introns in the Homeodomain that are indicated between residues 40/41 and 60/61 in figure 3.6. After divergence, the ancestral GSP1-like gene of the chlorophyta lost the intron between residues 40 and 41, while the streptophyta retained both introns. The ancestral GSP1-like gene in the streptophyta lineage underwent a gene duplication event to create the GSP1 and BELL classes. The GSP1 class gene may have been lost in the early ancestral embryophytes, but retained in the charophytes. Since charophyte genomes are not available, it is unknown whether BELL class genes exist in all extant lineages or only a subgroup of charophytes. Thus, it is unclear how early the gene duplication event of the ancestral GSP1-like gene occurred. When gene duplication occurs, it is possible that the duplicated gene takes on a new function. The residues indicated at position 66 and 70 in the Homeodomain of GSP1 and BELL in Mougeotia sp. are required for target DNA specificity (figure 3.6, sequence 6 and 7) (Damante et al., 1996; Viola and Gonzalez, 2006). The differences in the amino acid identity at residues 66 and 70 of the Homeodomain in Mougeotia BELL and Mougeotia GSP1 indicate that the encoded proteins possibly target different downstream genes. Lee et al. (2008) found that the residues at position 66 and 70 are T/V66 and A70 for chlorophyte GSP1, and I66 and V70 for embryophyte BELL. Similar to chlorophytes, Mougeotia GSP1 (figure 3.6, sequence 7) and C. constrictum GSP1 (figure 3.6, sequence 8) also have V66 and A70, while Mougeotia BELL (figure 3.6, sequence 6) shares the I66 and V70 residues with embryophyte BELL.  The modern day GSP1 is known to regulate genes that are crucial to diploid development (Lee et al. 2008). It is possible that the ancestral GSP1 may have already been involved in regulating developmental programs crucial for survival. Changes in residue 66 and 70 in the Homeodomain of the ancestral GSP1 would have affected the specificity of GSP1 to its target 42  DNA, resulting in disruption of developmental program regulation. When the ancestral GSP1-like gene in the early streptophyta underwent duplication, there was no selective advantage to retain residue 66 and 70 in the Homeodomain of the new copy of GSP1 (BELL class gene) because the original GSP1 was already regulating proper onset of developmental programs. After GSP1 duplication, the ancestral charophytes had an opportunity to use the extra copy of GSP1 to experiment with new developmental networks, without foregoing the well established algal developmental network. The extra BELL class in the charophyte ancestors allowed for new combinations of TALE heterodimers. New heterodimer partners may have played a role during the emergence of the early embryophytes by delaying meiosis after zygote formation, and turning on mitosis to create the multicellular sporophyte stage. The change in developmental program regulation might have allowed ancestral charophytes to escape the algal life cycle and elaborate the diploid body plan for survival on land.   43   Figure 3.7 Model of evolution for TALE homeobox classes in the Viridiplantae. The conserved domains and intron locations of KNOX, GSP1 and BELL classes are shown in the gene schematic diagram at the bottom. The model on the left assumes gain of intron in the ancestral GSP1-like gene, and the model on the right assumes lost of intron in the ancestral GSP1-like gene. KNOX evolution is the same in both models. KNOX class genes evolved from an ancestral KNOX gene present in the common ancestor of the Viridiplantae. A duplication event in the ancestral charophytes resulted in two classes of KNOX in the streptophyta lineage. After the duplication event, the ancestral KNOX I lost intron 3 (in Elk domain) and the ancestral KNOX II lost intron 1 (in Knox 1 domain). The chlorophyta lineage retained the ancestral intron locations. GSP1 and BELL class genes evolved from an ancestral GSP1-like gene in the common ancestor of the Viridiplantae. In the gain-of-intron scenario (left), the ancestral GSP1-like gene carried intron 2 in the Homeodomain. The streptophyta lineage gained a second intron (intron 1) within the Homeodomain of the ancestral GSP1-like gene. The intron gain event was followed by a gene duplication event to create the GSP1 homologs and BELL class genes. The GSP1 homologs class was lost in the embryophytes, but retained in the charophytes. The chlorophyta lineage retained the ancestral intron (intron 2) in the GSP1 homologs class. In the lost-of-intron scenario (right), the ancestral GSP1-like gene carried two introns. The chlorophyta lineage lost intron 1, while the streptophyta lineage retained both ancestral introns.    44  3.4 Materials and Methods 3.4.1 Cultures Axenic cultures of Mougeotia sp. CCAC0197, and Cosmocladium constrictum CCAC0217 were purchased from the Culture Collection of Algae at the University of Cologne (CCAC). The axenic culture of Picocystis salinarum CCMP1897 was purchased from The National Center of Marine Algae and Microbiota (NCMA). Mougeotia sp. was grown in Waris-H+3V liquid media, Cosmocladium constrictum was grown in liquid Bold’s Basal Media (BBM), and Picocystis salinarum was grown in L1 media. All cultures were kept under 24 hour light exposure that has an average light intensity of 169.1 lux.   3.4.2 Cloning Genomic Sequences of TALE Homeobox from the Transcriptome  Full length TALE homeobox genes were amplified from genomic DNA of Mougeotia sp. , Cosmocladium constrictum and Picocystis salinarum using primers listed in table 3.1. Primers were designed using transcriptomes of Mougeotia sp., Cosmocladium constrictum and Picocystis salinarum available from the 1KP project data base (https://www.bioinfodata.org/Blast4OneKP/). Each primer pair amplified regions which flank overlapping fragments in each TALE homeobox gene. The amplified regions were cloned into Promega’s pGEM T-easy vector and sequenced with M13R or M13F primers. The sequenced fragments were compiled in the Geneious 7.1 software to create the consensus genomic sequence. The full length KNOX II gene in Mougeotia sp. was found to have the 5’ end on transcript ZRMT-0008059 and a 3’ end in transcript ZRMT-0006388. As the size of the region between the two transcripts is unknown, primers were designed with two criteria: 1) primers are as close as possible to the ends of the transcripts to increase the chances that the GoTaq polymerase enzyme 45  (Promega) can complete the DNA replication process; 2) primers will amplify a region that spans both transcripts. The full length sequence of KNOX II gene was determined by compiling overlapping sequence fragments in the Geneious 7.1 software.  Table 3.1 Primer pairs used to amplify TALE homeobox genes from genomic DNA of Mougeotia sp., Cosmocladium constrictum, and Picocystis salinarum.   Gene Forward primer Reverse primer  Mougeotia KNOX I 5’-GAAGTTGCCCAAGGGAGCAAC-3’ 5’- GACACGTTCGTGCGCGTCAC-3’  5’- CCCGTTATATTCTGAGCTC-3’ 5’- GATATGAGCCTGCCACCACT-3’  5’- ATCTTGAGGACGCAGACGA-3’ 5’- GATATGAGCCTGCCACCACT-3’  5’-GCTCCACATGCCGCCATG-3’ 5’-CCATTTCTGGACTTGGGTTG-3’ Mougeotia KNOX II 5’-ATGAAACCTGGGCCTGGGAATG-3’ 5’-CATGGAAACGCTAGACAAG-3’  5’-CTCACACGAAACGCAAAGAA-3’ 5’-AGACATGGCAAGTCCATTCC-3’ Mougeotia BELL 5’- CGAATTCTCAGGGTGTGGCCTCTCAG-3’ 5’- TCAATTCTCTTTGCTCGCTTCATC-3’ Mougeotia GSP1 5’-CCTCCACATGCGCAAGTG-3’ 5’-TCCACTCATCACTTCTTCAC-3’ Cosmocladium KNOX I 5’-CCTCTCGATCCCAGCGGAC-3’ 5’-CATTCTACTCCACTAGTGG-3’  5’-CGAATTCTCGGACGGAATGCTGTTTC-3’ 5’-CTTGTACGTGTCCGCTGG-3’ Cosmocladium KNOX II 5’-CGAATTCATGAGGCTGGGTGGAGAAG-3’ 5’-CCACACCGTCAGGCTTCTG-3’ Cosmocladium GSP1 5’-CGAATTCATGCTACAGTCTCTGCCTCAG-3’ 5’-GTCAACTCCCTTCAAGACG-3’ Picocystis GSP1 5’ GCAATTGCATGCGACAAGTGGAGAG-3’ 5’-CAGATCTGAAAGCCGTCACTTTTGCTC-3’ Picocystis GSM (full) 5’-GCAATTGATGGATTTCCCATCGACTGAAG-3’ 5’-CGGATCCAATTACGGACGGTAGAG-3’ Picocystis GSM (truncated) 5’-CGAATTCACCCACGAAGGTAATTCAC-3’ 5’-CGGATCCAATTACGGACGGTAGAG-3’ Picocystis 4684  Exon 1 5’-GCATATGTCCAAGGTCGCAGACAG-3’ 5’-GCATTTTTTCGAACTGCTTCAAC-3’ Picocystis 4684 Exon 2 5’-GTTGAAGCAGTTTCGAAAAAATGC-3’ 5’-CGGATCCCTAGCTTGCGCGAATATTC-3’  3.4.3 Defining Introns, Exons and Conserved Domains The genomic sequence of the TALE homeobox genes were aligned with the corresponding cDNA sequence from the transcriptome to reveal the locations of introns and exons. Once all intron and exon locations were identified, the amino acid sequence was translated from the exon sequence. The translation frame was determined by looking for the conserved PYP and WFXN motifs within the Homeodomain. Conserved domains were found by querying the translated sequence against the Pfam database (http://pfam.xfam.org/). If the domain cannot be found in the Pfam database, pairwise sequence alignment with characterized 46  TALE Homeobox sequences from Chlamydomonas reinhardtii and embryophytes was done to find conserved domains. 47  Chapter 4: Y2H Analysis of TALE Homeobox from Picocystis salinarum 4.1 Introduction The third objective of this study is to test the possible interaction of TALE homeobox found in P. salinarum using the yeast two hybrid (Y2H) system. Charophyte TALE homeobox was not tested because the full length BELL gene containing the Pox and Homeodomain could not be isolated from the genomic DNA of Mougeotia sp., and the upstream domains in KNOX I and KNOX II class genes have not been found in the C. constrictum transcriptome (chapter 3). In embryophytes, the domains upstream of the Homeodomain are involved in increasing the affinity to a heterodimer partner (Bellaoui et al, 2001; Smith et al., 2002). However, the effect of removing upstream domains in the Y2H assay is unknown in charophytes. Thus, the full length sequence should be used in the Y2H assay to test interaction between charophyte TALE homeobox. Since the full length TALE homeobox genes have been isolated from P. salinarum, the Y2H analysis will only test the interaction of TALE homeobox from P. salinarum.  KNOX and BELL interactions have been observed to be selective in embryophytes (Bellaoui et al., 2001; Chen et al., 2003; Muller et al., 2001; Smith et al., 2002). The purpose of analyzing the heterodimerization network of TALE homeobox proteins from P. salinarum is to determine if selective interaction is also occurring in prasinophytes. P. salinarum will be used as a proxy for the ancestral state of TALE homeobox heterodimerization in the Viridiplantae. Different heterodimer partners can change the affinity to downstream target DNA (Bellaoui et al., 2001; Chen et al., 2003; Muller et al., 2001; Sharma et al. 2014; Smith et al., 2002). New combinations of heterodimers have the potential to turn on novel genetic networks downstream, which can alter developmental process. Lee et al. (2008) hypothesized that the ability to turn on new genetic networks allowed the algal ancestor to successfully escape from the algal life cycle.  48  4.2 Results To test which TALE homeobox from P. salinarum are interacting partners, the genes encoding different TALE homeobox proteins were cloned into pGBKT7 bait vector or pGADT7 prey vector. Each TALE homeobox was tested in the bait position, and the prey position. The yeast transformants expressing both bait and prey fusion proteins were selected using double drop out (DDO) media lacking leucine and tryptophan (figure 4.1A). Interaction between the bait and prey was selected using quadruple drop out (QDO) media lacking leucine, tryptophan, adenine, and histidine (figure 4.1B). The bait fusion proteins were assayed with empty prey vectors to test for autoactivation (figure 4.1B). Only the full length picoGSM 1-1001 showed autoactivation when tested with an empty prey vector (figure 4.1B). Due to the autoactivation by full length GSM, the N-terminal truncated picoGSM 217-1001 that still contained all KNOX protein domains was used to test for interaction activity. Homodimer interactions were not tested in this assay.  The results show interaction between picoGSP1-picoGSM 1-1001 and picoGSP1-picoGSM217-1001. The red algal homolog pico4684 shows interaction with both full length and truncated picoGSM when pico4684 was in the prey position (figure 4.1B). However, no interaction was seen when pico4684 was in the bait position (figure 4.1B). Similarly, pico4684 shows interaction with picoGSP1 when it is in the prey position, but no interaction was seen when pico4684 is in the bait position (figure 4.1B). 49   Figure 4.1 Yeast-2-hybrid assay of Picocystis salinarum TALE homeobox interactions with pGBKT7 fusion proteins (bait) and pGADT7 fusion proteins (prey). Y2HGold yeast strain (Clonetech) was co-transformed with one bait and one prey fusion protein; colonies that grow on DDO media (A) express both fusion proteins, and colonies that grow on QDO media (B) indicate interaction between the bait and prey fusion proteins. The TALE homeobox fused to the bait and prey proteins used are picoGSM 1-1001 (full ORF), picoGSM 217-1001 (truncated, but includes all domains), picoGSP1, and pico4684. Homodimer interactions were not tested.  4.3 Discussion The Y2H results show that picoGSP1 and picoGSM can interact (figure 4.1B). This result is similar to the GSM1 and GSP1 interaction observed in the chlorophyte, Chlamydomonas reinhardtii (Lee et al., 2008). As the role of GSM1 and GSP1 heterodimer is crucial for proper zygote development in C. reinhardtii (Lee et al., 2008), it was expected that the interaction of P. salinarum GSM and GSP1 would also be conserved.  The more surprising interaction is seen between the red algal homolog, pico4684, and picoGSM. Chapter 2 described the use of PCR to confirm that red algal homologs found in the P. salinarum transcriptome were indeed encoded in the genome. However, as the interaction between pico4684-picoGSP1 and pico4684-picoGSM were only seen when pico4684 is in the prey position (figure 4.1B), it cannot be concluded whether or not an interaction is truly occurring. It is possible that the interaction is only detected in one direction due to steric 50  hindrance in the pico4684 bait fusion protein. To overcome this problem, new bait fusion proteins could be made using truncated coding sequence for the pico4684 gene. The interaction patterns of the second red algal TALE homolog encoded by the               P. salinarum genome still need to be tested to conclude the function of the two red algal TALE homologs encoded in the P. salinarum genome.  4.4 Materials and Methods 4.4.1 Yeast Strain Y2HGold yeast strain from Clonetech was grown on YPDA plates in 30°C for 3 days. Red colonies were isolated on a new YPDA plate and grown in 30°C for 3 more days before using for transformation.   4.4.2 Y2H Constructs Full length Picocystis genes were amplified from genomic DNA using primers listed on the table 4.1. The primers were designed to add 5’ EcoRI and 3’ BamHI restriction enzyme sites to the ends of each gene. In genes where there was an internal EcoRI or BamHI restriction enzyme site, 5’ MfeI and 3’ BglII restriction enzyme sites were added to the ends. The overhanging ends of MfeI and EcoRI restriction enzyme sites are compatible, and the ends of BglII and BamHI restriction enzyme sites are compatible. The genes were cloned into the multiple cloning sites of pGBKT7 and pGADT7 vectors (Clonetech) at the EcoRI/BamHI locations.  The two exons of the full length Picocystis 4684 gene were separately amplified with PCR. The reverse primer of exon 1 and forward primer of exon 2 created overlapping ends on 51  each exon. To connect the two exons together, a second round of PCR was done with the forward primer of exon 1 and reverse primer of exon 2.  Table 4.1 Primer pairs used to amplify TALE homeobox genes from genomic DNA of Picocystis salinarum. Restriction enzyme sites were added to the ends of the gene using the primer sequences highlighted in bold underlined text. The Picocystis 4684 primers added regions to the 3’ end of exon 1  and 5’ end of exon 2, which over laps with each other (dashed underline).   Gene  Forward primer Reverse primer Picocystis GSP1 5’ GCAATTGCATGCGACAAGTGGAGAG-3’ 5’-CAGATCTGAAAGCCGTCACTTTTGCTC-3’ Picocystis GSM (full) 5’-GCAATTGATGGATTTCCCATCGACTGAAG-3’ 5’-CGGATCCAATTACGGACGGTAGAG-3’ Picocystis GSM (truncated) 5’-CGAATTCACCCACGAAGGTAATTCAC-3’ 5’-CGGATCCAATTACGGACGGTAGAG-3’ Picocystis 4684  Exon 1 5’-GCATATGTCCAAGGTCGCAGACAG-3’ 5’-GCATTTTTTCGAACTGCTTCAAC-3’ Picocystis 4684 Exon 2 5’-GTTGAAGCAGTTTCGAAAAAATGC-3’ 5’-CGGATCCCTAGCTTGCGCGAATATTC-3’  4.4.3 Yeast Competent Cells and Transformation  Yeast competent cells were made in a large batch and stored as a frozen stock prior to transformation. Competent cells were made by first growing a liquid culture in YPDA media. The culture was incubated overnight at 30°C on a VWR DS500 orbital shaker rotating at 200 rpm. The overnight culture was diluted with fresh YPDA to OD600 = 0.2, and allowed to grow to log phase (approximately OD600 = 0.4 – 0.6) by incubating at 30°C on a VWR DS500 orbital shaker rotating at 200 rpm. The growth was measured with a Beckman Coulter DU730 Life Science UV/Vis Spectrophotometer. Log phase cells were washed twice with ddH2O, and then resuspended into 5% (v/v) glycerol and 10% (v/v) DMSO solution. The resuspended competent cells were aliquoted into cyrotubes and frozen at a rate of -1°C per minute until the temperature reached -80°C.  Prior to transformation, the frozen competent cells were thawed in a 37°C water bath for 30 seconds. The glycerol-DMSO solution was removed and the yeast competent cells were resuspended into the transformation solution indicated in table 4.2. The yeast competent cells 52  were incubated with the transformation solution for 60 minutes at 30°C on a VWR DS500 orbital shaker rotating at 200 rpm. DMSO was added to the yeast competent cells after the incubation period, and the cells were heat shocked in a 42°C water bath for 15 minutes. The transformation solution was removed after the heat shock, and the cells were resuspended into TE buffer. Following transformation, cells were plated onto -2a media (-leu/-trp), and then replicated on -4a media (-leu/-trp/-ade/-his). Plate cultures were incubated at 30°C until colonies could be seen.    Table 4.2 Y2H transformation solution. Recipe indicated is for transforming one set of bait and prey plasmid constructs.  Reagent Volume (µL) 50% (w/v) PEG 3350 240 1.0M LiAc 36 10 mg/mL Boiled Salmon DNA 5 10X TE buffer 36 ddH2O 41 200 ng/μL prey fusion plasmid 1 200 ng/μL bait fusion plasmid 1  53  Chapter 5: Conclusion 5.1 Major Findings and Contributions to TALE Homeobox Research The objective for this study was to determine the changes that occurred in the genetic sequence and the heterodimerization interactions of TALE homeobox in the Viridiplantae. The results of the phylogeny and intron analysis show that the KNOX class from chlorophyta and streptophyta are a group of highly conserved genes originating from an ancestral KNOX. Similar to previously published phylogenies (Bürglin, 1997; Kerstetter et al. 1994; Lee et al., 2008), I found that the embryophyte KNOX is divided into KNOX I and KNOX II classes (figure 2.2). The charophyte KNOX was also found to have bipartite KNOX classes similar to embryophytes, while the chlorophyte KNOX falls into an algal KNOX cluster on the phylogeny (figure 2.2). Comparison of intron locations showed conserved introns in the Homeodomain and Knox 2 domain, suggesting KNOX I, KNOX II and algal KNOX shared common ancestry (chapter 3). An evolutionary model was proposed for KNOX class genes in the Viridiplantae (figure 3.7). The model predicts that the ancestral KNOX gene underwent gene duplication in the early charophytes to create two classes of KNOX in the streptophyta. The chlorophyta only retained one copy of the ancestral KNOX gene.  Prior to this study, it was known that embryophytes had BELL class genes, and chlorophytes had GSP1 homologs class genes (Bürglin, 1997; Lee et al., 2008). This study found that charophytes have both GSP1 homologs and BELL class genes (figure 2.2). To overcome the limitations of the phylogeny published by Lee et al. (2008), this study aimed to include more algal GSP1 homologs class and algal BELL class sequences in the phylogeny. However, the updated phylogeny also could not resolve the relationship between BELL and GSP1 homologs classes with good bootstrap support (figure 2.2). Intron comparison reveals that the BELL class 54  specific intron is also present in charophyte GSP1 homologs. This result suggested common ancestry between the two classes. As GSP1 homologs were found in both charophytes and chlorophyta, an ancestral GSP1-like gene is believed to have been present in the common ancestor of Viridiplantae. Two models of evolution were proposed for the BELL and GSP1 homologs classes (figure 3.7). The gain-of-intron model (figure 3.7, left) predicts the ancestral GSP1-like gene gained an intron in the streptophyta lineage, and the chlorophyta lineage retained the ancestral intron. The lost-of-intron model (figure 3.7, right) predicts the ancestral GSP1-like gene lost an intron in the chlorophyta lineage, and the streptophyta lineage retained both ancestral introns. Both models predict that the ancestral GSP1-like gene underwent gene duplication in the early charophytes to create the GSP1homologs and BELL classes. The GSP1 homologs class was later lost in the embryophytes.    Diversification of TALE Homeobox in the ancestral charophytes would have allowed more combinations of heterodimerization partners. Changes in TALE heterodimerization network led to the onset of new developmental networks, possibly resulting in delaying meiosis and initiating mitosis in the zygotes of charophyte-like ancestors. With the diversification of multicellular diploid body plan, the early charophyte-like ancestors could have developed specialized tissues to colonize land.  My study contributes to TALE homeobox research in the Viridiplantae by building upon the chlorophyte homeobox phylogeny by Lee et al. (2008), as well as adding a new phylogenetic analysis of TALE homeobox in charophytes. I also extended the intron analysis by Bürglin (1997) to include green algal GSP1 homologs and BELL classes. The combination of the phylogenetic tree, intron analysis, and heterodimerization network analysis allowed for more insights into the evolutionary history of TALE homeobox that previous phylogeny alone could not infer. 55  5.2 Future Directions  The future direction for this project is to sequence the genome of Mougeotia sp. and Cosmocladium constrictum. One of the limitations to this study is the lack of sequenced genomes for charophytes. Only a limited number of charophytes could be analyzed because the species were chosen based on transcriptome expression. When using the transcriptome to identify a full complement of TALE homeobox genes, unexpressed genes cannot be cloned for analysis. In this study, it was found that C. constrictum transcriptome does not express the BELL class gene. However, analysis of the genome would be necessary to conclude if C. constrictum encodes a BELL class gene. A completed genome will allow researchers to identify all TALE homeobox encoded by charophytes. In addition, genome sequences will aid in designing cloning schemes to isolate the full length gene of each TALE homeobox.  One of the restrictions of using the Mougeotia transcriptome to design the cloning strategy was that the full length sequences of two TALE homeobox genes were found on two separate contigs that were of unknown distance apart. In the case of the Mougeotia BELL gene, the primers could not amplify the full length gene. Having all the genetic sequence that encodes each domain of the TALE protein is necessary for a successful Y2H test.  56  References  Bellaoui, M., Pidkowich, M. S., Samach, A., Kushalappa, K., Kohalmi, S. E., Modrusan, Z., … Haughn, G. W. (2001). The Arabidopsis BELL1 and KNOX TALE Homeodomain Proteins Interact through a Domain Conserved between Plants and Animals. The Plant cell, 13 (November), 2455–2470. doi:10.1105/tpc.010161.2 Blackwell, W. H. (2003). Two Theories of Origin of the Land-Plant Sporophyte : Which Is Left Standing ? Botanical Review, 69 (2), 125–148. Bürglin, T R. (1997). Analysis of TALE superclass homeobox genes (MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domain conserved between plants and animals. Nucleic acids research, 25 (21), 4173–80.  Bürglin, Thomas R. (2011). Homeodomain Subtypes and Functional Diversity. In T. R. Hughes (Ed.), A Handbook of Transcription Factors (pp. 95–122). New York, USA: Springer. doi:10.1007/978-90-481-9069-0 Chen, H., Rosin, F. M., Prat, S., & Hannapel, D. J. (2003). Interacting Transcription Factors from the Three-Amino Acid Loop Extension Superclass Regulate Tuber Formation1. Plant Physiology, 132 (3), 1391–1404. doi:10.1104/pp.103.022434.TALE Damante, G., Pellizzari, L., Esposito, G., Fogolari, F., Viglino, P., Fabbro, D., … Di Lauro, R. (1996). A molecular code dictates sequence-specific DNA recognition by homeodomains. EMBO, 15 (18), 4992–5000. Graham, L. E., Cook, M. E., & Busse, J. S. (2000). The origin of plants: body plan changes contributing to a major evolutionary radiation. Proceedings of the National Academy of Sciences of the United States of America, 97 (9), 4535–40.  Kerstetter, R., Vollbrecht, E., Lowe, B., Veit, B., Yamaguchi, J., & Hake, S. (1994). Sequence analysis and expression patterns divide the maize knotted1-like homeobox genes into two classes. The Plant cell, 6 (12), 1877–87. doi:10.1105/tpc.6.12.1877 Langdale, J. A., & Harrison, C. J. (2008). Developmental transitions during the evolution of plant form. In A. Minelli & G. Fusco (Eds.), Evolving Pathways: Key Themes in Evolutionary Developmental Biology (pp. 297–314). Cambridge University Press. Lee, J.-H., Lin, H., Joo, S., & Goodenough, U. (2008). Early sexual origins of homeoprotein heterodimerization and evolution of the plant KNOX/BELL family. Cell, 133 (5), 829–40. doi:10.1016/j.cell.2008.04.028 57  Leliaert, F., Smith, D. R., Moreau, H., Herron, M. D., Verbruggen, H., Delwiche, C. F., & De Clerck, O. (2012). Phylogeny and Molecular Evolution of the Green Algae. Critical Reviews in Plant Sciences, 31 (1), 1–46. doi:10.1080/07352689.2011.615705 Leliaert, F., Verbruggen, H., & Zechman, F. W. (2011). Into the deep: new discoveries at the base of the green plant phylogeny. BioEssays, 33 (9), 683–92. doi:10.1002/bies.201100035 Lewis, L. A., & McCourt, R. M. (2004). Green algae and the origin of land plants. American journal of botany, 91 (10), 1535–56. doi:10.3732/ajb.91.10.1535 Müller, J., Wang, Y., Franzen, R., Santi, L., Salamini, F., & Rohde, W. (2001). In vitro interactions between barley TALE homeodomain proteins suggest a role for protein-protein associations in the regulation of Knox gene function. The Plant journal, 27 (1), 13–23. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11489179 Sakakibara, K., Ando, S., Yip, H. K., Tamada, Y., Hiwatashi, Y., Murata, T., … Bowman, J. (2013). KNOX2 Genes Regulate the Haploid-to-Diploid Morphological Transition in Land Plants. Science, 339, 1067–1070. doi:10.1126/science.1230082  Sharma, P., Lin, T., Grandellis, C., Yu, M., & Hannapel, D. J. (2014). The BEL1-like family of transcription factors in potato. Journal of experimental botany, 65 (2), 709–23. doi:10.1093/jxb/ert432 Smith, H. M. S., Boschke, I., & Hake, S. (2002). Selective interaction of plant homeodomain proteins mediates high DNA-binding affinity. Proceedings of the National Academy of Sciences of the United States of America, 99 (14), 9579–84. doi:10.1073/pnas.092271599 Viola, I. L., & Gonzalez, D. H. (2006). Interaction of the BELL-like protein ATH1 with DNA: role of homeodomain residue 54 in specifying the different binding properties of BELL and KNOX proteins. Biological chemistry, 387(1), 31–40. doi:10.1515/BC.2006.006  

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