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University of Iowa Iowa Research Online Theses and Dissertations 2012 Genes involved in asexual sporophyte development in Ceratopteris richardii and Arabidopsis thaliana Angela Ruth Cordle University of Iowa Copyright 2012 Angela Ruth Cordle This dissertation is available at Iowa Research Online: http://ir.uiowa.edu/etd/4598 Recommended Citation Cordle, Angela Ruth. "Genes involved in asexual sporophyte development in Ceratopteris richardii and Arabidopsis thaliana." PhD (Doctor of Philosophy) thesis, University of Iowa, 2012. http://ir.uiowa.edu/etd/4598. Follow this and additional works at: http://ir.uiowa.edu/etd Part of the Biology Commons GENES INVOLVED IN ASEXUAL SPOROPHYTE DEVELOPMENT IN CERATOPTERIS RICHARDII AND ARABIDOPSIS THALIANA by Angela Ruth Cordle An Abstract Of a thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Biology in the Graduate College of The University of Iowa May 2012 Thesis Supervisor: Associate Professor Chi-Lien Cheng 1 ABSTRACT The life cycle of land plants involves cycling between two multicellular phases, the haploid gametophyte and the diploid sporophtye. Asexual reproductive strategies, that bypass meiosis and fertilization, have evolved in many plants representing diverse taxa among the land plant phylogeny. Apogamy is an asexual reproductive strategy that produces a sporophyte directly from a gametophyte cell without the usual ploidy change associated with syngamy. The genes that drive the process of apogamy are almost completely unknown. Knowledge of these genes and their functions will provide insight into the evolution of asexual reproduction, and has the potential to provide insight into the evolution aspect of the development of the sporophyte body and the alternation of generations in land plants, two very important questions in plant science. My Ph. D. research has focused on elucidating the genes that are involved in the process of apogamy, and understanding the functions of the homologues of these genes in angiosperms. First, I induced apogamy experimentally from the semi-aquatic fern Ceratopteris richardii by culturing them on 2.5% glucose. I discovered that C. richardii gametophytes begin to become committed to the production of apogamous sporophytes after 10 days of culture on glucose. I then created a normalized, subtracted cDNA library that represents genes that increase in transcription in gametophytes during this commitment time. By comparing the Gene Ontology terms mapped to this cDNA library with that of the gametophyte transcriptome of the homosporous fern Pteridium aquilinum, I discovered that the C. richardii apogamy library is enriched in genes that are involved in stress response and metabolism. The C. richardii apogamy library also contains many sequences whose homologues in Arabidopsis are specifically expressed or upregulated in flower organs or seed structures, both of which are absent in ferns. One of these genes, UNC93-like, is also expressed in the eggs of C. richardii gametophytes, as evidenced by 2 whole mount in situ hybridization. Functional egg cells are implicated as necessary for C. richardii gametophytes to be induced to form apogamy. In Arabidopsis, a homozygous T-DNA line for the AtUNC93-like gene, which produces a partial AtUNC93-like transcript and likely retains partial functionality, has vegetative and reproductive defects. Embryo abortion and male and female gametophyte lethality contribute to a small seed set in these plants. However, reciprocal crosses indicate that the mutant allele does not affect gametophyte function. Instead, it appears to be a failure of the maternal plant to sustain the life of a percentage of the gametophytes that it produces. Thus, the AtUNC93-like gene has functions that are necessary for normal sporophyte vegetative growth and reproductive success, but is dispensable for the function of gametophytes. These studies have provided insight into the mechanisms that induce apogamy in the fern C. richardii. The apogamy cDNA library will provide a valuable resource for future investigations into the genes that are involved in apogamy. Abstract Approved: ____________________________________ Thesis Supervisor ____________________________________ Title and Department ____________________________________ Date GENES INVOLVED IN ASEXUAL SPOROPHYTE DEVELOPMENT IN CERATOPTERIS RICHARDII AND ARABIDOPSIS THALIANA by Angela Ruth Cordle A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Biology in the Graduate College of The University of Iowa May 2012 Thesis Supervisor: Associate Professor Chi-Lien Cheng Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL _______________________ PH.D. THESIS _______________ This is to certify that the Ph.D. thesis of Angela Ruth Cordle has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Biology at the May 2012 graduation. Thesis Committee: ___________________________________ Chi-Lien Cheng, Thesis Supervisor ___________________________________ Erin E. Irish ___________________________________ Daniel Eberl ___________________________________ Jan Fassler ___________________________________ Jackie R. Bickenbach To Mom, Dad, Craig, and Gracie ii ACKNOWLEDGMENTS I would like to express my gratitude to the following people: My advisor Chi-Lien Cheng for her patience, guidance and friendship. The current and past members of the Cheng and Irish labs including: Joshua Strable, Setu Vora, Ben Beydler, Amelia Hurst, and my good friend Linh Thuy Bui. My committee members including Jan Fassler, Dan Eberl, Jackie Bickenbach for their helpful suggestions, and especially Erin Irish and ChiLien who conceived of this project and provided guidance and direction while allowing me to mold it into a Ph. D. thesis. Thanks to Doug Houston for useful technical advice regarding in situ hybridization and RT-qPCR experiments, and also allowing me the use of his dissecting microscope. Thanks to Gery Hehman for technical advice and assistance. Finally, I thank my best friend and husband, Craig, who keeps me sane, and our beautiful daughter, Gracie, who is my favorite person in this world. iii ABSTRACT The life cycle of land plants involves cycling between two multicellular phases, the haploid gametophyte and the diploid sporophtye. Asexual reproductive strategies, that bypass meiosis and fertilization, have evolved in many plants representing diverse taxa among the land plant phylogeny. Apogamy is an asexual reproductive strategy that produces a sporophyte directly from a gametophyte cell without the usual ploidy change associated with syngamy. The genes that drive the process of apogamy are almost completely unknown. Knowledge of these genes and their functions will provide insight into the evolution of asexual reproduction, and has the potential to provide insight into the evolution aspect of the development of the sporophyte body and the alternation of generations in land plants, two very important questions in plant science. My Ph. D. research has focused on elucidating the genes that are involved in the process of apogamy, and understanding the functions of the homologues of these genes in angiosperms. First, I induced apogamy experimentally from the semi-aquatic fern Ceratopteris richardii by culturing them on 2.5% glucose. I discovered that C. richardii gametophytes begin to become committed to the production of apogamous sporophytes after 10 days of culture on glucose. I then created a normalized, subtracted cDNA library that represents genes that increase in transcription in gametophytes during this commitment time. By comparing the Gene Ontology terms mapped to this cDNA library with that of the gametophyte transcriptome of the homosporous fern Pteridium aquilinum, I discovered that the C. richardii apogamy library is enriched in genes that are involved in stress response and metabolism. The C. richardii apogamy library also contains many sequences whose homologues in Arabidopsis are specifically expressed or upregulated in flower organs or seed structures, both of which are absent in ferns. One of these genes, UNC93-like, is also expressed in the eggs of C. richardii gametophytes, as evidenced by iv whole mount in situ hybridization. Functional egg cells are implicated as necessary for C. richardii gametophytes to be induced to form apogamy. In Arabidopsis, a homozygous T-DNA line for the AtUNC93-like gene, which produces a partial AtUNC93-like transcript and likely retains partial functionality, has vegetative and reproductive defects. Embryo abortion and male and female gametophyte lethality contribute to a small seed set in these plants. However, reciprocal crosses indicate that the mutant allele does not affect gametophyte function. Instead, it appears to be a failure of the maternal plant to sustain the life of a percentage of the gametophytes that it produces. Thus, the AtUNC93-like gene has functions that are necessary for normal sporophyte vegetative growth and reproductive success, but is dispensable for the function of gametophytes. These studies have provided insight into the mechanisms that induce apogamy in the fern C. richardii. The apogamy cDNA library will provide a valuable resource for future investigations into the genes that are involved in apogamy. v TABLE OF CONTENTS LIST OF TABLES ...................................................................................................... viii LIST OF FIGURES ........................................................................................................ix CHAPTER I. INTRODUCTION ........................................................................................ .1 The Alternation of Generations Through Sexual and Asexual Means ............. 2 Sexual and Asexual Alternation of Generations in Ferns ................................ 7 Sexual and Asexual Alternation of Generations in Angiosperms .................. 10 Experimental Strategy .................................................................................. 16 II. THE INDUCTION OF APOGAMY IN CERATOPTERIS RICHARDII ....... 18 Introduction ................................................................................................. 18 Materials and Methods ................................................................................. 21 Plants and Growth Conditions ................................................................ 21 Apogamy Induction Experiments ........................................................... 23 Histology................................................................................................ 23 Results ......................................................................................................... 25 C. richardii Gametophytes Can Be Induced to Form Apogamous Sporophytes ............................................................................................ 25 C. richardii Gametophytes Become Committed to Apogamy After 12 Days of Induction ................................................................................... 35 Discussion.................................................................................................... 39 III. GENES UPREGULATED DURING APOGAMY INDUCTION IN C. RICHARDII ................................................................................................. 43 Introduction ................................................................................................. 43 Materials and Methods ................................................................................. 45 Plants and Growth Conditions ............................................................... 45 RNA extraction ..................................................................................... 45 Suppression Subtractive Hybridization .................................................. 45 Sequence Identification and Analysis .................................................... 46 Cloning, Sequencing, and RT-qPCR ...................................................... 47 Whole Mount in situ Hybridization ....................................................... 50 Results ......................................................................................................... 52 Strategy to Identify Genes Enriched During Apogamy Commitment ..... 52 Validation of the Apogamy Library ....................................................... 53 Composition of the Apogamy Library ................................................... 57 GO-slim Mapping and Comparative Analysis with the P. aquilinum Gametophyte Transcriptome .................................................................. 63 in silico Expression Patterns of Apogamy Library Closest Arabidopsis Homologues ....................................................................... 69 Whole Mount in situ Analyses in C. richardii gametophytes ................. 72 Discussion.................................................................................................... 78 IV. THE UNC93-LIKE GENE IN ARABIDOPSIS AND C. RICHARDII........... 84 vi Introduction ................................................................................................. 84 Materials and Methods ................................................................................. 88 Plant Materials and Growth Conditions ................................................. 88 Bioinformatics ....................................................................................... 89 Root Growth Assays, Fresh Tissue Weights and K+ in Apogamy .......... 90 Arabidopsis Seed Area and Seed Weight ............................................... 91 RT-PCR, 3’ RACE, PCR, and Degenerate PCR..................................... 91 Alexander Staining, Tissue Clearing, Optics and Photography............... 93 Results ......................................................................................................... 94 CrUNC93-like and AtUNC93-like Proteins are Conserved .................... 94 Vegetative Phenotypes of the Arabidopsis unc93-like Mutant ................ 99 Comparison of unc93-like Phenotypes with K+ Channel Mutants ........ 103 Reproductive Phenotypes of Arabidopsis unc93-like Mutant ............... 104 The Role of K+ in the Induction of Apogamy from C. richardii Gametophytes...................................................................................... 111 Discussion.................................................................................................. 112 V. SUMMARY AND FUTURE DIRECTIONS.............................................. 117 REFERENCES ............................................................................................................. 124 vii LIST OF TABLES Table 1. Primer sequences for semi-quantitative and RT-qPCR analyses ............................ 48 2. The 170 apogamy library clones identified with confidence .................................. 58 3. Primers used for RT-PCR, 3’ RACE, PCR and degenerate PCR ........................... 93 viii LIST OF FIGURES Figure 1. The dominant and reduced phases of the sexual life cycles of 3 representative land plants .............................................................................................................. 3 2. A phylogenetic tree depicting the relationships among extant land plants ................ 4 3. Ovule development in Arabidopsis ....................................................................... 11 4. The normal gametophyte and sporophyte forms of C. richardii ............................. 26 5. Various apogamous sporophyte forms of C. richardii ........................................... 29 6. The gametophyte-sporophyte junction in zygote-derived and apogamous plants ... 30 7. Apogamous sporophytes have typical sporophyte features .................................... 31 8. Confirmation of apogamy with ploidy comparisons .............................................. 33 9. Quantitative determinations of ploidy levels by Hoechst dye intensity .................. 34 10. Apogamy induction from C. richardii her mutant gametophytes ........................... 36 11. Apogamy induction from C. richardii fem mutant gametophytes........................... 37 12. Relative RT-PCR analyses of select sequences from the apogamy library ............ 54 13. RT-qPCR analyses of select sequences from the apogamy library ......................... 55 14. Top hit species and classifications ......................................................................... 64 15. Distribution of plant GO-slim categories mapped to the 170 unigenes in the apogamy cDNA library .......................................................................................... 66 16. Apogamy library enriched GO-slim terms ............................................................. 68 17. in silico expression patterns of Arabidopsis homologues of selected unigenes ....... 70 18. Whole mount in situs of nucleolar essential protein and RNA recognition motif clones ........................................................................................................... 73 19. The BTB and FTSH protease whole mount in situs show preferential expression in early embryo and gametophyte structures ........................................ 74 20. UNC93-like whole mount in situs show staining in egg cells of 12-day gametophytes ........................................................................................................ 75 ix 21. UNC93-like protein alignments and Arabidopsis UNC93-like gene structure ........ 95 22. Phenotypes of a homozygous T-DNA insertion mutant of AtUNC93-like .............. 97 23. Number of lateral roots from plants grown on medium containing varying concentrations of K+ ................................................................................ 98 24. Fresh root and shoot weights of unc93-like and WT plants................................... 100 25. Phenotypes of kco3 and tpk1 potassium channel mutants on 0 mM K+ differs from unc93-like and WT plants ........................................................................... 102 26. Seed sizes of unc93-like and WT plants ............................................................... 105 27. unc93-like plants have fewer seeds in their mature siliques than WT plants ......... 106 28. Examination of pollen viability in the unc93-like mutant ..................................... 107 29. Significantly fewer seeds produced from crosses performed with females heterozygous for the unc93-like mutant allele...................................................... 109 30. The effect of K+ concentration on apogamy induction in C. richardii ................. 110 x 1 CHAPTER I INTRODUCTION The alternation of generations is the defining feature of the life cycle of land plants. This life cycle delegates the major events of sexual reproduction, meiosis and fertilization, to two distinct, multicellular bodies: the diploid sporophyte and the haploid gametophyte. Meiosis in the sporophytic generation produces spores, which are the first cells of the gametophyte generation. The gametophyte generation produces gametes mitotically. Syngamy completes the life cycle by producing the zygote, the first cell of the sporophytic generation. This life cycle differs from that of animals, in which the products of meiosis do not execute a developmental program to form a haploid, multicellular body, but rather differentiate directly into unicellular gametes. There are also asexual reproductive strategies that have evolved in land plants that bypass fertilization and meiosis. A type of asexual reproduction that is important in ferns and angiosperms is the formation of an asexual sporophyte, from the cells of a gametophyte or surrounding a gametophyte, to continue the life cycle of the plant without syngamy. In this thesis I describe my investigations into the genes that trigger asexual sporophyte development in the fern Ceratopteris richardii and investigate the expression patterns and function of a subset of these genes in the angiosperm Arabidopsis thaliana. The genes that trigger asexual reproduction in land plants are unknown. Knowledge of the genes involved in this process has the potential to provide insight into the evolution aspect of asexuality in land plants, the alternation of generations, and the land plant sporophyte body. Knowledge of the genes involved in apogamy is also important in 2 agriculture, and could lead to the development of self-propagating superior hybrid varieties of crop plants. The Alternation of Generations Through Sexual and Asexual Means The extant descendants of the first plants to successfully colonize land comprise a monophyletic group called the Embryophyta (Nikas and Kutchera, 2009). “Embryophyte” refers to the fact that the developing sporophyte embryos are kept within the walls of an archegonium, where they are protected and nourished until they can fend for themselves (Walbot and Evans, 2003). Land plants have a heteromorphic alternation of generations, in which the gametophyte and sporophyte are distinctly different in size, morphology, and physiology. There are varying degrees of relative dominance of the phases (Fig. 1) in the different clades of the land plant tree (Fig. 2). The ancestral state of land plants, whether having a multicellular gametophyte, sporophyte, or both, is currently in question (Bennici, 2008; Becker and Marin, 2009). It is known that the Chlorophyta and streptophyte algae (green algae) are structurally and biochemically very closely related to land plants (Lewis and McCourt, 2004), thus the life cycle of the green algae sister taxon will be the most informative about the ancestral state of land plants (Haig, 2008; Niklas and Kutschera, 2009). Modern green algae include taxa in which gametophyte, sporophyte, or both generations are multicellular (Lewis and McCourt 2004; Becker and Marin, 2009), and the specific taxon that is the most closely related to embryophytes is unclear (Becker and Marin, 2009). However, currently, all sister taxon candidates are charophycean green algae in which the dominant gametophyte phase is 3 Figure 1 The dominant and reduced phases of the sexual life cycles of 3 representative land plants. The sporophyte phase of a) bryophytes, b) ferns, and c) angiosperms. The gametophyte phase of d) bryophytes, e) ferns, and f) angiosperms. The sizes of the gametophytes and sporophytes are not to scale. 4 Figure 2 A phylogenetic tree depicting the relationships among extant land plants. Model plants representing different clades are in parentheses. All model plants listed have sequenced genomes except the fern C. richardii. The lengths of branches do not indicate distance. Redrawn from Palmer et al., 2004. 5 multicellular and the sporophyte is unicellular (Karol et al., 2001; Turmel et al., 2006; Qiu et al., 2006; Qiu, 2008). Thus, current data suggest that extant land plants evolved from an ancestor that had a haploid multicellular generation, with meiosis proceeding directly from the zygote (Niklas and Kutschera 2010). This scenario dictates that the modern day land plant sporophyte generation is a derived condition that is the result of delayed meiosis and the introduction of mitotic cellular divisions after the formation of the zygote (Niklas and Kutschera 2010). In line with this, we see a transition from the basal lineage of extant land plants, the bryophytes, that have a dominant gametophyte generation and a tiny, dependant, ephemeral sporophyte, to the more derived state of sporophyte dominance and the progressive reduction of the gametophytic phase of the life cycle in higher land plants (Figure 1 and 2). Thus, all land plants engage in the alternation of generations, splitting the mechanisms of meiosis and fertilization between their two separate, multicellular bodies, but different clades differ in the dominance of the two phases. This difference may be regarded fundamentally as a difference in the timing of the process of meiosis (Niklas and Kutschera 2010). Despite the evolutionary benefits of sexual reproduction through the production of diverse progeny through meiotic recombination and syngamy, asexual reproductive strategies have evolved among diverse taxa of both animals and plants. This suggests that there are evolutionary benefits of asexuality, at least among some taxa in certain environments. Some basic assumptions about the benefits of asexuality are based on the costs of sexuality (Horandl 2009). The first are the costs of meiosis, in which favorable combinations of gene alleles may be recombined. Second, among non-hermaphroditic organisms the males do not reproduce. Finally, the time and energy that is associated with 6 meiosis and fertilization (Maynard Smith 1978; Bell 1982; Lewis 1987). In certain circumstances, then, asexuality may be favored because it would maintain combinations of gene alleles, circumvent the requirement for sperm, and allow escape from environmental requirements necessary for successful fertilization, such as individual density or, in the case of some plants, water films for the transport of flagellated sperm. Therefore, asexual strategies may at least provide short-term advantages, allowing less costly and quick colonization of certain environments. Asexual reproductive strategies of land plants involve the circumvention of fertilization combined with an abnormal meiosis to restore diploidy and continue the asexual life cycle. In ferns and mosses, the process of forming an asexual sporophyte from the cell or cells of a gametophyte is called apogamy. In seed plants, the various processes that circumvent fertilization and meiosis to form asexual sporophytes are collectively called apomixis (Asker and Jerling, 1992). Regardless of the asexual reproductive strategy employed, the switch from the development of one generation to the other remains, albeit in the absence of a change in ploidy. Thus, the genetic program for determining the development of sporophyte or gametophyte can be uncoupled from that for meiosis and fertilization. Defining the genes involved in these asexual processes has the potential to provide information regarding the evolution of asexual processes in land plants. Additionally, comparing and contrasting the genes and the mechanisms that drive asexual pathways in different plant taxa may provide insight into the evolution of the land plant life cycle. 7 Sexual and Asexual Alternation of Generations in Ferns The gametophyte and sporophyte generations of ferns are both free living and photosynthetic (Figure 2). Although the gametophytes of different fern taxa take various forms, there is a general similarity to their structures (Raghavan 1989). Male gametophytes have determinate growth, forming only a three-dimensional cluster of antheridia, the structures in which sperm develop. Hermaphrodites form a twodimensional prothallus, on which both archegonia, which contain egg cells, and antheridia develop. When gametophytes come in contact with water, mature antheridia swell and burst, releasing the flagellate sperm into an environment in which they can swim to the open channels of mature archegonia and fertilize the egg. After fertilization, the developing sporophyte remains attached to the gametophyte prothallus, drawing nutrients from it, until the root system reaches soil. Over a century ago it was noted that there were fern gametophytes in nature that did not form archegonia, yet produced sporophytes without fertilization (Farlow 1874, DeBary 1878). This process has now been termed apogamy. Apogamy, bypassing fertilization, involves the development of a sporophyte directly from the vegetative cells of a gametophyte. This obviously forms a sporophyte body that has the same chromosome number as the gametophyte, and if the life cycle is to continue, a mechanism is necessary to generate diploid spores of the next generation. There are 2 types of compensatory mechanisms to allow for the continuation of the life cycle for the approximately 10% of obligatorily apogamous ferns in nature (Sheffield and Bell, 1987). One involves a mitotic division that does not complete cytokinesis, and these cells proceed through meiosis to generate spores (Walker, 1979). In this scheme, the only 8 diploid cell in the life cycle is that which immediately undergoes meiosis. The other mechanism allowing for apogamous life cycle continuation involves a failed reductional division (meiosis I), and a normal meiosis II when generating spores (Klekowski, 1979). In this scheme, ploidy level (whether haploid or diploid) does not change in any cell of either generation. Both schemes produce spores containing the same number of chromosomes as the apogamous sporophyte. Gametophytes that develop from these spores produce sporophytes apogamously to complete the life cycle. Because this life cycle does not require a water film for sperm to swim for fertilization, it is thought to have evolved as an adaptation to drier environments (White, 1979). For some Chelianthoid ferns an apogamous life cycle allows life in xeric environments (Mickel, 1979), suggesting that the dry environment renders sperm motility moot, and over time the habit of developing sperm becomes degraded. Ferns that are apogamous in nature form nonfunctional gametes and are sterile. Some bear archegonia that are either abortive, or fail to open a channel sufficient for sperm entry (Laird and Sheffield, 1986). Not long after the discovery of ferns that reproduce apogamously in nature, the ability to induce apogamy from the gametophytes of fern species that normally reproduce sexually was recognized. This process is called “facultative” or “induced” apogamy and it was first accomplished by the prolonged culture of homosporous fern gametophytes in dry conditions that prevented the release of male gametes, or their mobility, so that fertilization is prevented (Lang, 1897). More efficient methods for inducing apogamy from fern gametophytes have been developed using axenic growth conditions in laboratory and involve the exogenous supply of sugars or other factors (Dyer, 1979; Raghavan, 1989; Bell, 1992). 9 In general, the factors that have been recognized as broadly apogamy-inducing in axenic culture systems are light, hormones, and sugars (Raghavan, 1989). The addition of high levels of sugars to the culture medium has been established as sufficient to induce apogamy from many fern species, and different sugars have different optimal concentrations for the induction of apogamy (Whittier and Steeves 1960, 1962; Whittier, 1964a; Mehra and Sulklyan, 1969; Kato, 1970; Padhya and Mehta, 1981; Kawakami et al., 2003). There is also evidence that light intensity plays a role in the induction of apogamy, because dark-grown gametophytes generally do not produce apogamous sporophytes, even when grown on sugars (Kato, 1970; Whittier, 1976; Whittier, 2004). Additionally, different wavelengths of light seem more effective at inducing apogamy from some ferns, suggesting a role for light signaling, rather than just photosynthetic flux, in apogamy (Whittier, 1964b; Whittier and Pratt, 1971). Plant hormones modulate plant growth patterns, and may induce the production of roots or shoots from gametophyte tissue that has formed a callus, or may actually increase the production of apogamous sporophytes in culture, but these effects only occur when combined with growth on a suitable carbohydrate. (Elmore and Whittier, 1973; Elmore and Whittier, 1975a, 1975b; Whittier, 1966; Kato, 1970). The phenomenon of apogamy has been thoroughly studied with respect to physiology and development, but the genes that are involved in the process of apogamy are largely unidentified. Recently the Physcomitrella patens CURLY LEAF homologue (PpCLF) was identified as being involved in apogamy in this moss. PpCLF is a member of the polycomb group complex (PCR2) and loss of this gene in P. patens causes apogamous sporophytes to form from the gametophytes (Okano et al., 2009). Other 10 members of PCR2 in angiosperms cause parthenogenic development of egg cells or autonomous endosperm development (Chaudhury et al., 1997; Guitton and Berger, 2005). Sexual and Asexual Alternation of Generations in Angiosperms Seed development in angiosperms requires the execution of several sequential, defined steps. The shoot apical meristem of the plant must first transition from the vegetative phase to the reproductive phase of organ production. The floral meristem produces the floral organs in which the male and female gametophytes (the anther and carpels, respectively) are eventually formed after meiosis. The mature male gametophytes are the three-celled pollen, containing two sperm cells and a generative cell, the latter eventually growing into the pollen tube, upon deposition on a stigma, to deliver the two sperm to the female gametophyte within the ovule. The ovules of angiosperm flowers are the structures that give rise to the seed after fertilization (Fig 3). The ovule also is the structure that gives rise to seeds derived from asexual reproduction. The mature ovule contains the female gametophyte, also called the embryo sac, which is typically a sevencelled structure including the egg cell (Fig 3C, shaded gray) and the binucleate central cell (Fig 3C). The development of the ovule begins with the ovule primordium, which has three regions, the nucellus, chalaza, and funinculus (Fig A). The meiotic initial, the megaspore mother cell, emerges from the nucellus, which divides to form the megaspore, which is the meiotic product closest to the chalazal region, and 3 spores that eventually degenerate (Fig 3B). The megaspore undergoes three rounds of mitosis to form an embryo sac with 7 cells and 8 nuclei (Fig 3C). From the chalazal region, the outer and 11 Figure 3 Ovule development in Arabidopsis. (A) The ovule primordium emerges from the placenta and differentiates into the funinculus (f), chalazal (c), and nucellar (n) zones. (B) The primordia of outer (oi) and inner integuments (ii) arise from the chalazal region and grow around the nucellar tissue, eventually becoming the seed coat after fertilization. A megaspore mother cell develops from the nucellar tissue (n) and undergoes meiosis. The funinculus (f) is the vascularized stalk that connects the ovule to the placenta. (C) A single megaspore mother cell (shaded gray), the meiotic product closest to the chalazal region, divides three times to form the 7celled female gametophyte, the embryo sac (es), which contains the egg cell (shaded gray). Redrawn from Park et al., 2005. 12 inner integument initials form (Fig 3B), which, during ovule development, grow around the nucellus (Fig 3C), protecting and nourishing it and the developing embryo sac. After fertilization the integuments develop into the seed coat. The funinculus is the vascularized stalk that connects the ovule, and by extension the female gametophyte and developing zygotic embryo, to the placenta (Figure 3C). Sexual reproduction proceeds when a pollen tube delivers two sperm to the ovule. One sperm fertilizes the egg and the other the binucleate central cell, forming the diploid zygote and the triploid endosperm, respectively. A functional, triploid endosperm is necessary for the normal development of a zygotic embryo and apomictic embryos in many species. Although the majority of angiosperms reproduce sexually to form seeds, approximately 400 species from 40 families of angiosperms have evolved the ability to reproduce via apomixis (Nogler, 1984; Carman, 1997). In fact, many apomictic plant taxa retain the ability to reproduce sexually, often producing sexual and apomictic embryos on the same plant. This makes it difficult to identify all plant taxa that are apomictic in nature, and the current count of apomictic species is likely a conservative one (Nogler, 1984; Bicknell and Kolunow 2004; Plitman, 2002). The process of apomixis involves the bypassing of meiosis during gamete formation, along with the absence of fertilization to form the embryo, instead producing embryos, without paternal contribution, from somatic cells (Tucker and Koltunow, 2009). The different types of apomixis differ in the cell that gives rise to the asexual embryo. Three types of apomixis have been described: adventitious embryony, diplospory, and apospory. Adventitious embryony produces multiple asexual embryos spontaneously 13 from the diploid, sporophytic cells of the nucellus or integument. This form of apomixis occurs alongside normal sexual reproduction (Asker and Jerling, 1992; Tucker and Koltunow, 2009). An apomictic embryo may invade the embryo sac to utilize the endosperm at the expense of the sexual embryo, or sexual and apomictic embryos may coexist and share the same endosperm (Asker and Jerling, 1992). Either way, the survival of the apomictic embryos appears to depend upon the successful development of a sexually derived endosperm, possibly because of the nutrients or growth signals it confers (Asker and Jerling, 1992; Koltunow et al., 1995; Tucker and Koltunow, 209). In diplospory, the megaspore mother cell, which is normally destined to undergo meiosis and form the first cell of the female gametophyte, fails to initiate or complete meiosis. Instead, mitosis forms a diploid, 8-nucleate embryo sac. The diploid, egg-like cell then initiates embryogenesis without fertilization. These apomictic embryos also depend upon an endosperm for their survival, whether the endosperm develops autonomously or arises from fertilization of the central cell (Tucker and Koltunow, 2009). Some species may also be facultative diplosporous apomicts, capable of sexual reproduction when necessity or opportunity arises (Grimanelli et al., 1998; Schranz et al., 2005). In apospory, the megaspore mother cell typically initiates meiosis, which it may or may not complete (Tucker and Koltunow, 2009). Meanwhile, in the vicinity of the megaspore mother cell, one or multiple somatic cells differentiate into aposporous initials, which then bypass meiosis, instead dividing mitotically to form a diploid embryo sac (Tucker and Koltunow, 2009). As in diplospory, the diploid egg-like cell initiates embryogenesis without fertilization, and the endosperm can be formed autonomously or 14 via fertilization. Additionally, in some aposporous apomicts for which sexual embryo sac development proceeds normally, both sexual and clonal embryos may be found on a single plant (Araujo et al., 2000; Araujo et al., 2005). All forms of apomixis have three features in common: the generation of a cell that has the capacity to form an embryo without first undergoing or completing meiosis, the ability to produce an embryo without fertilization, and the formation of an endosperm, either through fertilization or independent of fertilization, to assist in the development of the apomictic embryo (Tucker and Koltunow, 2009). The genes that control apomixis are unknown, but seem to be restricted to one or a few dominant loci in the majority of apomictic plants so far examined (Ozias-Akins and van Dijk, 2007). There are several genes that, when ectopically expressed, cause somatic embryogenesis (SE) from vegetative tissues, similar to apomixis phenotypes, but it is unknown whether these same genes are truly involved in apomixis. Genes with gain-offunction SE phenotypes when ectopically expressed include WUSCHEL (WUS), BABYBOOM (BBM) AND SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1). WUS encodes a homeodomain protein normally involved in regulating stem cell fate in the meristem, and recently has been found to be required for megasporogenesis (Laux et al., 1996; Mayer et al., 1998; Lieber et al., 2011). BBM, a transcription factor, is preferentially expressed in developing embryos and seeds (Boutillier et al, 2002). SERK1 is normally expressed throughout nucellar tissue during meiosis, and throughout the development of the embryo sac and zygotic embryo (Schmidt et al., 1997; Hecht et al., 2001), and confers competence for embryonic development. The LEAFY COTYLEDON 1 (LEC1) and LEAFY COTYLEDON2 (LEC2) 15 are master regulators of embryo development that are normally expressed during the morphogenesis and maturation phases of embryogenesis in seeds (Lotan et al., 1998; Stone et al., 2001; Braybrook and Harada, 2008). Recently, a role for RNA silencing has been implicated in apomixis with the recovery of the ARGONAUTE104 gene in a screen for mutations that form viable, unreduced female gametophytes, a phenotype that resembles diplospory, in maize (Singh et al., 2011). Recessive apomicitc genes, like FIS2, a member of the PCR2 complex that causes the initiation of embryo and endosperm development without fertilization similar to apomictic plants (Chaudhury et al., 1997) are also known. Mutations in a member of the PCR2 complex causes apogamy in the moss P. patens, suggesting that the processes of apogamy and somatic embryogenesis, if not apomixis, share genetic components, even though these plants are evolutionarily quite distant (Fig. 2). The obligate production of a haploid sporophyte from gametophyte cells is called androgenesis, rather than apogamy, in angiosperms (Segui-Simarro, 2010). Androgenesis occurs very rarely in angiosperms, and involves a process whereby egg fertilization occurs, but the female nucleus is somehow inactivated and a haploid sporophyte develops with only paternal contribution (Burke, 1962; Chase, 1969; Goodsell, 1961; SeguiSimaro, 2010). However, the production of apogamous sporophytes in angiosperms can be accomplished through inductive culture of pollen or embryo sacs. These inductive processes are called pollen embryogenesis or gynogenesis, depending on whether it is the male or female gametophyte being induced (Segui-Simarro, 2010). The genes involved in this inductive process are unknown, and inductive conditions vary, but, universally, 16 include stress treatment (Shariatpanahi et al., 2006). It seems that the pollen must first go through a stress response, which in turn triggers the embryonic response. Experimental Strategy My experimental strategy for elucidating the genes that are involved in asexual reproduction in land plants took advantage of the separate and independent generations of ferns, along with their unique ability to be easily induced to apogamy using laboratory culture conditions. I used Ceratopteris richardii, a fern with a normally diploid sporophyte that is a model fern system for the study of plant developmental processes (Hickock et al. 1995; Banks 1999; Chatterjee and Roux 2000). In chapter 2 of this thesis, I describe the induction of apogamy in C. richardii with culture on high levels of exogenous sugars, and describe the dramatic phenotypic changes of the gametophytes grown on sugar, as well as the apogamous outgrowths eventually produced. I also define a time period during which C. richardii gametophytes become committed to the production of apogamous sporophytes. In chapter 3, I describe the creation of a subtracted, normalized cDNA library, called the “apogamy library” that represents genes that increase in expression in C. richardii gametophytes grown on sugar, during the time that they are becoming committed to apogamy. This chapter also includes the list of genes from the apogamy library that were confidently identified using BLAST searches, including a broad overview of the function of these genes through Gene Ontology (GO) assignments. The GO summary of the apogamy library is compared to that of the gametophyte transcriptome of a homosporous fern to define the gene expression landscape of the unique genes during apogamy commitment. Also, the in silico anatomical expression 17 patterns of the Arabidopsis homologues of the apogamy library sequences are examined, and the expression patterns of a subset of these genes is also examined in C. richardii gametophytes undergoing apogamy induction. In chapter 4, I describe the characterization of one of these genes, UNC93-like, in Arabidopsis. This gene, when mutated, confers vegetative, root, and reproductive abnormalities. The defects include small, dark green leaves and slow development, an excess of lateral roots, and a reduced seed set due to embryo abortion and a failure of the parent plants to maintain gametophyte viability. In chapter 5, I summarize the findings in the thesis and discuss further lines of investigation that may be performed to more fully understand the functions of the UNC93-like gene, as well as the other genes in the apogamy library. 18 CHAPTER II THE INDUCTION OF APOGAMY IN CERATOPTERIS RICHARDII Introduction Both the sporophyte and gametophyte generations of land plants begin from a single cell, the zygote and the spore, respectively. Yet, the two generations develop into multicellular bodies that are remarkably different in morphology and habit. The mechanisms that trigger these two developmental pathways are unknown. While meiosis and fertilization normally precede the production of the zygote and spores, they are not necessary for the induction of sporophytic and gametophytic developmental pathways. This is evident from the development of sporophytes and gametophytes even in the life cycles of plants that engage in asexual life cycles that circumvent fertilization, meiosis, or both events altogether (Asker and Jerling, 1992; Tucker and Kolunow, 2009). Apogamy is a phenomenon in which a sporophyte is produced directly from the vegetative cells of a gametophyte without fertilization, thereby circumventing chromosome doubling (For a review of nonsexual cycles in plants, see Bell, 1992). This phenomenon has been well documented in ferns, and its existence provides evidence that the signal that determines which developmental program will commence, sporophytic or gametophytic, is not simply an assessment of ploidy by the single-celled zygote or spore. Apogamy is obligate in some homosporous fern species whose gametophytes produce nonfunctional gametes. To compensate for the lack of chromosome doubling, these apogamous sporophytes form restitution nuclei during sporogenesis, either through an 19 incomplete mitotic division or a failed first meiotic division, thereby allowing the production of spores with one set of chromosomes (Raghavan, 1989). In a process termed ‘‘induced’’ or ‘‘facultative’’ apogamy, several homosporous fern species that normally produce sporophytes through syngamy have been induced to form apogamous sporophytes through the manipulation of culture conditions. In general, sporophytes formed through induced apogamy have abnormal morphology compared with zygotederived sporophytes or sporophytes formed through obligate apogamy (Lang, 1898; Steil, 1939, 1951; Whittier, 1962); however, they do display one or more of the hallmarks of sporophyte development. These hallmarks, none of which are normally present in fern gametophytes, include leaves, stems, roots, vascular tissue, and stomata. The experimental conditions used to induce apogamy in ferns have already provided valuable information about the signals that induce the sporophytic developmental pathway. Two basic methods have been employed to induce apogamy in ferns. The first simply involves the extended culture of fern gametophytes while simultaneously preventing fertilization by carefully controlling water supply (Lang, 1898; Stei,l 1939, 1951; Duncan, 1941). Apogamy induction in this manner has generally been in combination with high light levels, suggesting that increased metabolic flux plays a role in the process of apogamy. Culturing fern gametophytes on medium containing high levels of exogenous sugars is sufficient to induce the apogamous production of sporophytes in many fern species (Whittier and Steeves, 1960, 1962). Exogenous sugars have also been found to induce the differentiation of sporophyte tissue from callus derived from the fern Pteris cretica, whereas sugar deprivation induces gametophyte differentiation from callus (Bristow, 1961). These studies suggest that sugar metabolism 20 and/or sugar signaling play a role in determining the gametophyte and sporophyte developmental pathways in ferns. The semi aquatic fern Ceratopteris richardii was chosen two decades ago in an effort to develop this fern into a model system for studying developmental processes (Hickock et al., 1995; Banks, 1999; Chatterjee and Roux, 2000). Two laboratory strains of obligately apogamous Ceratopteris have been described, both of which produce nonfunctional spermatozoids. One strain resulted from the aneuploid gametophyte of a triploid hybrid and the other from a diploid hybrid between C. richardii and C. pteridoides (Hickock, 1977, 1979). Here, I report that apogamy can be induced in the diploid, normally syngamous C. richardii by sugar. Apogamy was confirmed by measures of ploidy level and documented by the presence of the hallmarks of sporophytic tissues. I also documented the variation in morphology of apogamous sporophytes in this fern species. Most importantly, I have identified the minimum culture time that C. richardii gametophytes require the presence of exogenous sugar for sugar-induced apogamous sporophyte production. A differentiating cell in a gametophyte can follow one of several developmental pathways: vegetative gametophyte, gamete, or vegetative sporophyte (apogamy), depending on the developmental cues to which it responds. Focusing on initiation of the sporophyte from apogamy rather than from a zygote separates this direct switching between gametophyte and sporophyte from embryogenesis. It has been proposed that the conditions for sporophytic growth of the zygote have already been set in the mature egg (Bell, 1970). It has also been demonstrated in the homosporous fern Pteridium aquilinum that mature eggs in which RNA synthesis has been inhibited with thiouracil will, when egg and archegonium are 21 isolated from the prothallus, develop into structures whose morphologies are intermediate between gametophyte and sporophyte (Jayasekera et al., 1972; Jatasekera and Bell, 1972). In our view, once the zygote is formed, the identity of the sporophyte generation in normal alternation of generations has been determined. Therefore, the ability to generate sporophytes from fern gametophytes through induced apogamy, albeit abnormal in morphology, provides a tractable experimental system for understanding the gene network that controls the switching from one generation to another. Materials and Methods Plants and Growth Conditions The C. richardii plants used in these studies were of the mutant genotype hermaphrodite (her) (Carolina Biological Supply, Burlington, NC) and feminization1 (fem1) (a gift from J. Banks, Purdue). C. richardii her mutants are self-fertile, but are restricted to hermaphroditic development, because of their insensitivity to the maleinducing pheromone antheridiogen (Banks, 1994; Banks, 1997). Like other homosporous ferns, C. richardii gametophytes develop into hermaphrodites in the absence of the maleinducing pheromone antheridiogen (Naf, 1979; Naf et al., 1975; Banks et al., 1993). The hormone itself is secreted by hermaphroditic gametophytes when they are no longer competent to respond to it (Banks et al., 1993). Thus, the first spores to germinate in a wild-type population become hermaphrodites, and these same hermaphrodites induce the development of male gametophytes from the surrounding germinating spores by the secretion of antheridiogen into their surroundings. Fertilization results in the loss of the gametophyte notch meristem function and subsequent gametophyte senescence. The prevention of fertilization is therefore important both for the occurrence of apogamy and 22 for preventing sporophytes generated from fertilization. The absence of males in her gametophyte cultures reduces the likelihood of fertilization. The fem1 spores produce gametophytes that have archegonia but no antheridia, either in the presence or absence of the male-inducing pheroemone antheridiogen (Banks, 1994; Banks, 1997). The complete absence of sperm-producing structures from fem1 gametophytes ensures that all sporophytic structures that appear from these gametophytes are apogamous. Spore germination and gametophyte culture conditions were essentially as described by Hickock et al. (1995). Spores were sterilized for 5 min in 3% sodium hypochlorite with one drop of Tween 20, rinsed five times with sterile water, and imbibed in water in the dark for 3 days at room temperature to synchronize germination. Spores were inoculated at a density of 150–300 spores per 95 mm petri plate containing basal medium (1% agarose supplemented with 0.5x Murashige and Skoog salts at pH 6) and, where noted, rich medium (supplemented with 2.5% glucose). Medium supplemented with 1%–9% sucrose or 0.5%–4% trehalose was also employed in initial experiments to determine which sugar and at what concentration the best induction of apogamy is produced in this fern. Gametophyte culture plates were maintained in humidity domes at 28°C with constant illumination under Philips Agro-Lite fluorescent bulbs (Philips Lighting Company, Somerset, NJ) at 90–100 µM m2 s-1. Fifteen days after inoculation, gametophyte culture plates were inverted to reduce the incidence of fertilization by preventing water condensation on gametophytes when her mutant plants were used. Fertilization did not occur before this time under our culture conditions. All gametophytes were examined for the appearance of apogamous structures under sterile conditions every other day for 54 days using a Leica Wild MC3 dissecting microscope. 23 Apogamy Induction Experiments Sterilized, dark-treated her or fem spores were inoculated on petri plates either with rich medium or with basal medium. Every 2 days after inoculation, gametophytes growing on a rich medium plate were transferred in two cohorts, consisting of 120–169 gametophytes each, to fresh medium. The transfers were performed manually, with one cohort transferred to rich medium and one cohort transferred to basal medium. This was repeated every 2 days until day 16, the last day of transfer. In addition, every plate of gametophytes was moved to fresh medium every 14 d after the original transfer to ensure an adequate supply of nutrients at all times. In this manner, no gametophyte remained on medium for longer than 2 weeks except those that were on rich medium for 16 days before their first transfer. Histology For toluidine blue staining, four gametophytes, on which fertilization-derived sporophytes were growing, and six gametophytes, on which apogamous sporophytes were growing, were chosen randomly, fixed at 4°C overnight in FAA (10% formaldehyde, 50% ethanol, and 5% acetic acid), and then processed through an alcohol dehydration series and embedded in Paraplast Plus (Kendall, Mansfield, MA). Ninemicrometer sections were deparaffinized, hydrated, and stained for 10 min in 0.05% toluidine blue (pH 6). Sections were then dehydrated and cleared in xylenes. Coverslips were affixed with Permount (Fisher Chemical, Fair Lawn, NJ). For chromosome squashes, whole gametophytes or sporophyte shoot and root tips were fixed in 3:1 ethanol:acetic acid overnight. Samples were then rinsed in distilled water, placed on a slide in a drop of saturated acetocarmine stain beneath a coverslip, 24 heated briefly over an open flame, and squashed. Digital images of chromosome squashes were used to count chromosome numbers. Five chromosome counts of putatively apogamous outgrowths were performed, four from shoot tips and one from a root tip. One chromosome count each was made from squashes prepared from haploid gametophyte notch meristems and diploid sporophyte root tips to confirm chromosome numbers. Vascular tissue was imaged by bleaching unfixed samples in 95% ethanol for several hours to remove chlorophyll and mounting bleached samples onto slides in glycerol. For ploidy level determination using Hoechst dye, at least two putatively apogamous structures from each cohort of gametophytes were cleared in 95% ethanol overnight at 4°C. Tissue was then rinsed in water and immersed in 40 mg/mL Hoechst 33342 dye for 2.5 h, rinsed twice in distilled water, mounted on slides in glycerol, and imaged under DAPI filtered UV light. To ensure uniformity of fluorescence comparisons, Hoechst dye-stained samples were analyzed immediately after mounting, and compared images were captured at the same gain and exposure. Fluorescence intensity was quantified using ImageJ 1.37j software (http://rsb.info.nih.gov/ij/). The fluorescing nuclei of three Hoechst images, representing three different experiments, each containing a putatively apogamous tissue sample along with haploid and diploid controls, were measured to obtain relative fluorescence intensities. The images were converted to 8-bit gray scale and binarized using the intensity thresholding function. The selected fluorescing nuclei were regions of interest. Mean gray values were then obtained from the defined regions of interest and converted to relative intensities. Mean pixel intensities of 7 to 18 in-focus nuclei from each putatively apogamous, haploid standard, and diploid standard were compared to quantify ploidy of the corresponding tissues. To view 25 stomata, cleared tissue samples were incubated overnight in 40 mg/mL Hoechst 33342 dye, rinsed in distilled water, and mounted on slides in glycerol; images were captured through DAPI filter. Chromosome squash images were recorded with a Zeiss Axioskop 20 microscope with MC100 attachment camera system (Carl Zeiss, Thornwood, NY). Apogamous plants were photographed with a Zeiss stereomicroscope SR and MC100 attachment camera system. All other images were recorded using a Leica DMRBE upright microscope and DC300F digital camera system (Leica Microsystems, Bannockburn, IL). Results C. richardii Gametophytes Can Be Induced to Form Apogamous Sporophytes Under optimum conditions (described in ‘‘Material and Methods’’), C. richardii gametophytes reach sexual maturity after 12–15 d of culture. At this stage, hermaphroditic gametophytes have several antheridia and archegonia (Fig. 4a). After the gametophytes are flooded, spermatozoids are released from antheridia and are free to swim into mature archegonia to fertilize the eggs. Connected at the region called the foot to the senescing gametophyte, the embryonic sporophyte develops until the root and shoot are established (Fig. 4b). The first-formed sporophyte fronds are sterile and simple three lobed. Later-formed fronds are fertile and have a dissected morphology (Fig. 4c). Sporangia, the structures within which spores are produced through meiosis, are formed abaxially within the inrolled margins of fertile fronds. The C. richardii hermaphroditic prothallus cells normally divide in only two dimensions, producing a gametophyte that is one cell layer thick with a meristem 26 Figure 4 The normal gametophyte and sporophyte forms of C. richardii. a, An hermaphroditic gametophyte grown on basal medium for 20 days is sexually mature, with many antheridia (an) and several mature archegonia (ar) located underneath the notch meristem. b, After fertilization, a zygote-derived sporophyte (sp) emerges from the senescing gametophyte (ga) until the root and shoot systems are established. c, A 4-month-old dissected sporophyte with dissected, fertile fronds. Black bars represent 1 mm, white bar represents 10 cm. 27 in the notch between the two lobes of the prothallus. In the absence of fertilization, gametophyte notch meristem activity is maintained, and gametophyte growth will continue indefinitely if supplied with sufficient nutrients. When grown on a medium that lacks sugar (basal medium), the continued gametophyte growth takes the form of additional prothalli that originate from the notch meristem and are one cell layer thick (Fig. 5a). Extended gametophyte growth on a medium that contains sugars (rich medium) also results in additional prothallus growth, but those prothalli are thickened and are accompanied by a proliferation of ectopic rhizoids, seen as a transparent mass (Fig. 5b). We grew her mutant gametophytes on agar-solidified medium, supplemented with various concentrations of trehalose, sucrose, or glucose, to determine whether apogamy could be induced by sugar in C. richardii. Whereas gametophytes were not viable on concentrations of glucose at or above 4%, presumably because of osmotic effects, all other sugar concentrations from 1% to 3% successfully induced the formation of apogamous sporophytes (data not shown). We found that medium supplemented with 2.5% glucose was optimal for the induction of apogamy, with 10%–28% of gametophytes per plate (150–300 gametophytes per plate) displaying apogamous sporophytes after 2 months of culture. Apogamous sporophytes assumed various morphologies in C. richardii, none of them identical to normal, zygote-derived sporophytes. The most common forms were slightly lobed leaves (Fig. 5c) but with no accompanying roots as is the norm with zygote-derived sporophytes (cf. Fig. 4b with Fig. 5c), outgrowths displaying intermediate sporophyte-gametophyte morphology that were cylindrical (Fig. 5d) or flattened (Fig. 5e), and clusters of sporophyte leaves and leaf-like protrusions (Fig. 5f). Rarely (2 out of 28 249 total cases of sugar-induced apogamy) a solitary root appeared in the absence of any visible leaf or shoot development (data not shown). Ramenta are one-cell-thick scales that normally occur on the stem and petiole of zygote-derived fern sporophytes. Ramenta accompanied apogamous sporophyte structures, appearing at the base or along cylindrical outgrowths. Although they become brown and scale-like on mature C. richardii stems, they initially appear translucent and accompany every leaf of young, zygote-derived sporophytes (Fig. 5g). Ramenta also appeared in the absence of other sporophyte structures, appearing in clusters or as a single apogamous ramentum (Fig. 5h). The gametophyte-sporophyte junction differed between zygote-derived and apogamous plants. Normal gametophyte-sporophyte junctions of C. richardii are characterized by sporophyte haustorial cells that develop before the emergence of the first leaf and contain extensive wall ingrowths (Duckett and Ligrone, 2003). Toluidine blue– stained longitudinal sections of normal sporophyte-gametophyte junctions display these wall ingrowths in a deep blue color (Fig. 6a). These ingrowths are unique and serve as an easily identified marker for the junction of the gametophyte and the zygote-derived sporophyte. This boundary is not evident at the junction between apogamous sporophytes and the gametophyte from which the sporophytes are generated. These junctions contain no clear demarcations save a tangle of vascular tissue and occasional bits of ingrown cell wall with no discernible pattern (Fig. 6b). At the microscopic level, C. richardii apogamous sporophytes were observed to possess stomata and vascular tissue; both are sporophyte-specific features. Stomata, which were observed on 100% of zygote-derived sporophyte leaves (Fig. 7a), were also 29 Figure 5 Various apogamous sporophyte forms of C. richardii. Gametophyte proliferation in the absence of fertilization results in additional twodimensional prothalli growth after 45 d on basal medium (a), while 45 d on rich medium results in prothallus thickening (arrowhead) and rhizoid proliferation (arrow) (b). Apogamy took various forms, including slightly lobed leaves (c), cylindrical (d) or bladelike (e) growths that were of intermediate gametophyte and sporophyte morphology, and clusters of leaves and outgrowths (f). Ramenta (arrows) accompanied every emerging leaf of young, zygote- derived sporophytes (g) and were the most common apogamous structure seen, appearing alone (h) or in conjuction with almost all other apogamous structures. Bar = 1 mm. 30 Figure 6 The gametophyte-sporophyte junction in zygote-derived and apogamous plants. a, The junction of a gametophtye (ga) and zygote-derived sporophyte (sp) is characterized by sporophyte haustorium cells with extensive cell wall ingrowths (arrows). b, The typical apogamous junction contains disorganized vascular tissue and some evidence of extra cell wall deposits (arrow) but no defined boundary between the gametophyte (ga) and apogamous sporophyte (sp). Bar = 0.05 mm. 31 Figure 7 Apogamous sporophytes have typical sporophyte features. Hoechst DNA staining revealed the elongated nuclei of guard cells (arrow) flanking stomata on the leaves of zygote-derived sporophytes (a) and apogamous sporophytes (b). Vascular tissue, never present in normal gametophytes, could be seen in zygote-derived sporophyte leaves (c) and apogamous sporophyte leaves (d). Bar = 0.05 mm. 32 observed, albeit at a lower density, on 100% of apogamous sporophyte leaves and apogamous structures with intermediate morphology (Fig. 7b). Vascular tissue, which exists in all zygote-derived sporophyte leaves (Fig. 7c), was seen in apogamous structures with few exceptions (Fig. 7d). If the sporophyte organs generated from gametophytes that have been grown on rich medium are the product of apogamy, they should have the same (haploid) ploidy level as gametophytes. The apogamous nature of sporophyte structures was verified by Hoechst dye staining of DNA or chromosome counts. With Hoechst dye staining of DNA, the ploidy level of putatively apogamous tissue samples could be determined by comparing the intensity of nuclear fluorescence with the nuclear fluorescence of known haploid (Fig. 8a) and diploid (Fig. 8b) standards. Mitotic chromosome squashes were also performed where meristematic tissue was present. Comparisons of putative apogamous tissue (Fig. 8c) with known standards (Fig. 8d, 8e) indicated ploidy levels. Approximate chromosome counts were made of five squashes from five independent instances of apogamy as well as one haploid and one diploid standard to verify chromosome numbers. Approximate chromosome counts for putatively apogamous tissue varied from 35 to 39 chromosomes. The approximate haploid and diploid control counts were 36 and 64 (out of the 78 chromosomes, only 64 were visible for counting), respectively. The relative fluorescence intensities between the putatively apogamous, haploid, and diploid samples in figure 8 are shown in figure 9. Similar results were obtained in two other experiments. 33 Figure 8 Confirmation of apogamy with ploidy comparisons. Hoechst DNA dye intensity was used as a ploidy marker by comparing nuclear fluorescence of putatively apogamous tissue (ap in a) with known haploid (b) and diploid (a) standards. Chromosome squashes of mitotically dividing cells were also used to confirm ploidy of the putatively apogamous (c) by comparing to haploid (d) and diploid (e) standards. Ceratopteris richardii haploid n=39, diploid n=78. Bar = 0.05 mm (a, b); bar = 0.01 mm (c–e). 34 Figure 9 Quantitative determinations of ploidy levels by Hoechst dye intensity. The mean pixel intensity calculated from 7 to 18 in-focus nuclei from each of putatively apogamous, haploid, and diploid samples were compared, with the highest mean value set at 100. Bars represent the relative fluorescence intensities. Vertical lines represent standard deviation. 35 C. richardii Gametophytes Become Committed to Apogamy after 12 Days of Induction To determine when gametophytes become committed to apogamy by sugar induction, we cultured gametophytes on rich medium and transferred a subset of these gametophytes to basal medium every 2 days. As a positive control, cohorts of these gametophytes were transferred from rich medium to rich medium every 2 days. Thus, subsets of gametophytes, consisting of 120–169 gametophytes each, received the apogamy induction signal for increasing lengths of time. We then monitored these gametophyte populations for the production of apogamous sporophytes. In this way, we could determine the time point at which gametophytes are committed to sugar-induced apogamy development. Similar experiments were performed twice using the her mutant and twice with the fem mutant (Fig. 11), and very similar results were obtained in all three experiments, save the fact that much higher percentages of fem1 gametophytes undergo apogamy. This may be explained in part by the fact that the experiment with the fem mutant was conducted for 80 days, compared to 55 days for the her mutants, allowing more time for the gametophytes to form apogamous sporophytes. In one experiment, 54 days of continuous culture of the her mutant gametophytes on rich medium exhibited 14%-20% apogamous sporophytes (Fig. 10a, open bars; Fig. 10b). Surprisingly, gametophytes were found to produce a basal level of apogamous sporophytes in the absence of sugar induction, with approximately 2% of gametophytes displaying apogamous sporophytes after 54 d of growth on basal medium (Fig. 10a, lane 36 Figure 10 Apogamy induction from C. richardii her mutant gametophytes. Bar graph (a) shows the percentage of each cohort of gametophytes that displayed apogamy after 55 d. Filled bars indicate cohorts that were transferred to basal medium; open bars indicate cohorts transferred to rich medium. B indicates gametophytes cultured continuously on basal medium. R indicates gametophytes cultured continuously in rich medium. Line graphs illustrate the kinetics of the appearance of apogamous sporophytes on rich medium (b) and basal medium (c). Notice the different scales on the Y-axis in b and c. 37 Figure 11 Apogamy induction from C. richardii fem mutant gametophytes. Depicted is the percentage of gametophytes that displayed apogamous structures after 80 days of growth. “R” refers to those gametophytes cultured on glucose for the entire 80 days, “B” were grown on basal fern medium for 80 days. Gametophytes were transferred from rich medium to basal medium on the corresponding days after sowing. Graph represents two identical experiments, and error bars depict standard deviation. 38 B; Fig. 10c, B). Although apogamy could be seen in gametophytes cultured without sugar, its appearance was substantially slower compared with apogamy induced by sugar. Gametophytes that were maintained on rich medium for 2–10 days also displayed a similar low rate of apogamy, with 0%–2% of gametophytes displaying apogamy after 54 days of culture (Fig. 10a, lanes 2–10; Fig. 10c). When gametophytes were cultured on rich medium for longer than 12 days, the percentage induced to apogamy rose substantially when they were observed on day 54. Seven percent of gametophytes were induced to apogamy after 12 days (Fig. 10a, lane 12) of culture on rich medium, and this value rose to 12% after 16 days of culture (Fig. 10a, lanes 16; Fig. 10c). Similar experiments were conducted with fem mutant gametophytes to examine the timing of apogamy commitment in fern gametophytes that have no capacity for sexual reproduction (Fig. 11). Differently, in this experiment, total apogamy counts were not conducted periodically throughout the experiment, but tallied only at the end of an 80-day experiment. It was found that these gametophytes show a dramatic increase in the commitment to apogamous sporophyte production after 10 days of culture on rich medium (Fig. 11). Indeed, approximately 4 times as many fem gametophytes became committed to the apogamy after 12 days of culture on rich medium compared to her gametophytes. This difference may be attributed to the fact that the fem experimental observations continued for 80 days compared to 54 days. Alternatively, it may be that fem gametophytes are more competent for apogamy induction in general, perhaps indicating an apogamy-suppressing role for antheridia and/or sperm. However, the basal level of apogamy displayed from fem gametophytes is very similar to that displayed by the her 39 mutants (Fig. 10a, Fig. 11), which is evidence against the hypothesis that fem mutants are generally more competent for induction to apogamy. Discussion Here I have documented and characterized the sugar induction of apogamy in the fern C. richardii. The apogamous sporophytes of C. richardii take many forms, ranging from intermediate between sporophyte and gametophyte in morphology to clusters of sporophyte leaves. In agreement with previous descriptions of induced apogamy in other fern species (Lang, 1898; Whittier and Steeves, 1960, 1962), none of these forms are identical to normal zygote-derived sporophytes. Additionally, the gametophyteapogamous sporophyte junction lacks the extensive cell wall ingrowths that form in the “foot” of normal C. richardii gametophyte-sporophyte junctions (Duckett and Ligrone, 2003). In sexual reproduction, these wall ingrowths have been described as facilitating the transfer of nutrients from the gametophyte to the developing sporophyte (Gunning and Pate, 1969; Khatoon, 1986; Frey et al., 1994). How the apogamous sporophytes in this study are acquiring nutrients is unknown. Possibly, extensive cell wall ingrowths are unnecessary for the transfer of sufficient nutrients for apogamous sporophyte growth. A somewhat limited supply of nutrients through a poorly developed or non-existent placental region may explain why the apogamous sporophytes generated in this study fail to complete development and eventually die (data not shown). Alternatively, the apogamous sporophytes may acquire nutrients directly from the agar medium without the need for gametophytic transfer. This scenario may indicate that the failure of the apogmaous sporophytes to continue development is a side effect of their haploid nature, or the lack of a full complement of sporophytic development gene activities. 40 By removing exogenous sugar every other day during the induction period, we determined that C. richardii gametophytes begin commitment to apogamy development after growth for 12 d in the presence of sugar. At day 12, the gametophytes showed neither visible signs of apogamy nor any morphological difference under dissecting microscope compared with those grown on basal medium. However, the significantly higher frequency of apogamy in gametophytes that have been grown on rich medium observed at a later time indicates that a fundamental change at the molecular level has taken place by this critical time point (between day 10 and 12). It is this change that allows the gametophyte cells to commence, or to respond to the commencement of, a sporophyte-specific developmental program. The basal level of apogamy in the absence of exogenous sugars was surprising because it had not been reported previously. The implications of this intriguing result are exciting. The difference in kinetics of induced versus spontaneous apogamy suggests that different mechanisms are employed. It is possible that another aspect, in addition to exogenous sugar, of the culture conditions in our experiments was conducive to apogamy induction in C. richardii. One possible factor is the light conditions under which these gametophytes were grown, as high light intensity has been hypothesized to induce apogamy in other fern species (Lang, 1898; Duncan, 1941). Previously, C. richardii gametophyte cultures were grown under light intensities of 60–90 mM m2 s-1 (Hickock et al., 1995), which is slightly lower than our light conditions (90–100 mM m2 s-1). No matter what this factor may be, this result suggests a certain developmental plasticity of the gametophyte cells. This plasticity allows a small percentage of the gametophytes to produce apogamous sporophytes in conditions such as dryness that otherwise would not 41 allow for sexual production of sporophytes. The remarkable ability of infertile Ceratopteris strains to modify their life cycles and compensate for the inability to form a zygote (Hickock, 1977, 1979) is a testament to the developmental plasticity of this fern. Before our study in C. richardii, apogamy was experimentally induced in several species of homosporous ferns by culturing gametophytes on exogenous sugars (Whittier and Steeves, 1962). In addition, apospory, the formation of gametophytes from the vegetative tissue of sporophyte leaves can be induced in many ferns by culturing sporophyte leaves on basal medium (Raghavan, 1989). These two opposing culture conditions (exogenous sugar/no exogenous sugar) each induce an opposite alternative pathway, sporophytic development from gametophyte tissues, or vice versa, in many fern species (Whittier and Steeves, 1960, 1962; Raghavan, 1989). Moreover, sugar promotes sporophyte regeneration and lack of sugar promotes gametophyte regeneration from callus (Bristow, 1961). These results strongly point to the critical role of sugar, as metabolite and/or as a signal, in determining the two alternate developmental pathways in ferns. Sugar status, either through the sensing of metabolic flux or sugar signal transduction, is known to coordinate molecular and environmental cues in the control of plant growth and development (Sheen et al., 1999; Smeekens, 2000; Rolland et al., 2002; Rosa et al., 2009; Hanson and Smeekins, 2009). Furthermore, there is an extensive crosstalk between sugar-signaling pathways and the signaling pathways of ethylene, cytokinin, auxin, and abscisic acid in Arabidopsis (Smeekens, 2000; Rolland et al. 2002; Rosa et al., 2009). Some evidence shows that different hormone treatments, in combination with exogenous sugars, can promote the formation of apogamous sporophytes from fern gametophytes (Elmore and Whittier, 1973; Raghavan, 1989). 42 Despite the complex interplay between sugar signaling and hormonal signaling pathways, modulating sugar levels allowed me to establish the minimum time C. richardii gametophytes require an inductive cue to gain competence to undergo apogamy. The identification of genes whose expression specifically increased during this time is described in Chapter 3. 43 CHAPTER III GENES UPREGULATED DURING APOGAMY INDUCTION IN C. RICHARDII Introduction In the previous chapter and, Cordle et al., 2007, I showed that the homosporous fern C. richardii can be efficiently induced to form apogamous structures by culturing gametophytes on medium containing 2.5% glucose. C. richardii gametophytes also begin to become committed to the production of apogamous gametophytes between 10 and 12 days of growth on this apogamy-inductive medium. An alternative way of explaining this phenomenon would be that the gametophytes are not capable of responding to the inductive signal until at least 10 days growth on the inductive medium. Regardless, the identification of this response time provides an opportunity to identify genes that are differentially expressed during the commitment, or induction, of apogamy. Genes identified as differentially expressed during this time period may play an important role in the induction of apogamy in this fern. I used subtractive suppression hybridization (SSH; Diatchenko et al, 1996) to identify the genes expressed during this time period. This method allows for the comparison of two populations of mRNA and enables one to obtain clones that are present in one population while absent in the other, while at the same time normalizing the two populations, so that low copy transcripts will not be excluded from the library clones obtained (Diatchenko et al., 1996). The SSH method has been used to construct many cDNA libraries containing specifically expressed rare transcripts (for instance: 44 Jeong et al., 2004; Archambault and Stromvik, 2011; Landreville et al., 2011). The enrichment can be as high as 5000 fold (Diatchenko et al., 1996). In this chapter I describe how I utilized SSH to obtain cDNA clones that are present in a population of mRNAs from C. richardii gametophytes grown on inductive (rich) medium for 12 days, but absent or expressed at a low level from a population of mRNAs isolated from gametophytes grown on rich medium for 9 days. In this way, I cloned sequences that represent genes induced during the time period when C. richardii gametophytes are responding to an apogamy-inducing signal, or are becoming committed to the production of apogamy. I then mapped plant GO-slim terms to the identified genes in the apogamy library, and compared them to those mapped to the entire gametophyte transcriptome of Pteridium aquilinum, another homosporous fern (Der et al., 2011). I found that the apogamy library is enriched in GO-slim terms that are associated with stress response and metabolism, and includes several genes that are mapped to flower and embryonic development processes in plants. Additionally, I found that many of the Arabidopsis homologues of the apogamy library genes are specifically expressed or upregulated in Arabidopsis flower organs and seed structures, including embryos, based on in silico expression analyses. A subset of these genes was examined via whole mount in situ for their expression profiles in C. richardii gametophytes undergoing apogamy commitment, and 3 were identified as being upregulated in the sexual structures or the early embryo. 45 Materials and Methods Plants and Growth Conditions C. richardii hermaphrodite mutant spores were sterilized in 3% sodium hypochlorite and Tween 20, rinsed 5 times with sterile water, and plated on ½ strength MS medium supplemented with 2.5% glucose. Plates were incubated at room temperature in the dark for 5 days to synchronize spore germination, and then cultured as previously described (Cordle et al., 2007). RNA Extraction Gametophytes cultured for 9 days and 12 days on rich medium were removed from plates with sterile blades and flash frozen in liquid nitrogen. The frozen tissue was ground to a fine powder and total RNA was extracted using the guanidinium thiocyanate and acid phenol method (Chomczynski and Sacchi, 1987) with the following modifications. Before precipitation with isopropanol, 0.4 volume of a high salt solution (0.8M sodium citrate, 1.2M sodium chloride) was added to minimize the precipitation of polysaccharides. The RNA pellet was dissolved in freshly deionized formamide, heated to 70ºC for 10 minutes in the presence of 0.7M NaCl and 1% CTAB, and extracted twice with chloroform to remove precipitated polysaccharides. After adding 1 volume of DEPC-treated water, total RNA was recovered with standard ethanol precipitation. mRNA was enriched from the total RNA with the Oligotex mRNA mini kit (Qiagen) according to the manufacturer's instructions. Suppression Subtractive Hybridization The 12-day subtracted, normalized cDNA population was created according to the PCR-Select cDNA Subtraction Kit instructions (BD Biosciences; Diatchenko, et al., 46 2004), with minor modifications. First and second strand cDNA was created from 1µg of mRNA from 9-day-old and 12-day-old gametophytes. The synthesis of both first and second strands was monitored by incorporation of P-32 labeled dCTP in parallel reactions. The cDNA was digested with RsaI (New England Biolabs), and then precipitated with ammonium acetate and ethanol to remove unincorporated nucleotide triphosphates and primers. Completion of RSAI digestion was verified by agarose gel electrophoresis with pre- and post-digested cDNAs. Ligating equal proportions of 12-day cDNA with two different adapters created the tester cDNA population. The ligation reaction was performed overnight at 4° C with T4 DNA ligase (New England Biolabs). Ligation efficiency was tested with PCR using C. richardii hexokinase gene-specific primers along with adapter-specific primers. The 12-day tester cDNA populations were then hybridized with an approximately 30x excess of 9-day driver cDNA to normalize and enrich for differentially expressed sequences at day12. Another round of normalization and enrichment with approximately 10x excess of 9-day driver cDNA was then performed to create templates for PCR amplification of differentially expressed sequences. Using primers specific for the two adapter sequences, two cycles of PCR, the second with nested primers, were performed to amplify sequences that were present in the 12-day population and absent in the 9-day population. Sequence Identification and Analysis Sequences were uploaded in FASTA format into the Blast2GO program (Conesa et al., 2005; Conesa and Gotz, 2008) and were identified with BLASTx against the nonredundant NCBI protein database. Those sequences that could not be identified in this manner were identified manually via BLASTx at the Joint Genome Institute or BLASTn 47 searches followed by BLASTx of best hits. Those sequences that were identified with an E-value significance of less than 1E-3 were selected for further mapping and annotation with the Blast2Go program. The Genevestigator program (Zimmerman et al., 2004; Zimmerman et al., 2005) was utilized for in silico expression analysis of Arabidopsis homologues. Choosing only those probe sets specific for the genes of interest, 6100 high quality ATH1 22K array experiments were chosen to create a meta-profile. Gene hierarchical clustering was performed to facilitate identifying genes with similar expression profiles. The resulting heat map output was modified using Adobe Photoshop CS4 (Adobe Systems Inc.). Cloning, Sequencing, and RT-qPCR Amplified sequences were cloned directly into the pCRII-TOPO dual promoter vector (Invitrogen) and transformed into TOP10 F’ E. coli cells (Invitrogen). Clones were arrayed into 96-well plates and stored as glycerol stocks. Minipreps were performed with a Manual PerfectPrep Plasmid 96 Vac Kit (5 PRIME) and the resulting plasmids were sequenced with T7 promoter primers on an ABI 3730 DNA Analyzer (Applied Biosystems) at the Carver Center for Genomics (University of Iowa). For semi-quantitative RT-PCR analyses, C. richardii mutant gametophytes grown on 2.5% glucose or no glucose medium for 8 to 13 days were flash frozen in liquid nitrogen. Total RNA was extracted from these gametophytes as described above. Total RNA purity and integrity were verified via A260/A280 and A260/A230 readings (NanoDrop 2000, Thermo Scientific) and native gel electrophoresis, respectively. Specific primers were designed based on apogamy library clone sequences, and in the case of CrUBQ10, based on the publicly available sequence from a spore germination 48 Table 1 Primer sequences for semi-quantitative and RT-qPCR analyses Sequence Name Primer Sequence Glycine Rich Protein LP 5’-CTCGCTGTTCTCTTCCACTTG-3’ Glycine Rich Protein RP 5’-AGGACCAGGAACATGAATGC-3’ P3F12 LP 5’-GTGCTCCCTCATCTCGTTTC-3’ P3F12 RP 5’-CAAACTCAGGCTGGCTTCTC-3’ UNC93-like LP 5’-CACTTCTGCTGCTAAAAGTTATGC-3’ UNC93-like RP 5’-CATGCAACCAAGAAATGTAAGG-3’ PHD Finger LP 5’-CGTTCTTTGATTCTCCCTCTG-3’ PHD Finger RP 5’-CAGGACGAAAACGGACATAG-3’ RNA Recog. Motif LP 5’-GGCGGATTCGGAGTTATTG-3’ RNA Recog. Motif RP 5’-GCTGAAAGATCATGGGGAAC-3’ Snf2-like LP 5’-AGGAGGCATACGGATGAAGA-3’ Snf2-like RP 5’-GCTTCTTGGGGAGGTTATGA-3’ Sugar Transporter LP 5’-TCTGCACAGTTTACCAAAATGG-3’ Sugar Transporter RP 5’-TGTCACAGAAGATTAGTGTCTCCAG-3’ Leunig-like LP 5’-GAGAGGTACGGCTGAACTGC-3’ Leunig-like RP 5’-CACAATGGAAACGCACAGTC-3’ Glycine Clvage Syst. H LP 5’-CCTGTGGGTGGTAAGGTGAT-3’ Glycine Clvage Syst. H RP 5’-GCCCCTCATAATCAGAAGCA-3’ Nucleolar Essential LP 5’-GAAACAGCCAGTCACAAAACATC-3’ Nucleolar Essential RP 5’-GCACCAACCACAAATACCATC-3’ RPN9 LP 5’-TGGGTTCAGCCAAGAGTTTT-3’ RPN9 RP 5’-AGACGGACAAGGGTTTTATGC-3’ HMG Protein LP 5’-TGCCTTTTTCGTGTTCCTTG-3’ HMG Protein RP 5’-AAAGCCAATATGACAGAGGTTCC-3’ BTB-domain Containing LP 5’-AGGTCCCTGGAACAACTGATT-3’ BTB-domain Containing RP 5’-CAGCAGCAAATGATTGTGAGT-3’ 49 library (Salmi et al., 2005; Table 1). The exponential amplification range of all targets was determined by removal of aliquots of PCR amplifications every 2 cycles followed by gel electrophoresis and ethidium bromide staining. For each gene, the cycle at which amplicons became visible with ethidium bromide staining (using 12-day gametophyte cDNA as template) was used for subsequent PCR analyses. Both control and experimental primers were used in the same PCR reaction tubes, and final PCR products were run on 1% Agarose gels, stained with ethidium bromide and imaged under UV light. Relative expression was determined using ImageJ 1.43r (Abramoff et al., 2004). Briefly, after calibration, gel lanes were selected and plots created for each gel lane. Plot peaks, which represent each distinct PCR band within a gel lane, were specified and the area beneath each peak was calculated. For each gel lane, the ratio of control (UBQ10) area to experimental area was calculated. The highest ratio was arbitrarily set as 1, and all other ratios calculated relative to that. Graphs were created using Microsoft Excel for Mac 2004 (Microsoft corporation) and finished with Adobe Photoshop CS4 (Adobe Systems Inc.). For RT-qPCR, C. richardii hermaphrodite mutant gametophytes grown on 2.5% glucose or no glucose medium for 9 to 12 days were flash frozen in liquid nitrogen. Total RNA was extracted and its quality verified as described above. 1 µg of total RNA was used to create first strand cDNA using 9-nucleotide random primers. 2 µL of a 1/10 dilution of the resulting cDNAs was used for qPCR reactions. Specific primers were carefully designed based on the corresponding apogamy library clone sequences, and tested for Tm, the amplification of predicted products, and the production of primer dimers before qPCR analysis (Table 1). The primer concentration in the final qPCR 50 reactions was 1 µM. The qPCR experiments were executed using the LightCycler 480 SYBR Green I Master kit (Roche Applied Science) and the LightCycler 480 Real Time PCR System (Roche Applied Science). The cycling parameters were a 10-minute denaturing step at 95°C followed by 45 cycles of 10 seconds at 95°C, 20 seconds at 58° C annealing, and 15 seconds at 72° C for extension, with a single fluorescence read at the end of each extension. A melting curve analysis was performed, and analyzed using the Tm calling software module to generate melting peaks at the end of each run to verify the absence of significant primer dimers or other non-specific PCR products. Within each plate of qPCRs, pairs were set manually and raw data was analyzed using Crossing point (Cp) calculation and relative quantification using the advanced relative quantification software modules. Cp calling was obtained by the second derivative max method and standard curve settings were set to use the default PCR efficiency. The LightCycler 480 software calculated relative quantification automatically and the resulting data and bar graphs were exported. The highest ratio for each gene/reference pair was arbitrarily set to 1, and the results for all other gene/reference pairs relative to that, and standard errors were adjusted accordingly. The final bar graphs were created in Microsoft Excel for Mac and were finished using Adobe Photoshop CS4. The data for this experiment is a combination of 2 biological and 2 technical replicates. Whole mount in situ Hybridization For probe synthesis, plasmids were linearized overnight with BamHI (NEB) or XbaI (NEB). Complete linearization was verified with gel electrophoresis. 1 µg of purified, linear plasmid was subsequently used in probe synthesis with T7 (Stratagene) or SP6 (Stratagene) RNA polymerases and DIG RNA labeling mix (Roche) to generate 51 antisense and sense probes. Probes were precipitated with 3M LiCl to remove enzyme and template, and after resuspension in DEPC H 2 O, quantified with the NanoDrop 1000 (Thermo Scientific). C. richardii gametophytes were fixed under vacuum for 10 minutes in ice cold formaldehyde, acetic acid, and alcohol (FAA), then fixed overnight at 4°C in fresh FAA, and then stored at -20°C in 75% ethanol. For all steps, gametophytes were kept in small baskets made from 0.2 mL tubes for PCR, cut in half, with 70-micron nylon mesh glued to the bottom. These baskets facilitated the movement of gametophytes through the various treatments and washes, which in prehyb steps were performed in 96 well plates. For prehybridization, gametophytes were moved through a 3-step ethanol series into PBS pH 7.5, 20 minutes for each wash, permeabilized in 0.2M HCl for 20 minutes, neutralized with 2x washes in PBS, and treated with 0.1 mM Proteinase K for 10 minutes at room temperature. Proteinase K digestion was stopped with three 2 minute washes in PBS, 1% glycine, and PBS. Gametophytes were then postfixed in 4% paraformaldehyde for 20 minutes at room temperature. After a 90 min. prehybridization at 55°C in hybridization solution, hybridization was performed for 15-18 hours in screw cap tubes set in a 55° water bath with 100 µL hybridization solution (6x SSC, 3% SDS, 50% formamide, 0.1 mg/mL tRNA) and 1 ng/µL denatured probe. Posthybridization washes were performed in screw cap tubes. 2-10 minute low stringency washes (0.2x SSC, 0.1% SDS) at 55°C, and 1-2 minute high stringency wash (2x SSC) at room temperature with gentle agitation. RNase digestion was performed with 10µg/mL RNaseA in 2x SSC for 30 minutes at 37°C, and then gametophytes were rinsed for 2 minutes in 2x SSC at room temperature. Three final washes were performed: low stringency for 10 minutes at 55°C, high 52 stringency for 2 minutes at room temperature, and Tris-buffered saline (TBS pH 7.5) for 2 minutes at room temperature. Gametophytes were then incubated for 2 hours in 1% blocking solution (Roche) for 2 hours, then 2 hours in a 1/1000 dilution of anti-DIG AP fab fragments (Roche) in 1% blocking solution, washed 4 times for 10 minutes each with agitation at RT (100 mM Tris-base pH 7.5, 150 mM NaCl, 0.3% (v/v) Tween 20). Detection was performed with 1x color substrate solution (NBT/BCIP, Roche) in detection buffer (100 mM Tris-base pH 9.5,150 mM NaCl), with overnight incubations. Gametophytes were mounted in 50% glycerol and photos for each experiment were taken under the same light and exposure conditions on a Leica DM LB microscope with imaging system, and exported to Adobe Photoshop CS4. Results Strategy to Identify Genes Enriched During Apogamy Commitment Using the optimized conditions for apogamy induction in C. richardii, and the timing of apogamy commitment under the experimental conditions (Chapter 2; Cordle, et al. 2007), I designed a strategy to identify genes that may play important roles in apogamy commitment. I employed the SSH method to normalize over-represented mRNA sequences from, and amplify only those sequences that are enriched in, the 12day-old gametophyte transcriptome (the experimental cDNA population, after apogamy commitment) as compared to the 9-day-old transcriptome (the driver cDNA population, before apogamy commitment). I call this normalized, subtracted cDNA library the apogamy library. 53 The C. richardii genome is not sequenced, and sequence information is limited to approximately 3000 ESTs in public databases. Therefore, to learn more about the functions of the genes in the apogamy library, I chose to identify their closest Arabidopsis homologues using BLASTx of the apogamy sequences against the NCBI non-redundant Arabidopsis protein database, and examine the expression patterns of these Arabidopsis genes in silico by compiling vast publicly available microarray data using Genevestigator (Zimmerman et al., 2004; Zimmerman et al., 2005). Validation of the Apogamy Library The successful normalization and subtraction of the library was validated by colony hybridization using a probe which is specific to the C. richardii RuBisCo small subunit, which without normalization, would be expected to constitute approximately 50% of the cloned cDNA sequences (Ellis, 1981). I found that this sequence comprised 0.2% of the apogamy library clones (2 out of 902 total clones), representing a more than 200-fold normalization and/or subtraction. To confirm that the apogamy library represents transcripts enriched in day 12 gametophytes compared with day 9 gametophytes, 5 library clones whose homologues in Arabidopsis show flower or embryo-specific or embryo-enhanced expression (Fig. 17) and one clone that has no apparent homologue in other organisms (P3F12) were chosen for relative RT-PCR analysis (Fig 12). All of the sequences chosen showed higher expression in day 12 gametophytes compared to day 9 gametophytes in this experiment (Fig. 12). However, not all of these sequences increased steadily over that time period, for instance the sequences encoding a glycine-rich protein and an RNA recognition motif-containing protein appear to peak in expression in day 11 gametophytes, and have 54 Figure 12 Relative RT-PCR analyses of select sequences from the apogamy library. Expression levels are relative to a UBQ10 internal standard. Experiment was performed once. 55 Figure 13 RT-qPCR analyses of select sequences from the apogamy library. Results are the combination of 2 technical and 2 biological replicates. Asterisk indicates a statistically significant increase in gene expression (Student’s t-test; p < 0.05) 56 decreased in expression by day 12 (Fig. 12). Nevertheless, the expression levels of these genes, and all others, were higher at day 12 compared to day 9, which suggests that the apogamy library contains genes genuinely upregulated during this time period. The expression patterns of these same genes in gametophytes cultured on non-inductive basal fern medium are very different than the glucose-grown gametophytes (Fig.12). Indeed, on basal medium, they all appear to be expressed at a lower level in day 12 gametophytes compared to day 9 gametophytes (Fig. 12). Differing expression patterns between basal and rich medium would be expected of genes that are necessary for the induction of apogamy, or for those genes that are directly or indirectly regulated by glucose. Three of these genes, plus 7 other library clones, were chosen for RT-qPCR analysis (Fig. 13). These 10 genes represent diverse molecular function activities, based on their slimmed plant Gene Ontology mappings (Fig. 15). Specifically, the molecular functions mapped to these sequences are transcription factor activity, hydrolase, transporter, RNA binding, protein binding, and enzyme regulator activity. Also, the molecular function of the sequences that encode the senescence associated protein, UNC93-like protein, and nucleolar essential protein are unknown. The RT-qPCR experiments are the combination of 2 technical and 2 biological replicates, and all sequences examined show a significant increase in expression from day 9 gametophytes to day 12 gametophytes (Fig. 13). These experiments provide further evidence that the clones in the apogamy library represent genes that increase in expression during the time period when C. richardii gametophytes are undergoing apogamy commitment. 57 Composition of the Apogamy Library From 441 sequenced clones, 306 unique sequences (unigenes) were identified (Table 2). Among these unigenes, 156 had significant BLASTx hits (defined in this study as an E-value at or below 1E-3) against the NCBI non-redundant protein database using the Blast2GO 3000 program (Conesa et al., 2005; Conesa and Gotz, 2008). Among the remaining 150 unigenes, 14 could be identified with significant BLASTn hits against NCBI land plant reference RNA sequences or with BLASTx hits against JGI genome databases (Physcomitrella patens and Selaginella moellendorffii). The remaining 136 unigenes had no significant hits from any database. All but 4 of the sequences without significant hits were smaller than 200 nucleotides and/or contained long poly(A)+ strings. The 4 longer unigenes were between 374-595 nucleotides and, not having significant hits from any database, including the P. aquilinum gametophyte transcriptome (Der et al., 2011), are presumably C. richardii specific. Alternatively, they may be present in P. aquilinum but expressed only in the sporophyte, or expressed in the P. aquilinum gametophyte but not detected in the transcriptome analysis by Der et al. (2001). The 170 unigenes identified with confidence were chosen for further analysis. In the process of identifying these 170 genes, the top BLASTx hits for each sequence were collected and analyzed (Fig 14). To prevent bias, the top hits included in this table are restricted to those whose genomes are completely sequenced, in assembly, or that have large-scale EST projects completed (at least 25,000 ESTs in GenBank). 156 genes from the apogamy library had top blast hits among these organisms and 3 species dominate these top hits, the moss P. patens, the conifer Picea sitchensis (Sitka spruce), and the lycophyte S. moellendorffii. These three organisms comprise over 50% of the top BLAST 58 Table 2 The 170 apogamy library clones identified with confidence Clone name Clone Identity Clone length (nt) Best Hit E-value Query Coverage (%) P1A10 lipid transfer protein 398 8.10E-06 42 P1A11 casein kinase 2 subunit beta 129 1.50E-15 100 P1A12 heat shock 70 266 1.90E-15 82 P1A2 glycine-rich protein 803 8.30E-17 69 P1A7 transketolase 485 1.70E-24 41 P1A9 pyrophosphate-dependent phosphofructokinase beta subunit 383 2.40E-50 98 P1B11 60S ribosomal subunit 164 1.10E-07 98 P1B3 calcium-dependent phosphotriesterase-like 636 9.40E-13 48 P1B7 glutathione S-transeferase 388 5.30E-26 98 P1B8 nitrite reductase 306 5.30E-26 67 P1B9 uncharacterized protein 186 2.20E-11 100 P1C10 fructose bisophosphate class I 168 7.70E-17 98 P1C12 L-ascorbate peroxidase 236 5.50E-31 99 P1C2 thylakoid lumenal protein 112 2.00E-06 77 P1C4 catalase 117 1.50E-12 97 P1C6 60S L7A ribosomal protein 1103 3.30E114 70 P1C7 translation initiation factor 200 1.00E-08 99 P1C9 flavoprotein monooxygenase 247 1.20E-09 53 P1D1 chlorophyll a/b binding protein 570 3.60E-55 59 P1D11 calreticulin precursor 263 1.00E-37 99 P1D12 light rearvesting complex II protein LHCB6 320 1.2E-6 39 P1D4 UDP-glucoronosyl transeferase family protein 264 1.9-07 79 P1D5 glutathione S transferase 1039 60E-64 66 P1D9 delta tonoplast integral protein 516 3.00E-35 66 P1E10 serine esterase domain-containing protein 465 7.10E-10 43 P1E11 phosphoglycerate kinase 517 9.40E-53 66 P1E12 immunophilin 144 3.80E-11 79 P1E5 cytoplasmic hydroxyacylglutathione hydrolase 349 7.80E-25 91 P1E6 plastid transcriptionally active (PTAC17) 431 1.00E-06 20 59 Table 2 Continued P1E7 carbonic anhydrase 425 3.00E-29 84 P1F2 10 kd photosystem II polypeptide 295 3.90E-25 85 P1F4 cytochrome C 821 6.40E-44 48 P1F8 FTSH10 protease 279 7.90E-14 90 P1G10 multidrug resistance protein 153 1.70E-11 98 P1G4 augmenter of liver regeneration 327 1.00E-13 35 P1G7 hydroxycinnamoyl shikimate/quinate hydroxycinnamoyltransferase-like 486 2.30E-42 99 P1G8 uncharacterized protein 685 5.10E-72 84 P1H1 alpha-expansin 15 808 9.20E-64 69 P1H10 transducin family protein 249 2.60E-17 86 P1H11 eukaryotic translation initiation factor SUI1 family protein 314 2.30E-13 45 P1H9 rhodanese domain-containing protein 559 6.80E-64 92 P2A1 ribosomal protein L1 285 1.00E-05 31 P2A11 HMGB1 transcription factor 628 4.70E-15 22 P2A12 DEAD box RNA helicase 114 5.00E-06 60 P2A2 acyl-synthetase 946 4.80E-44 40 P2A4 cysteine protease 493 1.30E-19 43 P2A7 BTB domain-containing protein 405 1.90E-07 45 P2A9 cathespin B-like cysteine protease 217 1.6E-14 66 P2B1 60S ribosomal protein L17 334 1.6E-27 82 P2B12 24-sterol-C-methyltransferase 448 10E-16 50 P2B3 elongation factor TU 237 1.50E-28 98 P2B6 chloroplast light-harvesting complex II protein 125 1.50E-12 98 P2C2 small nuclear riboprotein F 630 1.80E-35 36 P2C5 LURP1-related protein 906 3.30E-39 68 P2C7 26S proteasome non-ATPase regulatory subunit 13 (RPN9) 447 3.20E-23 40 P2C8 26S proteasome regulatory subunit 814 8.10E100 97 P2D1 citrate synthase 230 7.80E-33 99 P2D10 glutaredoxin-related protein 538 8.20E-29 55 P2D12 elongation factor 1A 1040 1.90E137 72 P2D2 zeanthin epoxidase 98 2.00E-07 97 60 Table 2 Continued P2E12 flavanoid 3'-hydroxylase (transparent testa 7) 562 1.00E-35 98 P2E11 70 kD DNA-binding replication protein A70 415 2.80E-27 99 P2E3 chloroplastic NADPH-protochlorophyllide oxidoreductase 297 5.70E-44 94 P2E4 ribulose bisphosphate carboxylase oxygenase 1022 activase 2.70E104 65 P2E5 40S ribosomal protein 374 4.70E-06 69 P2E6 flavonoid 3-hydroxylase 562 7.70E-39 98 P2F1 GDSL esterase/lipase protein 227 7.10E-10 99 P2F4 boron transporter 191 3.60E-06 98 P2F5 ankyrin repeat domain-containing protein 854 2.70E-56 58 P2F9 peptidylprolyl isomerase 144 2.00E-14 79 P2F8 geranylgeranyl diphosphate reductase 203 1.00E-08 48 P2G10 HTPG chaperone-family protein 189 6.00E-25 100 P2G11 uncharacterized protein 488 5.90E-65 99 P2G12 snakin-1 (gibberellin-regulated protein) 375 3.20E-18 69 P2G4 cyclin-dependent kinase regulatory subunit 313 4.60E-22 77 P2G5 uncharacterized protein 189 3.60E-06 66 P2G9 sodium-bile acid cotransporter-like protein 327 2.00E-25 80 P2H6 23S ribosomal RNA 113 1.60E-06 90 P2H7 endo-beta-D-glucanase precursor 231 2.60E-12 97 P3A1 alpha-beta-hydrolase domain-containing protein 734 2.30E-93 91 P3A10 lanatoside 15-O-acetylesterase 223 1.90E-10 73 P3A2 chlorophyll a oxygenase 332 4.80E-11 47 P3A4 rubisco subunit binding protein beta 673 3.80E-24 32 P3A6 peroxisomal-2-hydroxy-acid oxidase 2 481 4.30E-20 47 P3A8 SCF ubiquitin ligase 233 3.50E-09 48 P3B1 alpha-glucan phosphorylase 317 7.30E-47 99 P3B10 LisH/CRA/RING-U-box domain-containing protein 159 4.90E-19 100 P3B12 60S ribosomal protein 398 1.10E-10 48 P3B4 senescence-associated protein 215 2.70E-14 97 P3B5 ubiquitin-conjugating enzyme E2 36 215 5.30E-18 62 P3B8 eIF2 beta subunit protein 605 2.40E-26 31 P3C1 early nodule-specific protein 392 8.40E-24 99 61 Table 2 Continued P3C10 long chain fatty acid CoA ligase 467 4.00E-42 40 P3C3 protochlorophyllide reductase 392 5.00E-06 19 P3D1 actin 405 9.30E-63 99 P3D12 protein phosphatase 236 4.40E-20 82 P3D5 sec61 transport protein 495 2.10E-70 82 P3E10 hypersensitive response protein 117 7.00E-13 97 P3E1 60S acidic ribosomal protein P0 216 1.30E-24 98 P3E11 fructose-bisphosphate aldolase 408 1.10E-44 70 P3E12 rRNA 2'-O-methyltransferase (fibrillarin 2) 418 3.60E-70 99 P3F3 dienelactone hydrolase family protein 231 4.0E-11 97 P3F1 actin depolymerizing factor 134 4.0E-51 21 P3F4 map 4 kinase alpha 1 563 90E-06 93 P3E3 non-yellowing protein 1 834 7.20E-06 21 P3F11 methionine synthase 112 9.30E-10 91 P3G2 phytochrome-associated serine/threonine protein phosphatase 193 7.80E-25 93 P3G3 uncharacterized protein 230 3.90E-16 97 P3G5 pyrophosphate-energized vacuolar membrane 455 protein 1.10E-63 99 P3H11 14-3-3 protein 197 1.80E-26 98 P3H3 DNAJ heatshock protein 249 1.00E-10 24 P3H4 photosystem I protein 159 2.30E-21 98 P4A6 hydroxymethylbutenyl 4-diphosphate synthase 649 2.70E-69 71 P4A8 macrophage migration inhibitory factor 94 2.00E-07 98 P4B2 superoxide dismutase 374 5.20E-53 95 P4B3 RNA recognition motif protein 322 8.80E-37 98 P4B12 copper transporter 215 2.00E-06 39 P4C2 thylakoid lumenal 29.8 kDa protein 1036 2.90E-56 53 P4C3 zinc-binding hydrolase 329 2.00E-09 17 P4C5 beta-glucosidase G3 315 1.90E-10 49 P4D1 plastidic glucose transporter 540 1.20E-22 37 P4D11 calcium-dependent phosphotriesterase-like 635 4.90E-12 48 P4D9 nucleolar essential protein 317 1.90E-42 98 P4E11 40S ribosomal protein 118 2.00E-08 71 P4E2 S-adenosyl methionine synthetase 2 487 5.90E-33 48 62 Table 2 Continued P4E7 ran-binding zinc finger domain-containing protein 800 2.30E-06 19 P4E9 adenosine kinase 330 2.60E-33 80 P4E10 aminocyclopropanecarboxylate oxidase 362 2.00E-04 9 P4F12 DNAJ heatshock N-terminal-domaincontaining protein 397 5.60E-23 92 P4F4 pentacopeptide repeat-containing protein 378 6.50E-24 71 P4F8 DNAJ heatshock protein 534 2.10E-13 50 P4G11 soluble starch synthase I 477 4.50E-33 54 P4G12 chlorophyll a/b binding protein 269 3.90E-37 84 P4G2 3,5-epimerase-4-reductase 207 1.20E-30 100 P4G3 magnesium cheletase subunit D 327 3.10E-42 99 P4G7 uncharacterized protein 97 2.00E-09 86 P4G8 histone H1 linker 818 3.00E-09 15 P4H3 transcription factore Hecate2 320 4.00E-04 43 P4H8 translation elongation factor I alpha 783 1.30E115 83 P5A10 peroxin 22 307 1.30E-13 44 P5A2 ferredoxin-NADP+ reductase 432 4.20E-15 30 P5A4 60S ribosomal protein 115 3.40E-12 96 P5A6 S-adenosyl methionine synthetase 664 7.00E-76 75 P5B12 BP5A protein 237 3.50E-09 50 P5B2 pentacopeptide repeat-containing protein 422 3.20E-26 88 P5B8 glycine dehydrogenase 264 7.00E-11 34 P5B9 photosystem I reaction center subunit IV-A 375 1.90E-10 28 P5C1 ATP-citrate lyase A-1 480 5.40E-47 74 P5C12 biotin carboxyl carrier protein 742 3.50E-40 70 P5C4 eukaryotic translation initiation factor 2gamma 494 4.50E-57 78 P5C5 30S ribosomal protein S13 428 5.20E-13 41 P5D12 glycine cleavage system H protein 222 2.40E-13 91 P5D5 monodehydroascorbate reductase 601 1.20E-65 93 P5D9 DNA photolyase protein 241 3.60E-14 75 P5E2 UNC93-like 237 6.80E-13 100 P5E4 RNA binding protein 47C 233 2.80E-19 97 P5E6 light harvesting complex 408 1.70E-24 74 63 Table 2 Continued P5E7 sugar transporter 280 5.00E-07 45 P5F10 chlorophyll a/b binding protein 1 243 6.50E-32 91 P5F11 delta-aminolevulinic acid dehydratase 107 9.30E-10 98 P5F12 ketol-acid reductoisomerase 471 2.70E-54 93 P5F2 plastid transcriptionally active 4 (PTAC4) 206 8.00E118 53 P5F7 malate dehydrogenase 2 348 2.20E-48 99 P5F9 transducin/WD40 domain-containing protein 258 3.60E-30 97 P5G11 chalcone synthase 142 1.70E-16 99 P5G9 senescence-associated protein 294 1.60E-46 98 P5H12 translation elongation factor TS 187 2.80E-14 83 P5H2 chlorophyll a/b binding protein 235 1.10E-18 63 P5H9 30S ribosomal protein S31 323 3.30E-07 62 hits for the 156 genes (Fig. 14a). The remaining top BLAST hits were a variety of angiosperms and, surprisingly, one gene in Carboxydothermus hydrogenoformans, a thermophilic bacterium (Fig. 14a and b). This sequence is predicted to encode a glycine cleavage system H protein, and, although this particular region of the sequence is most highly similar to an extremophile, it is unlikely to represent contamination. The sequence could be amplified from two separate biological samples of gametophytes in RT-qPCR analysis, and shows increasing expression over the 9-12 day apogamy commitment period (Fig. 13). Overall, though, the majority of the sequences identified in the apogamy library are related to dicotyledonous angiosperms (Fig. 14b). Monocots and dicots together comprise 38% of the top hit species from the sequence identifications (Fig. 14b). GO-slim Mapping and Comparative Analysis with the P. aquilinum Gametophyte Transcriptome The 163 unigenes were annotated with GO terms, enzyme codes, and InterPRO domain/motif information. Annotations were then mapped to their parent plant GO-slim 64 Figure 14 Top hit species and classifications. (A) The number of genes from the apogamy library with best E-value BLASTx best hits from the given organisms. Only organisms with genome sequencing completed, in assembly, or with large-scale EST projects were included. (B) The classifications of the species from (A) into their respective land plant phylogeny clades. 65 terms (Fig. 15). 58% of the evidence codes were inferred from electronic annotation, 16.5% from direct experimental evidence (direct assay), and 6.6% and 5.8% from sequence or structure similarity and reviewed computation analysis, respectively (data not shown), accounting for over 85% of the annotations. Within the biological process domain, the term mapped to approximately 13.5% of the apogamy library unigenes is response to stress (22 unigenes), followed by 12.3% involved in catabolic processes (20 unigenes), and 10.4% (17 unigenes) each involved in generation of precursor metabolites and energy, translation, and photosynthesis (Fig. 15a). 40.5% of the apogamy library unigenes are mapped to the plastid (66 unigenes), the term that dominates the cellular component domain, followed by approximately 12.3% (20 unigenes) each mapped to the mitochondrion and thylakoid (Fig. 15b). Over 50% of the terms under the molecular function domain are mapped to nucleotide binding (20.2%, 33 unigenes), protein binding (17.2%, 28 unigenes), and hydrolase activity (15.3%, 25 unigenes) (Fig. 15c). It would be interesting to compare the apogamy library plant GO-slim terms with those mapped to the whole C. richardii genome to learn which terms are overrepresented in the apogamy library. However, the C. richardii genome is not sequenced and the EST coverage is limited to 5125 clones representing approximately 4000 genes from sporophyte (sporophyll) tissue and germinating spores (Salmi et al., 2005). Therefore, I performed a statistical assessment of the differences in GO-slim terms between the apogamy library and the gametophyte transcriptome of the homosporous fern P. aquilinum (Der et al., 2011), the only large sequence data set for any fern species. I found a number of plant GO-slim categories that are over-represented (Fisher’s Exact Test, p < 0.05) in the apogamy library compared to the P. aqulinum gametophyte 66 Figure 15 Distribution of plant GO-slim categories mapped to the 170 unigenes in the apogamy cDNA library. 67 transcriptome. These categories are shown in Figure 16. The biological process categories over-represented are mainly involved in metabolism (secondary metabolic process, catabolic process, and photosynthesis) and stress response (response to stress, abiotic stimulus, biotic stimulus, and external stimulus) (Fig. 16a). This is consistent with previous reports that suggest an increase in metabolic processes and stress response mechanisms underlie the induction of haploid sporophytes from gametophyte cells (Raghavan, 1989; Segui-Simarro, 2010). Also over-represented in the apogamy library, compared to the P. aquilinum gametophyte transcriptome, are the embryonic development and flower development plant GO-slim terms (Fig. 16a). This is interesting because the source tissue for the apogamy library is, of course, fern gametophytes, and these two GO terms are mapped to genes that function in early sporophyte development and the development of angiosperm sporophytic sexual structures. Obviously, flowers are structures that are not present in ferns, but structures that are homologous to flowers may be present in the gametophytes of ferns. For instance, the antheridia may be analogous to the anthers of flowers because the end product of both structures is sperm. The plastid and thylakoid are among most highly over-represented GO-slim cellular compartment terms mapped to the apogamy library compared to the P. aquilinum transcriptome (Fig. 16b). These proteins are likely involved in photosynthesis, which is also an overrepresented biological process term. Nucleolus and ribosome components and translation/nucleic acid binding functions are also over-represented, consistent with an increase in the production of ribosome and translation factor biological activity (Fig16a). Components of the vacuole are also over-represented. The vacuole is an organelle that enables plants to respond to changing environmental conditions (including stressors), 68 Figure 16 Apogamy library enriched GO-slim terms. (A) Biological process plant GO Slim categories over-represented in the 12-day cDNA library (grey bars) compared to the Pteridium aquilinum gametophyte transcriptome (black bars). (B) Cellular compartment and molecular function plant GO-slim categories over-represented in the 12-day cDNA library compared to the Pteridium aquilinum gametophyte transcriptome. Statistical assessment of annotation differences was performed using the Fisher’s Exact Test (p < 0.05) corrected for multiple testing. 69 store excess nutrients, and maintain optimal metabolic conditions in the cytosol (Martinoia et al., 2006). in silico Expression Patterns of Apogamy Library Closest Arabidopsis Homologues Using BLASTx against the NCBI Arabidopsis non-redundant protein database, the closest Arabidopsis homologues of the 170 unigenes of the apogamy library were identified. Only those sequences that returned BLASTx hit scores of 1E-5 or lower were considered for this analysis; 8 sequences did not make this cutoff, so 162 Arabidopsis genes were considered. The locus tags for these 162 genes were entered into the Genevestigator program (Zimmerman et al., 2004; Zimmerman et al., 2005), and a metaprofile analysis was performed. Genevestigator mines high quality microarray data from public repositories and integrates that data into biologically meaningful gene expression profiles (Zimmerman et al., 2004). All gene profiles were hierarchically clustered, and from those clustered genes I isolated the profiles of those that are expressed mainly, or upregulated in, the floral organs and seed structures (Fig. 17). These genes could be separated into 4 general categories: those that have increased expression in the seed and organs of the flower but are absent from the stamen and anthers (Fig 17A), those that have increased expression in the stamen and anthers and may or may not show elevated expression levels in the seed and other flower organs (Fig 17B), those genes that show elevated or specific expression in the embryo (Figure 17C), and genes that show specific or highly elevated expression in other flower organs or seed structures such as the seed coat, suspensor, ovule, petal, or sepal (Fig 17D). Among those genes 70 Figure 17 in silico expression patterns of Arabidopsis homologues of selected unigenes from the apogamy library. Anatomical meta-profile heat maps were generated with data mined from over 6000 high quality microarray experiments and clustered using the Genevestigator program. 71 expressed mainly in the seed and flower organs and the genes that are specific or upregulated in the embryo, are interspersed various ribosomal proteins and translation initiation and elongation factors (Fig 17A and C). This is indicative of the importance of de novo protein synthesis for these tissues and organs. Additionally, specific transcriptional profiles for different ribosomal proteins are in line with previous reports of developmental and tissue-specific expression for many proteins associated with ribosomes (McIntosh and Bonham-Smith, 2006). In addition to ribosomal proteins, there are also proteins that are associated with the nucleolus, such as the snoRNA binding protein fibrillarin, and the nucleolar protein, both upregulated in the Arabidopsis embryo (Fig. 17C). There may be novel developmental functions of the nucleolus that are not fully understood (Pendle et al., 2005). Suggestive of this is the TORMOZ gene of Arabidopsis, which is involved in the specification of the first planes of mitosis in the developing embryo, whose homologues in S. pombe is predicted to function in 18S rRNA biogenesis (Griffith et al, 2007). Genes in the apogamy library whose homologues showed elevated or specific expression in Arabidopsis flower and seed organs and tissues were chosen for whole mount in situ analysis in C. richardii gametophytes. The reason for this approach was two-fold: to investigate whether, as in Arabidopsis, these genes also showed increased expression in homologous structures in the fern gametophytes, and to find those genes that show a specific or increased expression in the mature C. richardii egg and/or archegonia. I have made 3 observations which suggest that perceiving the inductive signal for the development of apogamy in C. richardii gametophytes requires a functional archegonia or egg. First, under my culture conditions, the first functional archegonia 72 begin to develop on C. richardii gametophytes between 8 and 10 days of culture. The timing of apogamy commitment (10 days on inductive medium) approximately coincides with this, occurring after 10 days of culture on inductive medium, suggesting the need for mature eggs and/or archegonia, or perhaps “post-functional” eggs in the process of degenerating after not being fertilized. Second, I have never successfully induced apogamy from wild-type male gametophytes of C. richardii. Finally, and perhaps the most convincingly, I have never successfully induced apogamy from the fem1-1tra2 mutant C. richardii gametophytes which posses an “intersex” phenotype, producing normal antheridia with functional sperm, but abnormal archegonia and non-functional eggs (Strain et al., 2001). Thus, it appears that the induction of apogamy depends upon a functional egg and/or archegonium, suggesting that some genes that are expressed in these structures are necessary for the inductive process. Whole Mount in situ Analyses in C. richardii Gametophytes Whole mount in situ analyses were performed in C. richardii gametophytes for 13 sequences from the apogamy library whose homologues in Arabidopsis were preferentially expressed in the seed or flower structures: glycine rich protein, heat shock 70, and RNA recognition motif-containing protein (Fig 17A); FTSH protease, WD40 repeat, 14-3-3- protein, protein kinase CK2 regulator (Fig 17B), UNC93-like protein, nucleolar essential protein, zinc finger (Fig 17C). Also included in the in situ analyses were two apogamy library sequences with interesting identity but were not identified with high confidence: a sequence weakly similar to LEUNIG, a co-repressor of the floral homeotic gene AGAMOUS also involved in lateral organ patterning in Arabidopsis (Liu 73 Figure 18 Whole mount in situs of nucleolar essential protein and RNA recognition motif clones. Nucleolar essential protein antisense probes against 9-day (A) and 12day (B) gametophytes, in which a slight increase in staining intensity is evident. Nucleolar essential protein sense probe control against a 12-day gametophyte is depicted in (C). (D) Antisense RNA recognition motif antisense probe against 9-day (D) and 12-day (E) gametophytes, and sense probe control against a 12-day gametophyte (F). Very little difference in staining intensity is detectable in these samples probes with the RNA recognition motif probe. 74 Figure 19 The BTB and FTSH protease whole mount in situs show preferential expression in early embryo and gametophyte structures. An antisense BTB probe (A) shows staining of an early sporophyte embryo (white arrow) 72 hours post-fertilization compared to sense control (D). Antisense FTSH protease probe against 12-day gametophytes shows intense staining of developing antheridia (B, white arrows) compared to mature antheridium (B, black arrow). Antisense FTSH protease probes also show intense staining of the archegonial cells (E, thick white arrows), but not the egg cells at the base of the archegonia (E, thin white arrow). Rubisco small subunit antisense (C) and sense (F) against 12-day gametophyte prothalli are included here for signal to background comparisons. 75 Figure 20 UNC93-like whole mount in situs show staining in egg cells of 12-day gametophytes. UNC93-like antisense probes against 9-day (A) gametophyte grown on rich medium has little signal, while antisense probes against 12-day gametophyte (B) grown on rich medium shows intense signal in the archegonial cells and the egg cells (white arrows), as well as throughout the prothallus. UNC93-like sense probe against 12-day gametophyte (C) shows no staining in prothallus, archegonia or eggs (white arrows). 76 and Meyerowitz, 1995; Connor and Liu, 2000; Stahle et al., 2009), and a SNF2-like sequence, a family of genes involved in chromatin remodeling and other functions in plants (Knizewski et al., 2008). Additionally, a sequence very similar to a BTB domaincontaining gene in the moss P. patens, with no obvious homologue in Arabidopsis was examined. 10 of these sequences showed no specific expression in gametophyte structures, instead showing signal over the entire prothallus of gametophytes grown on rich medium, and when detectable, an increase in expression from gametophytes cultured for 9 days to gametophytes cultured for 12 days (Fig 18 A-F). The whole mount in situ results for 6 representative sequences, including the 3 that show enhanced expression in specific gametophytic structures, are shown in Figures 18-20. The whole mount in situ results for most of the genes tested had very low levels of staining, presumably due to the low expression levels of the genes, thus were difficult to distinguish from background staining levels. A slight increase in staining could be detected for most genes between 9 day and 12 day gametophytes, as can be seen in the in situ expressions for the nucleolar essential protein clone (Fig. 18A-C) and the RNA recognition motif clone (Fig. 18D-F). However, 3 clones showed specific expression patterns in the structures of the gametophytes, or in early sporophyte embryos developing from gametophytes grown on basal fern medium (Fig. 19 and 20). It is conceivable that genes involved in triggering the process of apogamy may also play a role in embryogenesis derived from syngamy. One sequence examined, encoding a BTB-domain containing protein, was upregulated in the early embryo, 72 hours after fertilization (Fig. 19A). Antisense (Fig. 19C) and sense (Fig. 19F) staining for the Rubisco small subunit transcript are included in this figure to aid the reader in 77 comparisons of positive staining from background staining. There are many BTB domain-containing proteins in Arabidopsis, and it is unclear whether this C, richardii sequence is homologous to any of the genes that encode BTB domain-containing proteins in Arabidopsis. However, this sequence shows 25% amino acid identity and 40% similarity to proteins in both the moss P. patens and the lycophyte S. moellendorfii. This information, together with the evidence of its upregulation in the early embryo suggests a role for this gene in early embryo development, perhaps specifically for lower plants. Another sequence, encoding an FTSH protease, was found to be upregulated in the young antheridia (Fig. 19B) and the archegonia cells (Fig. 19E, thick arrows), but absent or expressed at a lower level in egg cells (Fig. 19E, thin arrows) of 12 day gametophytes grown on 2.5% glucose. Interestingly, the Arabidopsis homologue of this sequence is upregulated in the anthers, pollen and endosperm of Arabidopsis flowers and seeds (Fig. 17B). The possibility that antheridia of ferns and anthers of flowers are homologous structures is not surprising because both are structures in which sperm (or pollen) develop. Perhaps more interestingly, FTSH protease expression in the endosperm of Arabidopsis may suggest an evolutionary relationship between fern archegonial cells and endosperm in angiosperm seeds. Staining for the UNC93-like clone was observed throughout the prothallus of 12 day gametophytes grown on rich medium, compared to 9 day gametophytes (Fig. 20A and B) Also in 12 day gametophytes, intense staining for the UNC93-like probe was detected in the archegonia and the egg cells that sit at the base of the archegonia (Fig 20B, arrows point to eggs) compared to the sense probe (Fig. 20C). This is the only probe that showed whole mount in situ staining of egg cells. 78 Discussion In this chapter I described the creation and analysis of a normalized, subtracted cDNA library that represents genes that are upregulated during apogamy commitment in the gametophytes of the fern C. richardii. Among the clones in this library, 170 sequenced clones could be identified with confidence through BLAST searches of NCBI and JGI databases. I found that the 3 highest represented species in the compiled BLAST top hit data were a moss, P. patens, a gymnosperm, P. sitchensis, and a lycophyte, S. moellendorffii. It is perhaps not surprising that so many genes identified from the apogamy library from C. richardii are most closely related to genes in a moss, lycophyte and gymnosperm, considering the fact that within the land plant phylogeny, the position of the ferns is nested between the branches of these three clades (Chapter 1, Fig. 2). Angiosperms were the clade that represented the most top BLAST hits, with over 38% of the apogamy library genes showing the highest homology. Among the land plant phylogeny, angiosperms are the most divergent from the basal lineages, and the most distant relatives of the ferns (Chapter 1, Fig. 2). The fact that angiosperms comprise the highest percentage of top BLASTx hits may be explained by the overrepresentation of angiosperms, especially dicots, among those land plants that have sequenced genomes, which can bias evolutionary comparisons (Jackson et al., 2006). In fact, 11 out of the 13 land plant families represented among plants with sequenced genomes are angiosperms, which is likely a side effect of, historically, the almost exclusive emphasis on sequencing of plants with small genomes and of economically important plant species (Jackson et al., 2006). There are currently no ferns with sequenced genomes, and only recently P. aquilinum with transcriptome data (Der et al., 2011). It is likely that, if this analysis were 79 repeated in the future when the genome sequences of more lower plants are completed, the results would be dramatically different, showing a greater percentage of sequences most highly related to other ferns and “lower” plants. The GO-slim terms mapped to the apogamy library genes showed a preponderance of terms involved in stress response, metabolism, translation and photosynthesis. A comparison to the GO-slim terms mapped to the entire gametophyte transcriptome of the homosporous fern P. aquilinum, proved these processes significantly enriched in the apogamy library. In addition to these biological processes, the cellular compartments and molecular functions that may be associated with these processes, such as the plastid (metabolism), thylakoid (photosynthesis), vacuole (stress and metabolism), and ribosome (translation), are also over-represented. An over-representation of genes associated with the nucleolus may be interpreted as necessary for rRNA transcription and processing for the ribosomes, but the nucleolus is also implicated in a multitude of RNA processing functions and also the sensing of stress in plants (Brown and Shaw, 2008). It would be ideal to compare the apogamy library to the transcriptome of the C. richardi gametophyte, but the latter is not available for comparison. The only fern gametophyte transcriptome available for comparison is that of P. aquilinum (Der et al, 2009). P. aquilinum is an homosporous fern, and its gametophytes are amenable to apogamy induction and the process of apogamy has been thoroughly studied in this fern using tissue culture techniques (Steeves et al., 1955; Whittier and Steeves, 1960, 1962; Whittier, 1964; Whittier and Pratt, 1971; Whittier, 1975). Additionally, P. aquilinum is relatively closely related to C. richardii, within the very large order of Polypodiales. P. aquilinum is a member of the Dennstaedtiaceae family within the Polypodiales (Smith et 80 al., 2006; Der et al., 2009), which is the sister family to Pteridaceae (Smith et al., 2006). C. richardii is a member of the Pteridaceae family (Hasebe et al., 1995; Pryer et al., 1995, Schneider et al., 2004; Smith et al., 2006; Schuettpelz et al., 2007) and its sister taxa among these ferns are the Acrostichum, the only other aquatic species in this family (Smith et al., 2006; Schuettpelz et al., 2007). The in silico expression analysis of the apogamy library Arabidopsis homologues uncovered a number of genes that were induced or specifically expressed in the flower organs and/or seed structures. It is interesting to consider what those genes are doing in the fern gametophytes. The common ancestor of land plants is likely to have had a multicellular and dominant gametophyte generation (Niklas and Kutschera, 2010), and the current hypothesis is that the evolution of the sporophyte body of land plants occurred through the recruitment of genes involved in gametophyte function of their ancestors. In line with this is the finding that nearly 70% of the genes in the genome of Arabidopsis thaliana have homologues that are expressed in the gametophyte tissues of the moss P. patens (Nishiyama et al., 2003). Of course, the genes in the apogamy library were isolated from a gametophytic system that had been induced to produce sporophytes, so it is not surprising to see genes from this library whose homologues are specific for sporophyte structures in Arabidopsis. The interesting aspect of these observations is the fact that these genes are being expressed in Arabidopsis structures that do not exist in ferns. This may be evidence supporting the scenario that these genes have been recruited from their ancestors to perform functions in the unique sexual structures of flowers in angiosperms. It would be interesting to examine the expression patterns of the P. patens and Arabidopsis homologues of genes showing preferential expression in the C. richardii 81 early embryo (BTB), developing antheridia and archegonial cells (FTSH protease) and egg cells (UNC93-like). This does not preclude the possibility that the flower organs and seed structures of angiosperms have homologous structures in ferns. When examining the expression patterns of the FTSH protease clone, I found that this sequence is induced in the developing antheridia and archegonial cells of 12-day gametophytes grown on 2.5% glucose. The homologue of this gene in Arabidopsis shows upregulation in the anther and pollen as well as in the endosperm of the seed. Pollen is the male gametophyte of an angiosperm. Each pollen grain contains 2 non-flagellated sperms and one generative cell. The pollen initial is formed via meiosis in the anther of the flower, followed by two rounds of mitosis to form the three-celled pollen grains. The antheridia of fern gametophytes is where the sperm develop via mitosis, and in C. richardii each antheridium produces 32 coiled and flagellated sperms after 4 rounds of cell division and subsequent development. These similarities in function suggest that the pollen (and perhaps the anthers) of angiosperm flowers, and the antheridia of fern gametophytes, are homologous structures, and the similarity in genes expression of FTSH protease between the two organisms supports this view. However, while the final products for both are sperm, antheridia and anthers may not go through homologous developmental pathways to reach that end. The FTSH protease may be a gene that is generally involved in mitosis, or some other process that is not specific for the production of sperm. One could also consider the possibility that the archegonia, which form a canal through which the sperm swim to fertilize the egg, which lies at the base of the canal, and the endosperm of seeds, are homologous structures. The archegonium of ferns cradles the 82 developing embryo as it develops, providing nutrients and protecting it until it is independent. Angiosperms lack a morphologically distinct archegonium, but their embryo sacs contain the binucleate central cell, which, as a product of double fertilization, forms the triploid endosperm that nourishes the developing embryo upon seed germination. Upon first comparison it may seem like the nucellar tissue or the integuments surrounding the embryo sac are the analogous structures to the archegnium in ferns (Chapter 1, Fig. 3). But these structures are sporophytic in origin, and as such seem unlikely be analogous to the gametophytic archegonia of lower plants. The origins of double fertilization and the triploid embryo of modern angiosperms has long been an unresolved question in plant biology (Friedman and Williams, 2004). The major questions are: what was the ancestral state of the female gametophyte? Did the earliest flowering plants have double fertilization, and if so, did it create a triploid or a diploid endosperm? The answer to these questions has changed as the resolution of the basal lineages of the angiosperms has become clearer (Williams and Friedman, 2002; Friedman and Williams, 2004). The current view is that the ancestral state of the embryo sac was a 4 celled structure that produced a diploid endosperm upon double fertilization, and that the 7-celled, 8 nucleate embryo sac of modern angiosperms is a reiteration of the 4-celled ancestral structure (Friedman et al., 2003; Friedman and Williams, 2003; Friedman and Williams, 2004). In this scenario it is possible that the embryo sac itself may have evolved from egg cell and a three-tiered archegonium, typical of most archegonial structures in extant ferns (Bell, 1960; Raghavan, 1989). Indeed, the cell that comprises the neck canal of the archegonium, which eventually breaks down to allow sperm to enter, is generally binucleate in extant ferns, but there are some primitive ferns which 83 have mononucleate neck canal cells (Nishida and Sakuma, 1961; Raghavan, 1989). Thus, one can imagine the canal cell as the progenitor of the central cell of the modern day angiosperm embryo sac. The whole mount in situ assays revealed the increased expression of the UNC93like gene in the eggs of the gametophyte prothallus undergoing apogamy commitment. The expression profile of this gene fit the expectation of a gene that is likely involved in apogamy induction in C. richardii, i.e., upregulated during apogamy commitment, specifically expressed or upregulated in the egg cell, and having an embryo-specific expression pattern in Arabidopsis. Furthermore, despite its ubiquitous distribution, the function of this gene is unknown in plants. As such, I chose the UNC93-like gene to investigate further in Arabidopsis. The details of these investigations are in Chapter 4. 84 CHAPTER IV THE UNC93-LIKE GENE IN ARABIDOPSIS AND C. RICHARDII Introduction In the previous chapter I presented evidence of a gene, UNC93-like that has increased expression during apogamy commitment in C. richardii gametophytes. This gene also shows expression in the mature antheridia and eggs of gametophytes grown on 2.5% glucose. My hypothesis is that mature and functional eggs are necessary for C. richardii gametophytes to become capable of apogamy (see discussion in Chapter 3). The temporal and spatial expression pattern of the UNC93-like gene in C. richardii fits the profile of a gene that may be fundamentally involved in the process of apogamy in this fern. Additionally, in silico expression pattern analysis of the UNC93-like homologue in Arabidopsis indicates that this gene is most highly expressed in the embryo, which is the young sporophyte. This suggests a role for AtUNC93-like in early sporophyte growth and/or development. While this does not directly implicate UNC93-like as a gene involved in triggering asexual sporophytes in C. richardii, many genes involved in zygotic embryo development, when mutated or ectopically expressed, induce asexual embryo development (Mayer et al., 1998; Lotan et al., 1998; Hecht et al., 2001; Boutiliier et al., 2002; Okano et al., 2009), so a gene induced during apogamy induction may also play a role in zygotic embryo development. Another possibility is that the UNC93-like gene product is essential for a mature egg and the latter indirectly controls apogamy. It is equally possible that the UNC93-like gene product is essential for the early development of both sexual and apogamous sporophytes. In either case, a better understanding of the 85 function of the UNC93-like gene in Arabidopsis will provide crucial information for how it may function in apogamy in C. richardii. In this chapter I present the characterization of the UNC93-like gene in Arabidopsis thaliana. Its function in plants is unknown. The UNC93 gene was first discovered in Ceaenorhabditis elegans as a gene that encodes a novel transmembrane protein involved in the regulation of muscle contraction (Greenwald and Horvitz, 1980; Levin and Horvitz, 1992). A rare semi-dominant allele of unc93 in C. elegans gives the characteristic sterile, sluggish, and uncoordinated phenotype for which the gene is named, while unc93 null alleles have no phenotype (Greenwald and Horvitz, 1980; Levin and Horvitz, 1992). Suppressors of the unc93 phenotype in C. elegans, sup9 and sup18, encode a two-pore potassium channel and a novel transmembrane protein, respectively, suggesting that unc93 is a component of a multiprotein complex involved in potassium transport (Greenwald and Horvitz, 1980, 1986; Levin and Horvitz, 1993; De Stacio et al., 1997, de la Cruz et al., 2003). Two other suppressors of the unc93 dominant mutation encode a chromatin remodeling factor and an inorganic phosphatase (Long and Horvitz, 2006). An UNC93::GFP fusion protein is found in the membranes of the C. elegans body wall, vulval, anal, and intestinal muscles, as well as in dense bodies, which are structures that perform structural and signaling functions in muscle cells (de la Cruz et al., 2003; Lecroisey et al., 2007). The UNC93::GFP construct is also found in 4 muscle neurons in the head (de la Cruz et al., 2003). In humans, there are two UNC93 paralogs. UNC93A is expressed in the testis, ovary, small intestine and spleen, but the function of this gene has not yet been elucidated (Liu et al., 2002). The UNC93B1 gene is upregulated in the heart and antigen-presenting 86 cells (Isnardi et al., 2008) This gene is involved in immunity by regulating nucleotidesensing Toll-like receptors. UNC93B1 transports a subset of these receptors from the ER to endolysosomes where they activate signaling cascades and engage pathogens. (Isnardi et al., 2008). Potassium is the most abundant cation in plants, and unlike other soil cations, it is not incorporated into the organic matter of the plant, instead serving as a major osmoticum (Ho and Tsay, 2010). The physiological responses to potassium starvation include growth arrest, impaired phloem transport of sucrose, and reduced water content (Ho and Tsay, 2010). The reduced water content decreases turgor pressure, which is required for cell expansion in the elongation zone of roots (Dolan and Davies, 2004). Plants, therefore, have a number of root morphology responses to potassium starvation including reduced primary root growth, decreased lateral root length and number, and increased root hair elongation (Jung et al., 2009, Shin and Schachtman, 2004). How plants detect and respond to changing potassium levels is not fully understood, but some components of the potassium deficiency response have been elucidated. Under low potassium conditions, an influx potassium channel, AKT1, and a high affinity potassium transporter, HAK5, are responsible for potassium uptake (Rubio et al., 2008; Gierth et al., 2005). ROS production by NADPH oxidase and a type III peroxidase mediates the transcription of the HAK5 gene (Shin and Schachtman, 2004; Kim et al., 2010). ROS production, the activation of HAK5, as well as the promotion of root morphological responses, are eliminated in the absence of ethylene and in ethylene insensitive mutants, indicating an important role for this gaseous plant hormone in potassium sensing (Jung et al., 2009). 87 The internal transport of potassium relies on other potassium channel proteins that are incorporated into the membranes and envelopes of organelles. The Arabidopsis genome contains 15 genes whose proteins are capable of forming potassium channels (Lebaudy et al., Szczerba et al., 2009). Based on their structure, there are 3 classes of proteins: 9 relatively well characterized Shaker-like K+ channels, 5 tandem pore K+ (TPK) channels, and 1 K ir -like (KCO3) channel (Maser et al., 2001; Very and Sentenac, 2003; Ward et al., 2009; Voelker et al., 2010). The latter 6 proteins are targeted to the vacuolar membrane (Czempinski et al., 2002; Schonknecht et al., 2002), with the exception of TPK4, which is targeted to the plasma membrane and the ER (Voelker et al., 2006; Latz et al., 2007). The proteins likely form homodimers (Voelker et al., 2006) and most of them interact with 14-3-3 proteins in a phosphorylation-dependent manner (Latz et al., 2007), which is also true for their animal orthologues (Plant et al., 2005). Many details of potassium sensing, signaling, and transport in plant cells have yet to be elucidated. In this chapter I describe two transcripts that originate from the sole AtUNC93like gene in the Arabidopsis genome, and examine their spatial distribution in various Arabidopsis tissues. I also describe the phenotypes of an AtUNC93-like T-DNA insertional mutant line, detailing a number of vegetative, root, and reproductive defects. Based on the prediction that the C. elegans UNC93 protein regulates a two-pore potassium channel, I performed a number of experiments to investigate the possibility that AtUNC93-like is involved in K+ uptake or sensing, and whether K+ plays a role in the induction of apogamy in C. richardii gametophytes. My experiments and observations reveal little evidence that the AtUNC93-like gene is involved in K+ uptake 88 in Arabidopsis, or that K+ plays a role in apogamy beyond supplying an essential nutrient for growth. I do find, however, that the AtUNC93-like gene is involved in embryo development, as a proportion of seeds undergo abortion before maturity, and that the homozygous T-DNA plants are unable to maintain the viability of a proportion of their gametophytes, whether male or female, but AtUNC93-like does not appear to play a critical role in gametophyte function. Materials and Methods Plant Materials and Growth Conditions Arabidopsis wild-type plants utilized in this study were Columbia ecotype. Seeds for the AtUNC93-like homozygous T-DNA line, SALK_010430 were acquired from the Arabidopsis Biological Resource Center (ABRC; Alonso et al., 2003). Homozygous TDNA lines for the 6 tandem-pore potassium channel genes in the Arabidopsis genome, two of which are putative orthologs to sup9 and sup10 in C. elegans, were obtained from ABRC. These lines are: SALK_146903 (TPK1), SALK_007975 (TPK2), SALK_049137 (TPK3), SALK_053858 (TPK4), SALK_123690 (TPK5), SALK_048607 (KCO3). The C. richardii her mutant gametophytes were used for 3’ RACE and degenerate PCR, and the self-sterile fem mutant was used for the apogamy induction experiments (Banks, 1994). When grown in soil, Arabidopsis seeds were cold-treated at 4°C for 2-3 days on filter paper soaked with 10 mM KNO3, and then sown on plant mix # 1 (Beautiful Land Products, West Branch, IA). When cultured on agar medium, unless [K+] was varied, both Arabidopsis plants and C. richardii gametophytes were grown on ½ strength MS medium (pH 5.7 for Arabidopsis and pH 6 for C. richardii), supplemented with 0.5% 89 sucrose and 50 µg/mL ampicillin for the prevention of contamination. All Arabidopsis growth conditions were under a 16-hour light, 8-hour dark schedule at 21-25°C. C. richardii gametophytes were cultured under continuous light (100 µM m2 s-1) in a 28°C incubator. For Arabidopsis seed sterilization, seeds were agitated in 0.1% Tween 20 for 30 minutes, soaked in 100% ethanol for 2 minutes, 1.5% Na hypochlorite for 5 minutes, rinsed 5 times in sterile H 2 O, and plated on the appropriate medium. Plates were stored in the dark at 4°C for 2 days to stratify seeds before placing under the light conditions described. C. richardii spores were sterilized as previously described (Cordle et al., 2007), and plates dark treated for 3 days at room temperature before moving plates to the incubator conditions described above. Bioinformatics The Arabidopsis UNC93-like protein sequences were acquired from the NCBI protein database, and the AtUNC93-like predicted alternate transcript sequences are from The Arabidopsis Information Resource (TAIR). Amino acid alignments were generated with ClustalW2 (Larkin et al., 2007) with default parameters, and the UNC93-like domain boundaries were defined with information from the Conserved Domain Database (Marchler-Bauer et al., 2011). Transmembrane domain predictions and N- and C-terminal domain conformations were generated with the PredictProtein program (Rost et al., 2003). Potential protein interactions with Arabidopsis UNC93-like protein were generated with the GeneMania program (Warde-Farley et al., 2010). 90 Root growth assays, Fresh Tissue Weights and K+ in Apogamy For the root growth assay, Arabidopsis seedlings were grown on agar plates as described above, with plates held steady at a 90° angle from horizontal. Photographs were taken on the given days with the DinoXcope 1.4 (AnMo Electronics Corporation). For obtaining fresh weights of shoots and roots, Arabidopsis seeds were sterilized, plated on appropriate medium, and cold-treated for 2 days. After moving to light, plates were positioned vertically and plants were grown for 15 days before removing tissue for weighing. Aerial tissue was excised from roots; all tissues were blotted gently to remove any moisture, and immediately weighed in batches. The experiment examining the role of K+ in apogamy induction from C. richardii gametophytes was performed on 0.8% agar and ½ strength MS medium, either with or without the addition of 2.5% glucose. Gametophytes were transferred to fresh plates every 14 days to prevent drying conditions, and total apogamy counts were made after 45 days of culture. Modifications were made to the [K+] by replacement of KNO 3 and KH 2 PO 4 in the MS medium with NH 4 NO 3 and NH 4 H 2 PO 4 , respectively. KCl was added to the medium to raise [K+] above that found in ½ MS medium (above 10 mM). There is a trace amount of K+ in the agar used to solidify the medium (Xu et al., 2006), so the 0 mM K+ plates refer to the lack of K+ in the MS medium, but not a complete lack of K+ altogether. K+ - depleted medium for Arabidopsis plants was made in the same fashion except 0.5% sucrose was added and the pH was adjusted to 5.7. 91 Arabidopsis Seed Area and Seed Weight For data on seed size, photographs of mature WT and unc93-like seeds were taken with the DinoXcope camera (AnMo Electronics). Images were processed with ImageJ. Briefly, images were converted to binary, threshold adjusted, and areas of outlined seeds were measured. Seed area measurements are an average of 25 seeds for each WT and unc93-like. Seed weights were obtained by measuring 10-20 mg of mature seeds, counting the number of seeds, and dividing the weight by the number of seeds. Over 400 WT seeds and 800 unc93-like seeds are represented in this experiment. RT-PCR, 3’ RACE, PCR, and Degenerate PCR For RT-PCR, degenerate RT-PCR, and 3’ RACE, RNA extraction was performed by grinding frozen tissues in liquid nitrogen to a fine powder and extracting RNA using the guanidinium thiocyanate and acid phenol method (Chomczynski and Sacchi, 1987) Before precipitation with isopropanol, 0.4x volume of a high salt solution (0.8M sodium citrate, 1.2M sodium chloride) was added to minimize the precipitation of polysaccharides. The RNA pellet was dissolved in freshly deionized formamide, heated to 70ºC for 10 minutes in the presence of 0.7M NaCl and 1% CTAB, and extracted twice with chloroform to remove precipitated polysaccharides. After adding 1x volume of DEPC-treated water, total RNA was recovered with standard ethanol precipitation. RNA quality and quantity was analyzed with the NanoDrop 1000 (Thermo Scientific) and native gel electrophoresis. 300 ng of total RNA was used for cDNA synthesis with the Superscript III enzyme (NEB) using 9-nucleotide random primers, for RT-PCR reactions, and with the modified oligo (dT) primer for 3’ RACE (Table 3). 1 µL of the resulting cDNA was used undiluted in subsequent 25 µL PCR reactions. For 3’ RACE, a 92 subsequent PCR was performed with nested primers and a 1/500 dilution of the original PCR products. The degenerate RT-PCR product and 3' RACE products were cloned into the pCRII vector (Invitrogen) and transformed into TOP10 (Invitrogen) E. coli cells. Plasmid DNA was isolated and purified with the Wizard Plus Minipreps DNA Purification System (Promega), and submitted for sequencing on the ABI 3730 DNA analyzer (Applied Biosystems) at the Center for Comparative Genomics (University of Iowa). For the determination of the genotypes of the reciprocal cross progeny, genomic DNA was extracted from progeny. A single leaf was ground in a centrifuge tube with a pestle and 600 µL of extraction buffer added (200 mM Tris-Hcl pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) and briefly vortexed. Plant debris was pelleted in a tabletop centrifuge for 1 minute and supernatant transferred to a fresh tube. 500 µ of isopropanol was added and the mixture incubated at room temperature for 2 minutes. DNA was spun down in a table-top centrifuge for 5 minutes. The pellet was dried gently and resuspended in 100 µL TE. 1µl of the resulting DNA was used in 2 separate PCR reactions for each plant: one using the AtUNC93-like gene specific RP and LP primers (amplicon indicates a WT allele present in plant) and one using the AtUNC93-like RP and a primer specific for the T-DNA border (amplicon indicates a T-DNA allele). PCR products were separated on 1% agarose gels and visualized with ethidium bromide staining and UV imaging. 93 Table 3 Primers used for RT-PCR, 3’ RACE, PCR and degenerate PCR Sequence Name Primer Sequence 3’RACE gene specific 5’-CAGTCTGGGAAGGTCGATTC-3’ 3’RACE nested gene specific 3’RACE modified oligo (dT) 3’RACE adapter specific 1 3’ RACE adapter specific 2 5’ gene specific 5’-CCTGTTACAACTTGGCCTCTT-3’ 5’ degenerate 5’-TGGCATATCATGCAGCAcaraayytnsa-3’ AtUNC93-long LP 5’-CAGTTGAAGAAACACATTTCACG-3’ AtUNC93-long RP 5’-TCGAATCTGAATGACATGTTGAG-3’ AtUNC93-short LP 5’-GAATGTATTTACCCACGACGAA-3’ AtUNC93-short RP 5’-AGTTGCATACTTCCCAAGTCTC-3’ AtUNC93-both LP 5’-GCTGTTTGGGAATCTCATCAC-3’ AtUNC93-both RP 5’-AACTCAGCCCAAACAAATGC-3’ 5’-CAGTGATAACGCAGCATACAGGTGAGATGAG(T)5-3’ 5’-CAGTGATAACGCAGCATACAG-3’ 5’-GCAGCATACAGGTGAGATGAG-3’ 5’-TTCCAATTATCTGGTTCGATGC-3’ Alexander staining, Tissue clearing, Optics and Photography Alexander staining was utilized for detecting pollen abnormalities. The anthers of freshly opened flowers were removed from plants and placed on microscope slides. 3 drops of Alexander stain (20% ethanol, 0.05% malachite green, 0.25% Fuchsin acid, 0.025% orange G, 10% (w/v) phenol, 10% (w/v) chloral hydrate, 0.7 M glacial acetic acid, 50% glycerol) were placed on top of the anthers and gentle pressure was applied to 94 push the pollen out of the anthers. Slides were imaged after a 15-minute incubation at room temperature. For Hoechst-dye staining of pollen grains was performed as described in Park et al., 1998. Briefly, freshly opened flowers were placed into an eppendorf tube with 300 µL of extraction buffer (0.1% Triton X-100, 0.1 M sodium phosphate, 1 mM EDTA, 0.8 µg/mL Hoechst 33342), vortexed briefly, and then spun in a tabletop centrifuge for 1 minute. The pollen pellet was transferred to a slide and a coverslip added. After a 30-minute incubation in the dark, pollen was imaged via brightfield and UV epiillumination with a Leica DMRB microscope. Images were imported into and processed in Adobe Photoshop CS4 (Adobe Systems Inc.). For imaging, siliques of various ages were removed from plants, fixed under vacuum for 15 minutes in ice-cold FAA. Fresh FAA was added, and siliques were kept overnight at 4°C in this solution. Siliques were then moved through an ethanol series at room temperature and finally immersed in chloral hydrate solution (2.5 g chloral hydrate in 1 mL of 30% glycerol). Siliques were allowed to clear in this solution for 2 days, then pictures were taken with a DinoXcope camera (AnMo Electronics). Results CrUNC93-like and AtUNC93-like Proteins are Conserved The original CrUNC93-like clone from the apogamy library was 220 nucleotides long. After performing 5’ RACE and amplifying the 3’ region of the transcript using a sequence-specific primer along with a degenerate primer (see Materials and Methods), a CrUNC93- like transcript sequence of 1149 nucleotides was obtained that spanned the UNC93 domain through the 3’ portion of the transcript (Fig. 21A). From the Arabidopsis 95 Figure 21 UNC93-like protein alignments and Arabidopsis UNC93-like gene structure. The C. richardii UNC93-like predicted protein sequence aligned with the Arabidopsis “Short” and “Long” UNC93-like proteins from alternative splicing (A). The black bar in alignments indicates the UNC93 domain, amino acids in red are predicted transmembrane domains, and “I” and “O” designate predicted inside and outside plasma membrane domains for the Arabidopsis (top) and C. richardii (bottom) proteins. The blue dashes at the N-terminal region of the C. richardii protein sequence indicate the region for which no amino acid sequence may be inferred because the transcript sequence is unknown. (B) is a diagram of the AtUNC93-like gene and transcripts. Blue boxes are exons, light blue boxes are 5’- and 3’- 96 UNC93-like gene, two alternate transcripts are produced, of which the longer transcript is predicted to code for a protein with 11 transmembrane domains, and a shorter transcript that is predicted to code for a protein with 12 transmembrane domains (Fig. 21A and B). Based on the conformation of the proteins in C. elegans and humans (Levin and Horvitz, 1992; Isnardi et al., 2008), and the PredictProtein algorithm (Rost et al., 2003) the larger AtUNC93-like protein likely is inserted into the membrane with its N-terminal domain inside the membrane, and the C-terminal domain on the outside (Fig. 21A). In contrast, the smaller AtUNC93-like protein likely has both its N-terminal and C-terminal domains inside the cell or organelle (Fig. 21A). The predicted amino acid sequence of the CrUNC93-like protein aligns with the large and the small Arabidopsis proteins with 37.9% identity, 52.1% similarity, and 39.1% identity, 54.3% similarity, respectively (Fig. 21A). Within the UNC93 domain, the C. richardii proteins are 56.9% identical and 83.7% similar, indicating a high degree of conservation for this domain. The CrUNC93like protein is predicted to encode a protein with 10 transmembrane domains, with both N-terminal and C-terminal domains outside of the cell (Fig. 21A). Note that the entire 5’ sequence of the CrUNC93-like transcript has not been elucidated (Fig. 21A). It is likely that this 5’ sequence would encode another transmembrane domain, as is present in both the small and large Arabidopsis UNC93-like proteins. It can be surmised, then, that the CrUNC93-like protein encodes a protein with 11 transmembrane domains, with the Nterminal domain inside the membrane and the C-terminal domain outside the membrane, which is a topology more similar to the large UNC93-like protein in Arabidopsis (Fig. 21A). 97 Figure 22 Phenotypes of a homozygous T-DNA insertion mutant of AtUNC93-like. (A) Primers specific for the long and short AtUNC93-like transcript amplify both (black arrows) from the flower (F), leaf (L), and root (R) tissues of WT plants, but none in those same tissues from unc93-like (T-DNA). Primers that amplify both transcripts, that hybridize upstream of the T-DNA insertion, detect UNC93-like transcript in both flower and root of WT and unc93-like (black arrows) (B) unc93-like plants are smaller, darker green, and have shorter petioles (arrows) than WT. (C) unc93-like roots produce more lateral roots than WT plants. (D) wavy root phenotype, compared to WT, of unc93like roots grown on 90° vertical plates. Bar = 1 cm in all pictures except (C) where white bar = 1mm 98 Figure 23 Number of lateral roots from plants grown on medium containing varying concentrations of K+. Bars are standard error, and asterisks indicate significance (Student’s t-test, p < 0.05) 99 Vegetative Phenotypes of the Arabidopsis unc93-like Mutant The Arabidopsis T-DNA insertional mutant line SALK_010430 is homozygous for a T-DNA insertion into the AtUNC93-like gene near the junction of the 8th exon and 8th intron (Figure 21B). This T-DNA insertion disrupts the coding region of both the large and small transcript for this gene. Accordingly, using two sets of primers specific for the large and small transcripts, respectively, reverse transcriptase PCR failed to detect either transcript in this mutant. Notably, both sets of primers are downstream of the TDNA insertion. However, using a set of primers upstream of the T-DNA insertion, in a region that is shared by both transcripts, an amplicon was detected with RT-PCR (Figure 22A). This may indicate that this T-DNA line is not a knockout mutation, instead generating a truncated transcript and possible truncated protein that may provide partial function or dominant negative effects. This T-DNA mutant line displays a number of vegetative and reproductive phenotypes (Figure 22). The mutant plants have smaller leaves and shorter petioles thus have a smaller circumference than wild-type (WT) plants (Figure 22B). Additionally, the T-DNA plants are dark green compared to WT plants (Figure 22B), and are 1-2 weeks delayed in development compared to WT plants, having on average the same number of leaves at flowering time as WT plants (unc93 11.5 ± 0.63, WT 11.8 ± 0.85, n = 10; t = 0.896, p > 0.05). When grown on sterile agar plates, unc93-like plants produce more lateral roots than WT plants (Figure 22C). Therefore, the mutant plants, while having less aerial tissue than WT plants, were proliferating more root tissue than WT plants. This was verified in 100 Figure 24 Fresh root and shoot weights of unc93-like and WT plants. Bars indicate standard error and asterisks indicate statistical significance (Student’s ttest, p < 0.05). 101 two ways, by directly counting number of lateral roots emerged from the main root of plants grown vertically for 12 days on agar medium with varying concentrations of K+ (Fig. 23) and by measuring the fresh tissue weight of WT and unc93-like shoots and roots after 16 days of growth on agar medium with 10 mM K+ (Fig. 24). It was found that unc93-like plants develop significantly more lateral roots than WT plants when grown on agar medium for 12 days (Fig. 23). Furthermore, varying amounts of K+ in the medium does not appear to affect this phenotype, with unc93-like plants consistently developing more lateral roots than WT plants in all conditions except with no added K+, on which neither unc93-like nor WT plants developed roots (Fig. 23). The fresh root and shoot weight of unc93-like and WT plants were determined after 16 days of growth on agar medium (Fig. 24). WT plants have significantly more aerial tissue at this time period than unc93-like plants, but the root tissue weights did not differ significantly (Fig. 24). Since it is visually clear that unc93-like plants have more extensive lateral roots than WT plants after 10 or more days of growth (Fig. 22C), it is surprising that the difference is not significant with regard to weight. Roots are very thin and delicate structures, and this type of analysis may not be sensitive enough to detect the putative difference in weight between the approximately 40 plants of each measured. Alternatively, while WT roots do not grow longer than unc93-like roots (data not shown), they may be thicker, which would explain the discrepancy between these two experiments. In addition to having less aerial tissues and more lateral roots, unc93-like roots consistently grew in a wavy pattern on vertical agar medium (Figure 22D). Root waving, as well as other unusual root growth patterns, can be induced from WT roots by tilting agar plates 30° from vertical (Migliaccio et al., 2009). This is a well-characterized root 102 Figure 25 Phenotypes of kco3 and tpk1 potassium channel mutants on 0 mM K+ differs from unc93-like and WT plants. (A) kco3 and (D) tpk1 mutant plants grow roots in the presence of extreme K+ starvation. This phenotype differs from that of unc93-like plants (B and E) or WT plants (C and F). 103 response to the mechanical stimulation (Migliaccio and Piconese, 2001) of penetrating the agar medium. However, my growth conditions had the agar plates completely vertical, such that roots did not penetrate the agar medium, and WT plants grown in parallel or on the same plates as unc93-like plants did not show wavy root growth (Figure 22D). Moreover, this was a phenotype observed consistently when growing the plants on agar medium, even when WT and unc93-like were cultured on the same plates (data not shown). This suggests that this is a phenotype caused by the unc93-like mutation rather than a normal thigmomorphogenic response to growth conditions. Comparison of unc93-like Phenotypes with K+ Channel Mutants I also obtained homozygous T-DNA lines for the 5 TPK and KCO3 potassium channel-encoding genes in the Arabidopsis genome, to investigate whether any of these putative knockout lines phenocopy the unc93-like mutant. I grew plants in soil as well as on agar medium with optimal and suboptimal potassium concentrations. I found that the plants of all 6 potassium channel mutant lines have phenotypes indistinguishable from WT plants (data not shown). Except in the case of growth on 0 mM potassium agar plates, where tpk1 and kco3 produced more roots than the unc93-like mutant or WT plants (Fig. 25). The significance of this is unknown, but it may indicate that these 2 potassium channel mutants have a defect in potassium signaling, and produce roots even in the absence of K+. Potassium signaling is normally required for root growth (Dolan and Davies, 2004). Other than this phenotype in tpk1 and kco3, all of the potassium channel mutant lines had phenotypes that resembled WT plants, thus did not phenocopy unc93like. 104 Reproductive Phenotypes of Arabidopsis unc93-like Mutant The unc93-like plants also exhibit abnormal reproductive phenotypes. Seeds from unc93-like plants are significantly smaller in size than WT seeds (Fig. 26). Accordingly, the unc93-like seeds, at 61.4 seeds/mg are also almost 27% lighter in weight than WT seeds, which contain 45 seeds/mg. This could be indicative of the retardation of embryo or endosperm development. Additionally, siliques from unc93-like plants have 27.6% fewer seeds than WT plants (Fig. 27A). While some of those missing seeds are caused by failed fertilizations, suggesting a possible gametophytic effect for this gene, there is also evidence of seed abortion in unc93-like mutant siliques (Fig. 27B), also implicating a role for the AtUNC93-like gene in early sporophyte development. To examine whether there is a male gametophyte defect in the unc93-like mutant, I examined pollen viability with Alexander staining (Alexander, 1969). This method stains the pollen coats green and the cytoplasm of viable pollen dark red. Aborted pollen appears as empty green spore coats. Alexander staining of pollen from unc93-like plants showed a large fraction of pollen in this line that has aborted, as indicated by empty green pollen coats lacking functional (red) cytoplasm (Fig. 28B), while WT pollen did not display any obvious defects (Fig. 28A). Arabidopsis pollen normally contains 2 sperm cells and a generative cell. To examine whether there was any nuclear defect in the pollen of the unc93-like mutant, I stained WT and unc93-like pollen with Hoechst 33334 dye and imaged the pollen under epi-fluorescence (Fig. 28C and D). I found no obvious nuclear abnormalities in the viable pollen grains of WT or unc93-like mutants, as indicated by the 105 Figure 26 Seed sizes of unc93-like and WT plants. Bars indicate standard error and asterisk indicates significance (Student’s t-test, p < 0.05). 106 Figure 27 unc93-like plants have fewer seeds in their mature siliques than WT plants. (A) The seeds from 16 siliques for both WT and unc93-like plants were examined, representing 5 separate plants for each. Bars indicate standard error and asterisk indicates significance (Student’s t-test, p < 0.05) (B) Siliques of unc93-like plants have empty regions (white arrow) indicative of failed fertilizations, and shriveled seeds (black arrow) indicative of embryo abortion. 107 Figure 28 Examination of pollen viability in the unc93-like mutant. (A) WT pollen stained with Alexander stain showing no defective pollen grains. (B) Pollen abortion in the unc93-like mutant plant revealed by the presence of empty spore coats. (C) Staining of WT pollen nuclei with Hoechst dye showing two, brightlystaining sperm cells and one diffuse vegetative nucleus in each. (D) Mature unc93-like pollen does not have any obvious nuclear defects. 108 presence of 2 densely staining sperm nuclei and one diffusely staining vegetative cell nucleus in all pollen imaged (Fig. 28C and D). Based on the Alexander stain evidence that there is pollen abnormality and failed fertilization events in unc93-like siliques, I investigated the possibility that the mutant unc93-like allele affects female and/or male gametophyte function. I crossed WT plants to homozygous T-DNA plants to generate plants heterozygous for the T-DNA allele (TDNA +). Then, I performed reciprocal crosses with flowers from these heterozygous plants and those of WT plants. If gametophyte function is not affected, then I would expect to see a 1:1 ratio of T-DNA +: ++ plants in the next generation for both crosses. If female gametophyte function is negatively affected by the T-DNA allele, I would expect to see fewer heterozygous plants from the cross using heterozygotes as female. If male gametophyte function is affected, then I would expect to see a reduction in the 1:1 ratio of T-DNA+: ++ plants from the cross that uses heterozygotes as the male donor. The genotypes of these F 1 plants from these crosses were determined with PCR. It is important to note that siliques generated from the cross using heterozygotes as females resulted in approximately 2 times fewer seeds than those crosses using heterozygotes as males (Fig. 29). There is no obvious difference in flower architecture between heterozygous plants and WT plants to explain this difference (data not shown), so it suggested a female gametophyte deficiency. However, this is not the case. The T-DNA allele is passed with equal efficiency through both the Arabidopsis female and male gametophytes, with a ratio of 46:50 (T-DNA+: ++) F1 plants from the heterozygous female cross, and a 51:55 (T-DNA+: ++) F1 plants from the heterozygous male cross. Neither of these is significantly different than a 1:1 ratio (X2 test; p > 0.05). These results 109 Figure 29 Significantly fewer seeds produced from crosses performed with females heterozygous for the unc93-like mutant allele. Bars indicate standard error, asterisk indicates significance (Students t-test, p < 0.0001) 110 Figure 30 The effect of K+ concentration on apogamy induction in C. richardii. Gametophytes were grown on medium with varying concentrations of potassium in the presence or absence of 2.5% glucose. Asterisk = statistical significance (Student’s t-test, p < 0.05). Glc refers to rich medium, BFM refers to basal fern medium. The agar naturally contains a small amount of K+, and 0 mM indicates no added K+ to the medium. Empty set symbol indicates that all gametophytes died. 111 seem to be in conflict with the following two observations. First, there are significantly fewer seeds in the siliques from the crosses of heterozgous females to the WT males and second, that I have already detected a pollen defect in unc93-like plants, suggesting that the unc93-like allele affects pollen function. Based on the reciprocal cross results, the AtUNC93-like gene does not appear to be necessary for gametophyte function. Instead, just the presence of a mutant allele in the parent plant (of either pollen or embryo sac) renders equal numbers of the gametophytes of both sexes inviable, regardless of which allele the gametes carry. This suggests that the unc93-like allele creates a maternal environment that negatively affects the viability of both male and female gametophytes. The Role of K+ in the Induction of Apogamy from C. richardii Gametophytes The UNC93 protein in C. elegans is predicted to associate with components of a two-pore potassium channel, perhaps performing a regulatory role for potassium uptake (de la Cruz et al., 2003). Based on this information, I performed an experiment with C. richardii gametophytes to determine if potassium plays a role in apogamy induction in this fern. I cultured fem mutant gametophytes, from which there is no risk of selffertilization because they do not form sperm, on 2.5% glucose or no glucose plates, varying in the concentration of potassium. I then tallied the percentage of gametophytes that formed apogamous structures after 45 days of growth. I found that, grown on medium containing 2.5% glucose (previously shown to be inductive for apogamy), in the absence of potassium, an average of 8% of gametophytes produce apogamy after 45 days (Fig. 30). Note that there is a trace amount of potassium in the agar used for solidifying the media, enough for the gametophytes to survive and proliferate, albeit to a much lesser 112 extent than at higher potassium levels. This percentage increases significantly to 55% and above 70% for 5mM potassium and 10mM potassium, respectively (Fig. 30). 10mM potassium is the optimum concentration of potassium under normal growth conditions of 1/2x MS medium. Potassium concentrations 2 times higher than 10 mM produce a slightly lower percentage of apogamy in the presence of 2.5% glucose, and gametophytes grown on glucose with potassium concentrations higher than 20 mM do not survive, presumably due to osmotic effects (Fig. 30). On basal fern medium, with no glucose, approximately 5% of gametophytes produce apogamy after 45 days of culture. This percentage increases to between 15% and 30% for gametophytes grown on medium containing potassium concentrations between 5 mM and 20 mM, and reduces on 50 mM potassium, and gametophytes do not survive on higher concentrations of potassium (Fig 30). Therefore, while there is a significant increase in the incidence of apogamy with increasing potassium concentration on rich medium, there is no corresponding difference in the incidence of apogamy on basal medium. This suggests that potassium does not play a role in the induction of apogamy, or that potassium plays a role in apogamy when combined with glucose. An alternative view would be that potassium is necessary for the induction of apogamy by glucose, although this effect would be difficult to distinguish from the role of potassium in general plant cell growth. Discussion I have described in this chapter experiments investigating the role of the UNC93like gene in Arabidopsis. This gene was isolated from C. richardii gametophytes committed to apogamy, and identified as being induced in the embryos of Arabidopsis 113 based on in silico analysis. In C. richardii gametophytes, UNC93-like is induced in the female reproductive structure, the archegonia, and in egg cells. The AtUNC93-like gene produces two transcripts, and the expression level of the large transcript is increased in Arabidopsis embryos according to in silico analysis using Genevestigator (Zimmerman et al., 2004; Chpt. 3, Fig. 17C). Conflicting results were obtained using another probe on the ATH genome microarray chip. Both transcripts of the AtUNC93-like gene are detected with this second probe set which indicates expression in nearly all tissues of the plant with upregulation in the roots (data not shown). The CrUNC93-like protein more closely resembles the protein encoded by the large AtUNC93-like transcript based on transmembrane domain predictions. It is unknown whether the CrUNC93-like gene has multiple mRNA splice forms. If it does, the alternate forms are unlikely to be expressed in the gametophytes undergoing apogamy commitment because 3’RACE and degenerate PCR experiments amplified only one sequence from this fern tissue. It is also unknown whether CrUNC93-like is represented by only one gene in the fern genome. The Arabidopsis genome contains only one gene, At3g09470, which encodes a protein with an UNC93 domain. Because the UNC93 protein namesake in C. elegans is predicted to regulate a multi-component two-pore potassium channel, I examined how the unc93-like mutant is affected in responses to K+ starvation. I found that the unc93-like vegetative phenotypes are unchanged when the plants are K+ challenged, including the production of excess lateral roots. Furthermore, the unc93-like plants do not phenocopy tpk1-5 or kco3 mutant plants, which resemble WT plants in all respects except the ability to form roots on medium lacking potassium. The fact that these two mutants, tpk1 and kco3, have a 114 phenotype that differs from both WT and unc93-like plants suggest that the UNC93-like protein is not involved in the regulation of these two potassium channels. However, all five of the TPK proteins are likely to form heterodimers in vivo (Voelker et al., 2006), and the expression patterns of most of these genes significantly overlaps (Deeken et al. 2003; Philippar et al. 2003). The WT phenotypes of these mutants may suggest that they can compensate for each other when one of the genes is mutated. The creation of double or more mutant lines for these genes may reveal phenotypes that are similar to the unc93like mutant. Lateral root formation and length is inhibited in WT plants when exogenous K+ levels are limited (Jung et al., 2009, Shin and Schachtman, 2004). The fact that the unc93-like mutant continues to make excessive lateral roots, compared to WT plants, even in K+ starvation conditions is consistent with UNC93-like playing a role in K+ uptake, sensing, or signaling. Additionally, the small aerial tissues of unc93-like plants may be an indication that the mutant plants are not receiving enough K+ to proliferate. The unc93-like mutants have approximately 27% fewer seeds in their mature siliques than WT plants. Analysis of developing seeds in young siliques revealed that this reduced seed set is due to fertilization failures and seed abortion combined. In contrast, reciprocal crosses between plants heterozygous for the T-DNA insertion showed equal transmission efficiency through both male and female gametophytes. Combining these two seemingly contradictory results, the most plausible explanation is that, either one or both, fertilization and seed development was impaired when the female donor was the heterozygote. In agreement with this explanation, a fraction of the pollen grains of unc93like homozygous mutants aborted. These data may be explained by UNC93-like protein 115 being involved in the ability of a parent plant to maintain viable gametophytes, while not being essential for gametophyte function, per se. It is conceivable, but unlikely, that a reciprocal cross of this type would provide evidence of a low overall functional pollen count for a male donor. The procedure for manually crossing Arabidopsis flowers involves the copious application of donor pollen to a stigma—more than enough to fertilize the ~50 embryo sacs within a given gynoecium. Therefore, fewer embryo sacs will result in fewer seeds, regardless of how much pollen is applied to a stigma. An experiment was performed in which the role of potassium concentration on the induction of apogamy in C. richardii gametophytes was examined. There was a slight increase in the percentage of apogamy induced from gametophytes on basal medium as the potassium concentration increased to 20 mM. However, this increase is not statistically significant. A much larger increase in the percentage of gametophytes producing apogamy was obtained with increasing potassium concentration in the presence of 2.5% glucose. The peak percentage of apogamy was reached, around 70%, at10 mM K+ in medium supplemented with glucose, which is the optimal concentration of K+ in standard plant media. C. richardii fem1 gametophytes have displayed a similar peak level of apogamy in previous experiments when cultured on glucose (Chpt. 2, Fig. 11). In those experiments, basal levels of apogamy (induced from medium that does not contain sugars) never exceeded 5%, so it is notable that over 20% of gametophytes exhibited apogamous structures in this experiment on basal medium with potassium concentrations between 5 and 20 mM. Previous experiments were conducted on 10 mM potassium-containing medium. The high level of apogamy on basal medium may be attributed to some other cultural conditions between the experiments. Possible sources of 116 variation include different light bulbs in the incubation chamber, or the freshly acquired MS medium that was used for the experiments in this chapter. Varying light conditions and nutrient levels are known to affect apogamy induction in some ferns (Raghavan, 1989). Alternatively, it may be that potassium is necessary for the induction of apogamy by glucose. Because potassium is necessary for plant cell growth, this effect would be difficult to distinguish from the role that potassium plays in the normal growth and development of plant cells. If potassium is necessary for apogamy induction by glucose, and the UNC93-like protein is in fact involved in potassium uptake or transport, it would be important to induce it during apogamy commitment. To my knowledge, there is no published evidence of potassium playing a role in the induction of apogamy in other plants. Overall, this experiment may suggest that potassium is necessary for the induction of apogamy by glucose or that potassium concentration does not play a role in apogamy beyond providing the necessary nutrients for growth. 117 CHAPTER V SUMMARY AND FUTURE DIRECTIONS Land plants partition the two major events of a sexual life cycle, meiosis and fertilization, to two different multicellular generations, the diploid sporophyte and the haploid gametophyte. Despite the obvious evolutionary benefits of meiotic recombination and fertilization, many plants have evolved modes of asexual reproduction that avoid the processes of meiosis and fertilization altogether. These plants will produce asexual sporophytes directly from the cells of or around the gametophyte. The genes that drive these asexual processes are unknown, but are likely to at least partially overlap with those involved in zygotic embryogenesis. Defining and characterizing genes involved in these asexual processes is of interest not only agriculturally, but also for the insight they will provide on the evolution of asexuality, the alternation of generations, and the sporophyte body of land plants. In this thesis, the genes that are induced during the process of apogamy induction in the fern C. richardii are elucidated and examined. The process of apogamy occurs in approximately 10% of ferns in nature and may also be induced in the laboratory from many fern gametophytes that normally reproduce sexually (Sheffield and Bell, 1987; Raghavan, 1989). I discovered that 2.5% glucose medium is optimal for the induction of apogamy in the fern C. richardii (Ch. I). In Chapter II, I described the haploid sporophyte structures that these gametophytes produce in response to culture on this medium. While obligate apogamy had been previously described from hybrid and aneuploid strains of this fern (Hickock, 1977, 1979), the induction of apogamy from this fern had not previously been described in the literature. 118 As is the case with induced apogamy in other ferns, the apogamous sporophytes induced from C. richardii do not have the normal morphology of zygote-derived sporophytes but do share some or all of the features of zygotic sporophytes including vascular tissue and stomata. I found that fern gametophytes begin committing to the production of induced apogamous sporophytes after 10 days growth on 2.5% glucose medium. This commitment timing provided an opportunity to isolate the genes induced during the process of apogamy induction in this fern. I created a subtracted, normalized “apogamy” cDNA library that contains approximately 900 clones representing genes induced in C. richardii gametophytes at the time of apogamy commitment. Among the 306 unigenes identified from the sequenced clones in this library, 170 could be identified with sufficient confidence to examine as a representation of the library as a whole (Chapter III). 306 unique genes gives 3-fold coverage of the 902 clones in the apogamy library, which is equivalent to 95% confidence that each apogamy library gene is represented at least once in these sequence data. The apogamy library is enriched in genes that are involved in stress response and metabolism compared to the entire transcriptome of the fern P. aquilinum. This suggests a role for nutrient flux, and perhaps sugar sensing or signaling, in apogamy induction in C. richardii. Additionally, inductive microspore embryogenesis in angiosperms universally requires a stress treatment to induce an embryogenetic program from the haploid spores (Shariatpanahi et al., 2006; Segui-Simarro, 2010). Induction of stress response genes may be a requirement for induced apogamy from both angiosperm and C. richardii gametophytes. 119 The apogamy library contains many sequences whose homologues in Arabidopsis show enhanced or specific expression in flowers and seeds, two structures that do not exist in ferns. It is tempting to speculate on the function of these genes in ferns. It may be that these are sporophyte-specific genes in C. richardii that were induced to express in the gameotphyte through apogamy induction, or they could be involved in gametophyte function and/or development, or both. The prevailing hypothesis for the evolution of alternation of generations is that the modern sporophyte of the land plants evolved from an ancestor that had a dominant, multicellular gametophyte with meiosis proceeding directly from the zygote (Chapter I). As such, achieving development of the sporophyte body plan would likely involve the recruitment of gametophyte genes for related or novel functions in the sporophyte. The Arabidopsis homologues of the apogamy library sequences that have sporophyte-specific expression patterns are interesting for investigations into asexual processes and the evolution of the alternation of generations, both subjects of particular interest in plant biology. The apogamy library will be a rich resource for future investigations in this laboratory. One of the apogamy library genes with enhanced expression in Arabidopsis embryos was the UNC93-like gene. Whole mount in situ analyses in C. richardii gametophytes undergoing apogamy commitment suggested that this sequence was highly expressed in the egg cells of gametophytes. Based on my observations of induction of apogamy in C. richardii, I had hypothesized that mature, functional eggs or archegonia were required for the ability of C. richardii gametophytes to undergo apogamy (Chapter III). Therefore, the UNC93-like gene was chosen for further functional analysis in Arabidopsis. UNC93-like is a homologue of the unc93 gene in C. elegans, a membrane 120 bound protein that is predicted to regulate a two-pore potassium channel during the coordination of muscle contraction (Levin and Horvitz, 1992; de la Cruz et al., 2003). This gene is uncharacterized in plants. A homozygous T-DNA insertional line in the AtUNC93-like gene shows a number of vegetative and reproductive phenotypes, including small plant stature, retarded growth and delayed flowering. Siliques have a reduced seed set compared to WT plants and this is probably due to a combination of embryo abortion and gametophyte lethality (Chapter IV). The mutant allele does not appear to play a critical role in gametophyte viability, per se, because the allele is passed with equal efficiency as the WT allele through both male and female gametophytes to the next generation. RT-PCR experiments indicated that plants of this T-DNA line produce a partial UNC93-like transcript from upstream of the T-DNA insertion site, which may be translated into protein. If this is the case, and the protein has partial function, this mutation may be recessive or may cause dominant negative effects. Evidence for one or the other may be obtained by examining the phenotypes of plants heterozygous for the mutation. A dominant or semi-dominant negative effect will produce heterozygous plants with mutant phenotypes similar or identical to those homozygous for the allele. I have preliminary evidence that heterozygous plants have retarded growth resulting in a later flowering time than WT, suggesting that the T-DNA mutant allele is a semi-dominant mutation. These observations will have to be quantified before a conclusion can be made regarding the nature of this allele. Additionally, it would be informative to examine the phenotypes of plants belonging to a true knockout line for the AtUNC93-like gene. There are several insertional mutant lines available for this gene. If a knockout of the AtUNC93-like gene is 121 not lethal, and the phenotype resembles the phenotype of the current T-DNA line, this would be evidence that the allele of the current T-DNA line is recessive. While I found in silico expression evidence that the two transcripts of the AtUNC93-like gene are differentially expressed, my RT-PCR analysis found the presence of both transcripts in all tissues examined (Chapter IV). Accordingly, the first experiments that should be completed regarding the further characterization of the AtUNC93 gene are in situ hybridizations to examine at the tissue level and where the two transcripts of this gene are being expressed in the WT tissues of Arabidopsis. Additionally, it would be very interesting and informative to use this methodology to examine the locations of the AtUNC93-like transcripts in Arabidopsis tissues that are undergoing somatic embryogenesis. Multiple gain- and loss- of function Arabidopsis mutations which produce somatic embryos in developmental processes that resemble apomixis (Chapter I). If the expression of AtUNC93-like gene is found to be significantly altered in these mutant backgrounds compared to WT, or specifically expressed in or around the somatic embryos themselves, this would be strong evidence of a role for the UNC93-like gene in asexual reproduction. I have acquired a fis2 mutant line, from which 60% of embryo sacs produce endosperm and embryogenic growth in the absence of fertilization (Chaudhury et al., 1997). These tissues undergoing somatic embryogenesis will be used for in situ analyses with the AtUNC93-like probes. These in situ experiments are ongoing in our lab. Of course, the transcriptional profile does not always match the location of the encoded protein. The UNC93-like protein is predicted to be a transmembrane protein. There are a number of ways to identify the tissue and subcellular location of the 122 AtUNC93-like gene products. For the tissue specific location of the AtUNC93-like protein a gene trap line is preferable to transformation with a reporter construct, as it retains the native promoter elements and genetic milieu. I have already acquired a gene trap line from the TRAPPER collection (Sundaresan et al., 1995) that is annotated as being inserted into the AtUNC93-like gene immediately after the start codon. Unfortunately, this line does not have any appreciable GUS expression, and both the long and short AtUNC93-like transcripts are detected in the seedlings of plants homozygous for this construct, so its usefulness for mutant-phenotype analysis is limited as well. Alternatively, one could create GFP and YFP fusion constructs with the coding sequences of the two transcripts, driven by a native promoter region, transform plants with this construct and then examine the tissue-specific and subcellular location of the two proteins with confocal microscopy. Antibodies specific to each of the two proteins could be used in immunolocalization analyses. There is evidence of embryo abortion occurring in the unc93-like homozygous TDNA mutant. It will be important to follow up on this observation by observing embryo development in the seeds of this mutant to find the exact cause of embryo death. The seed abortion in this line could be evidence of a developmental defect affecting the embryo or the endosperm, or it could simply be the result of the failure of the parent plant to transfer sufficient nutrients to the developing embryos so that a portion of the embryos abort. This later scenario is supported by evidence of the failure of parent plants to maintain the viability of a proportion of their respective male and female gametophytes (Chapter IV). The ultimate test of the role CrUNC93-like role in apogamy is to create C. richardii lines that under-, over-, and ectopically express the protein. In order to achieve 123 this, an efficient and reliable transformation protocol needs to be developed for C. richarii. The only reported transformation method for C. richardii is direct bombardment of DNA into gametophyte cells (Rutherford et al., 2004). We have found this method to be expensive and inefficient (data not shown). Our lab is currently developing an Agrobacterium-mediated, stable transformation protocol. Once available, C. richardii gametophytes will be transformed with the CrUNC93-like full-length cDNA under the control of the strong, nearly ubiquitously expressed 35S promoter. Our previous experiments indicate that the 35S promoter works efficiently in this fern. The phenotype of the transformants, especially their ability to apogamously produce sporophytes, will be examined. Other interesting genes in the apogamy library, such as FTSH protease, BTB domain, and LEUNIG-like can also be tested using this transformation protocol. In addition, it can be used to test ectopic expression of those genes with gain-of –function, somatic embryogenesis phenotypes in Arabidopsis, such as WUSCHEL, BABYBOOM, and SERK1. 124 REFERENCES Abramoff MD, PJ Magalhaes, SJ Ram (2004) Image processing with ImageJ. 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