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Analysis of a seed coat mutant (patchy) in Arabidopsis thaliana Popma, Theodore Mark 2000

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ANALYSIS OF A SEED COAT MUTANT (PATCHY) IN ARABIDOPSIS THALIANA by Theodore Mark Popma B.Sc, University of Dalhousie, 1996 A THESIS SUBMITTED IN PARTIAL FULFULMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BOTANY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July 2000 © Theodore Mark Popma, 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date ^—^^7 X% I DE-6 (2/88) Abstract During seed development, many plants synthesize, secrete and extrude large quantities of pectic polysaccharides in the form of mucilage from the epidermal cells of the seed coat. These cells also undergo unique morphological changes throughout their development which have been well described. Phenotypic, genetic and molecular characterization of the patchy {pty-1) mutant in Arabidopsis thaliana was undertaken to further determine the genetic factors controlling the development and morphology of this cell type and its production of mucilage. The pty-1 mutation was found to be the result of a T-DNA insertion event and was mapped to distal locus on chromosome 5. Analysis of double mutant created between pty-1 and mum1 (another known seed coat mutant in Arabidopsis) indicates that pty-1 and mum1 interact synergistically with respect to the extrusion of mucilage, and their mutant phenotypes may be due to the altered chemical composition of pectins. Phenotypic characterization of pty-1 ,a second stronger pty-2 allele, and the pty-1/pty-2 heteroallelic mutant was performed using both light and scanning electron microscopy. These studies suggested that rather than merely affecting pectin composition, mutations at the PTY locus may control the general morphogenesis of this cell type. ii Table of Contents Abstract ......... ii List of Tables ......... v List of Figures ......... vi 1. Introduction: ........ 1 1.1 Seed Coat 1 1.2 Myxospermy ....... 4 1.3 Pectins ........ 6 1.4 Secretion and Assembly of Pectins. .... 7 1.5 Seed coat of the Brassicaceae 8 1.6 Genetic Analysis of the Mucilage and the Seed Coat in Arabidopsis thaliana ..... 9 1.7 Thesis Objectives 11 2. Materials and Methods:. 3 2.1 Plant material 12.2 Growth conditions2.3 Reagents and techniques: ..... 14 2.4 Ruthenium Red Assay . .12.5 Scanning Electron Microscopy (SEM) ... 14 2.6 Sample Preparation for Light Microscopy 15 2.7 Analysis of Seed Coat Development. ... 16 2.8 Generation of F2 Population for Genetic Mapping 12.9 Preparation of Plant Genomic DNA for Mapping 6 2.10 Polymerase Chain Reaction Amplification of DNA for mapping . 17 2.11 Preparation of DNA Probes for Southern Analysis 21 2.12 DNA extraction and Southern Blotting ... 22 2.13 Sequencing ....... 23 3. Results 24 3.1 Phenotypic Analysis of pry-7 ..... 24 3.1.1 Extrusion of mucilage in Ruthenium Red stain 24 3.1.2 Development of the seed coat epidermis in wild type and in pty-1 seeds 26 iii 3.1.3 Analysis of mature, dry pty-1 and wild type seeds 29 3.2 Genetic Analysis: ...... 32 3.2.1 Inheritance of pty-1 33.2.2 Complementation tests .... 33 3.2.3 Double mutant analysis .... 33 3.2.4 Allelism test with scm2 .... 36 3.3 Molecular Analysis ...... 38 3.3.1 Co-segregation of pty-1 with NPT2 33.3.2 Southern Analysis ..... 39 3.3.3 Genetic mapping of pty-1 .... 43 4. Discussion ........ 47 4.1 The function of the PTV gene 44.2 The pty-1 phenotype ...... 47 4.3 PTV and MUM1 both affect the extrusion of mucilage 48 4.4 The T-DNA insert in pty-1 is intact .... 49 4.5 pry-7 maps to distal chromosome 5 .... 49 Conclusions ......... 51 Future directions ........ 51 Bilbliography ........ 53 iv List of Tables Table 1. Summary of all CAPS markers used in genetic mapping of pty. 19 Table 2. Summary of PCR conditions used for different CAPS markers. 21 Table 3: Numbers of different phenotypes seen in F2 seed of 34 ptyx mum1 cross and the numbers of each phenotype expected if the novel non-staining seed phenotype represents the double mutant class. Table 4. Results of Complementation Tests between mum and 36 scm mutants. "c"=complements, "nc" = does not complement Table 5: Observed and Expected band sizes for Southern blots 41 of Hind\\\ and EcoRI digests. Table 6. Summary of genotypes for various CAPS marker loci of 44 F2 pty mutants from pty-1 (WS) x Col and pty-1 (WS) x Ler crosses. v List of Figures Figure 1. Components of the Angiosperm Seed 1 (transcribed from Boesewinkel and Bouman,1984). Figure 2. Mature seed coat of Brassica sp. Showing 3 different layers of cells derived from different integumental layers (transcribed from Esau, 1977). Fig. 3. Structure of the major components of Pectins: 7 Polygalacturonic Acid (un-substituted horizontal chain) and Rhamnogalacturonan-I (with R-groups which may consist of arabinans, galactans or arabinogalactans) (transcribed from Driouich et. al., 1993). Figure 4: Appearance of pty-1 and wild type (WS ecotype) seeds 25 after staining with Ruthenium Red for different time intervals. Fig 5. Developing cells of the wild type seed coat. Developing 27 seeds (WS ecotype) were harvested at different times following pollination (days after pollination =dap), sectioned, stained with Toluidine Blue and observed by light microscopy (a-d) or observed by scanning electron microscopy (e-h). Figure 6. Wild type (WS ecotype) and pty-1 developing seeds at 17 29 days after pollination sectioned, stained with Toluidine Blue and observed by light microscopy. Fig 7. Comparison of wild type (WS ecotype) and pty-1 mature 30 seed sections stained with Toluidine Blue and observed by light microscopy. Fig. 8 Scanning electron micrographs of wild type (a,c) and pty (b,d) 31 mature dry seed when dry-mounted (a, b) or fixed in aqueous glutaraldehyde to allow the potential extrusion of mucilage (c, d). Figure 9. mum1 phenotype and pty-1 x mum1 double mutant seed 33 phenotype after staining in Ruthenium Red Figure 10. mum1 and pty-1, mum1 double mutant seed sections 35 stained with Toluidine Blue and dry-mounted SEM pty-1, mum1 double mutant. Figure 11. pty-2 seed phenotype. 37 vi Figure 12. Phenotype of pty-1lpty-2 heteroallelic mutant seed. 38 Figure 13. Structure of the T-DNA used to transform the population 40 from which pty-1 was derived (c). The positions of the Hind\\\ (a) and EcoRI (b) restrictions endonuclease sites are shown. The numbers indicate the size of fragments. The bars above the T-DNA (c) indicate the position of the probes used. Gray shaded areas indicate flanking Arabidopsis genomic DNA for pty-1 as deduced from this study. Figure 14. Southern Blots showing fragments of pty-1 genomic 42 DNA which hybridize to three T-DNA probes: pBR322 (a), the Right Border region (b) and NPT2 (c). No hybridization was observed for wild type control genomic DNA. Fig. 15. CAPS banding patterns of 4 different markers for 45 individual pty-1 F2 plants. All lanes shown are results for pty F2 mutants selected from either a pty-1 x Col or a pty-1 x Ler cross . A preponderance of the parental pty-1 genotype (WS ecotype) indicates linkage to a particular marker. The WS is similar to Columbia (c), or Landsberg (L), depending upon the cross, as indicated below, c = plant homozygous for Columbia allele. L =plant homozygous for Landsberg allele. H = plant heterozygous for Columbia and Landsberg alleles. Map positions from Lister and Dean Rl map (Jarvis, et. al., 1994). Figure 16. Proposed map location of pty-1 in relation to four CAPS 46 markers on chromosome 5. vii 1. Introduction 1.1 The Seed Coat: Angiosperm seeds consist of three genera! components: the embryo, the endosperm and the seed coat (Figure 1, Boesewinkel and Bouman,1984). The diploid embryo is an immature sporophyte and is a product of fertilization of the egg by one of the sperm nuclei. The endosperm is triploid tissue derived from fertilization of polar nuclei by the other sperm nucleus and serves to provide the embryo with nutrients following germination. This study will focus upon the third component of the seed: the seed coat, or testa. Figure 1. Components of the Angiosperm Seed (transcribed from Boesewinkel and Bouman,1984). The primary functions of the seed coat are the maintenance of dormancy before gemination, the nutrition of the developing embryo and the protection and dispersal of the seed. Dormancy may be imparted either by action of the embryo, testa, or a 1 combination of the two (Debeaujon et. al, 2000). The seed coat acts to restrict the permeation of water and oxygen into the seed as well as to prevent the protrusion of the radicle (Jacobsen, 1984). The result is the inability of the seed to germinate unless exposed to environmental or chemical factors conducive to the eventual growth of the embryo (Boesewinkel and Bouman,1995). Although the seed coat is not the main storage tissue in the seed, it is essential in providing and transporting nutrients to be stored either in the endosperm or the cotyledons (Taiz and Zeiger, 1998). Since there is no vascular tract connecting the embryo to the parent plant, it is dependent largely upon nutrition from the endosperm as well as the seed coat. The testa has been shown to transport sucrose and oxygen to the endosperm and to the embryo sac to provide nutrients to the developing embryo (Boesewinkel and Bouman,1995). This has been shown to occur both by diffusion and through the action of a sucrose transporter located in specialized transfer cells. These cells are found at the contact point between the embryo and the inner layer of the seed coat (Weber et. al. 1997). To avoid eventual competition with the parent plant upon germination, a variety of adaptations in the seed coat have evolved to ensure the dispersal of the seed. These are extremely varied and include the development of air sacs, hairs and wings from the testa. Finally, the presence of phenolics, polysaccharides and other chemicals in one or several integumental layers of the seed coat provide protection for the developing embryo. Mucilage, a complex pectic polysaccharide, is one such compound (reviewed by Boesewinkel and Bouman, 1995). 2 The structure of the mature seed coat, as described by Esau (1977) may differ greatly between species, although certain general characteristics are conserved. The mature seed coat of a typical Brassica is shown in Fig. 2. The seed coat is usually dry at maturity and consists of several cell layers. The number of layers present depends on the initial morphology of the ovule integuments and on the development of these Figure 2. Mature seed coat of Brassica sp. Showing different layers of cells derived from different integumental layers (transcribed from Esau, 1977). layers after fertilization. The outer epidermal layer may eventually increase in thickness or secrete various substances such as mucilage involved in the protection or dispersal of the seed as described above. Underlying the outer epidermis are cells which give strength to the seed coat and are resistant to physical and osmotic stresses. These cells make up the palisade layer and may develop a columnar shape and deposits of thickened, lignified secondary cell walls. Other cell layers may be present in this region such as parenchymatic or schlerenchymatic cells which also lend structural support to 3 the seed coat. The innermost cell layers of the seed coat underlie the palisade layer and cells may contain pigments, giving the seed its color and possibly reducing the edibility and digestibility of the seed. The cells in the pigmented layer may often be crushed by other developing cell layers and therefore may not be present at maturity (Fig. 2). Description of the cell layers of the seed coat becomes more precise when considering their development from the integuments of the ovule. This has been described in a variety of genera including Asparagus, Lycopersicon, Phaseolus and Brassica (Kelly et. al., 1992) The ovule is the site of formation of the female gametophyte and consists of three parts: the nucellus, the integuments and the funiculus. The nucellus is composed of vegetative cells enclosing the sporogenous cells. The integuments are composed of cell layers which enclose the nucellus. The funiculus is the stalk which attaches the ovule to the ovary. After fertilization, differentiation of the ovule into a mature seed is initiated. Taking the Brassicaceae as an example, the inner layer of the inner integument (endothelium) develops to become the pigmented layer and the inner and outer layers of the outer integument develop into the palisade and outer epidermal layers of the mature seed coat, respectively (Figure 2) . (Van Caeseele 1981, Leon-Kloosterziel 1996, Esau 1977). 1.2 Myxospermy: In an aqueous environment, mucilage is released from the epidermis providing a sticky sheath around the seed. Plants which do this are said to be myxospermous. Mucilage is thought to aid in the protection, germination and dispersal of the seed by 4 increasing bouyancy in water, by decreasing diffusion of oxygen in environments unsuitable for seed germination, and by adhering to animal vectors (Mauseth,1988). Examples of myxospermous plants can be found throughout the plant kingdom in a variety of families including the Brassicaceae, Solanaceae, Linaceae and Plantaginaceae. The cytology of mucilage production in the seed coat epidermis, has been recently extensively described in Arabidopsis thaliana of the Brassicaceae (Western et al., 2000). In addition to its production by seeds, mucilage plays important roles in other tissues of the plant. It is essential in sexual reproduction for its role in aiding pollen tube growth through the transmitting tract of the pistil (Bystedt and Vennigreholz, 1991), and has also been shown to be produced in root cap tissue, presumably to allow the root tip to pass through the soil medium more easily (Northcote and Gould, 1989). In addition, mucilage has been shown to act as a chemical attractant for pollinators (Slater, 1991) and a protective "gum" which is secreted from wounded tissues (Fahn 1979, Goodwin and Mercer 1972). Less extensively described mucilage-producing cell types include the leaf epidermis and hypodermis, xylem and phloem as well as cortical and parenchymatic cells (Gregory and Baas, 1989). Mucilage is important for industrial purposes such as creating thickening agents (hydrocolloids) for food products such as jams and jellies (Cui, 1993). It has also been suggested that the presence of mucilage on certain canola seeds is an indicator of higher yield of oil and protein and has therefore been selected for by canola breeders. Conversely, a layer of adhesive polysaccharide on the surface of such seeds may provide a suitable substrate for pathogenic fungi (Fahn, 1979; Goodwin and Mercer, 5 1972). That mucilage may act as a substrate for pathogens has been shown in maize where different cell surface proteins on the pathogen were able to bind mucilage selectively (Gould and Northcote, 1986). Various studies have been undertaken regarding the development of mucilaginous cells, especially in the Brassicaceae, given the economic importance of many types of mustards as crop plants (Hirst et. al, 1965; Grant, 1969). Mucilage has also been found to have various ethnobotanical medicinal properties and has been used in the treatment of everything from intestinal irritation to hiccups. (Morton, 1990). 1.3 Pectins: Seed mucilage is composed almost entirely of pectin (Tomoda and Ichikawa, 1987). This has been confirmed in a variety of species including Sinapis alba, Plantago ovata, Zea mays and Psychotria) (Smith and Montgomery 1959, Hyde 1970). Pectin forms a heterogeneous mixture of hydrophilic.acidic polysaccharides which aggregate into a gel-like state, giving mucilage its "slimy" characteristics. Pectins are generally composed of polygalacturonic acid (PGA) and rhamnogalacturonan I (RG-1) (Fig. 3). PGAs are helical homopolymers comprised of up . to 200 repeating units of the monosaccharide galactoxyluronic acid (GalA). RG-1 is a contorted rod-like heteropolymer comprised of repeating units of the disaccharide rhamnosyl-GalA. Other branching pectic polysaccharides such as arabinans, galactans or arabinogalactans may also form side chains attached to RG-1 (Carpita and Gibeault, 1993). 6 Pectins are not only the primary constituents of mucilage, but are also approximately one third (35%) of the dry weight of the primary plant cell wall. The hydrophilicity of pectin contributes to the fact that the mature cell wall is approximately 30% water (Carpita et. al., 1996), and therefore is not a rigid structure, but rather dynamic as is required for cell division and expansion. It is the gel-like nature of pectin which supports the rest of the constituents of the cell wall and is thought to contribute to plasticity and porosity (Cosgrove, 1997). Fig. 3. Structure of the major components of Pectins: Polygalacturonic Acid (un-substituted horizontal chain) and Rhamnogalacturonan-I (with R-groups which may consist of arabinans, galactans or arabinogalactans) (transcribed from Driouich et. al., 1993). 7.4. Secretion and assembly of pectin: Pectins are synthesized and processed in the Golgi apparatus and then packaged into secretory vesicles for secretion across the plasma membrane. The building blocks of pectic polysaccharides generally take the form of UDP-glucose, which is taken into the lumen of the Golgi by an antiport mechanism (reviewed by Carpita, 1996). D- fftoamiiose D-grycosidic bnnd 7 Individual sugar residues are then linked together through the action of polysaccharide synthetase and glycosyltransferase enzymes. Some of these enzymes have been localized to specific locations within the Golgi apparatus, and several of these enzymes may be required for the synthesis of a single polysaccharide (Driouich et. al. 1993). The plant Golgi apparatus consists of four main compartments: the cis, medial trans-cistemae and trans Golgi network. Using antibodies to various epitopes of PGA and RG-1, it was found that enzymes involved in PGA/RG-I sugar-backbone synthesis are localized primarily in the cis- and medial- Golgi, whereas the addition of R-groups such as arabinans to RG-I occurs in the frans-cisernae. Processing proceeds in the cis-to trans- direction where mature polysaccharides are packaged into vesicles budding from the frans-Golgi network for secretion outside the cell. These studies suggest that specificity of polysaccharide synthesis depends on enzyme specificity for a target, and that enzymes and targets are also co-localized within the cell (Moore et. al., 1991, Staehelin and Moore, 1995). 1.5 Seed coat of the Brassicaceae: This study will focus upon the seed coat epidermis of Arabidopsis thaliana, of the Brassicaceae. This is an interesting system for morphological and genetic reasons. First, the cell type itself completes a series of diverse functions through its maturation including the formation of secondary cell walls, cytoplasmic rearrangements and the secretion and extrusion of mucilage (a gel comprised of complex polysaccharides). Second, Arabidopsis is amenable to genetic analyses necessary to explore the genes 8 controlling these functions due to its short generation time, high fecundity and nearly completely sequenced genome. The cells of the seed coat epidermis of Arabidopsis thaliana contain a raised column (columella) in the center of the cell, with mucilage filling the remaining space between the columella and the outer cell wall. Upon hydration of the seed, the mucilage inside these cells expands, ruptures the outer wall and is extruded into the environment, forming a capsule which completely surrounds the seed. The development of these mucilagenous cells was shown to follow a well-defined series of events (Western ef. al. 2000) After an initial growth phase, the accumulation of starch-containing amyloplasts can be seen approximately four days following fertilization. Approximately 7 days following fertilization, the cytoplasm recede and the vacuole shrinks away from the outer tangential cell wall. As the remaining cytoplasm and vacuole recedes towards the inner tangential wall, a portion of cytoplasm remains in the center of the cell producing a raised column of cytoplasm. Simultaneously, mucilage begins to accumulate in the extracellular space between the plasmalemma and the outer tangential cell wall. After approximately 10 days, a secondary cell wall begins to form between the plasmalemma and the mucilage deposits. As this occurs, the cytoplasm is pushed down towards the inner tangential cell wall and eventually disappears. The secondary cell wall eventually occupies the entire space of the cytoplasmic column thus forming the columella. (Western, et. al. 2000). 9 1.6 Genetic Analysis of Mucilage and the Seed Coat in Arabidopsis thaliana: Several genes involved in the development of seed coat epidermal cells in Arabidopsis have previously been identified and characterized through the analysis of mutants. The glabra! (gl2) and transparent testa glabral(ttgl) mutants fail to extrude mucilage from the cells of the outer layer of the seed coat and have incomplete development of their columellae (Szymanski et. al. 1998). The apetala2 (ap2) mutant has altered seed shape, abnormal development of the seed coat and lacks mucilage (Bowman et. a/.1989, Jofuku et. al., 1994). Finally, the aberrant testa shape (ats) mutants were also found to have reduced amounts of mucilage (Leon-Kloosterziel et. al. 1996). All these genes function in other tissues in addition to the seed coat. Indeed, mutants defective in these genes were originally isolated based on phenotypic analysis of tissues such as trichomes (ttg and gl2), ovules {ats) or flowers (ap2) (Bowman er. al., 1989). While TTG and GL2 have been cloned and are suspected to be transcription factors, they may be general regulators of the development and differentiation of epidermal cells. If this is the case, deficiencies in trichomes or mucilagenous cells are indirect results of a general and potentially complicated process (Rerie et. al., 1994). In an attempt to isolate genes required specifically for seed coat epidermal development, mutagenized populations of seeds were screened for their ability to extrude a mucilage capsule in aqueous solution as it becomes evident upon staining with the pectin-specific dye, Ruthenium Red. These mutants, called mucilage modified (mum), were grouped into 5 complementation groups by T. Western (unpublished), mum1 through mum5, all with slightly different phenotypes. Most intriguing were mum1, 10 and mum4. Both the mum1 and mum4 seeds stain red and fail to extrude mucilage, while mum4 seeds additionally have abnormally shaped cells and columellae. Also, they do not display any obvious defects in other tissues such as trichomes or flowers and so these genes are believed to be seed-specific (Western, Ph.D Thesis 1998). The same mutant screen which resulted in the isolation of the mum mutants also succeeded in isolating the mutant which will be the focus of this study: patchy (pty-1) (Western and Haughn, unpublished results). From the screen, it could be seen that pty-1 fails to form a complete sheath of mucilage around the seed, but rather forms discrete patches seemingly randomly upon the seed coat. There are several possibilities for the nature of this defect, including abnormal development of the outer layer of the outer integument of the seed, or improper synthesis, secretion or extrusion of mucilage. It is also possible that abnormal cell wall composition may prevent the cell wall from rupturing thereby preventing the extrusion of mucilage. Part of the goal of this study is to determine which of these hypotheses is most likely correct, thereby narrowing the possibilities as to the function of the PTV gene. Whereas the mum mutants were isolated from an ethylmethane sulfonate (EMS) mutagenized population, pty-1 was isolated from a T-DNA mutagenized population. This means a fragment of DNA of known sequence was inserted into the genome at random, generating mutations by disrupting genes. The DNA fragment which has commonly been used for insertional t-DNA mutagenesis is a version of the Agrobacerium tumefaciens T-DNA (Koncz et. al., 1990; Yanofsky et. al, 1990) that has been engineered to impart kanamycin resistance to plants and ampicillin resistance to bacteria (Feldmann, 1991). Because of these properties of the T-DNA, cloning a gene 11 which has been "tagged" in such a way can become a simpler task than cloning by position as is the case for EMS mutants. 7.7 Thesis Objectives: It is the goal of this study to understand the function of the PTY gene. The first step toward this goal is to analyze the patchy phenotype in detail to determine the nature of defects in mucilage synthesis, secretion, extrusion and/or cell wall biosynthesis and the time at which they occur. The second portion of this study will focus on the genetic aspect of this phenotype. This will address the inheritance pattern of the pfy-7 phenotype, whether this phenotype has been caused by T-DNA insertion and whether pfy-7 interacts with any other genes known to affect seed coat morphology and function. Finally, in an effort to clone this gene, Southern analysis of the T-DNA and of flanking genomic DNA will be undertaken to determine the orientation of the T-DNA and the presence of restriction sites in that region. The map position of the PTY locus will also be determined in order to provide a potential starting point for positional 4 cloning. 12 2. Materials and Methods 2.1 Plant material: The mum mutants (Col-2 ecotype) and the pty-1 mutant (Wassilewskija [WS] ecotype) were isolated by Dr. Tamara Western and Dr. Joanne Burn from ethylmethane sulfonate (EMS) and T-DNA mutagenized populations (Feldmann, 1991), respectively. The scm mutants were a gift from Dr. Scott Sattler from the University of Minnesota, Dept. of Plant Biological Sciences. ttg1-1 and g/2-7 mutants were obtained from the Arabidopsis Biological Resource Centre at Ohio State University. 2.2 Growth conditions: Seeds were sown on Terra-Lite Redi Earth prepared soil mix (W.R. Grace and Co. Canada Ltd., Ajax, Ontario, Canada) and then transferred to growth chambers at 20°C under continuous light (90-120 uE m-2 sec-1 photosynthetically active radiation conditions). Alternatively, seeds were germinated on AT medium (5 ml/L 1 M KN03, 2.5 ml/L 1 M KH2P04, 2.5 ml/L 20 mM Fe EDTA, 2 ml/L 1 M MgS04, 2 ml/L 1 M CaN03x4H20, 1 ml/L micronutrients (70 mM H3BO3, 14 mM MnCI2x4H20, 0.5 mM CuS04, 1 mM ZnS04x7H20, 1.2 mM NaMo04, 10 mM NaCl, 0.01 mM CoCI2x6H20) . Antibiotics such as kanamycin or ampicillin were added accordingly to a final concentration of 50 mg/L). Seedlings were then transferred from AT medium to soil at the two true-leaf stage. 13 2.3 Reagents and techniques: Restriction enzyme (Gibco-BRL or New England Biolabs) digestion and agarose gel electrophoresis were performed as described by the manufacturer or by Sambrook et al. (1989). 1% agarose gels were used for all experiments except in genetic mapping where separation of fragments differing in size by less than 30 bases was required. In these cases, 1.5% agarose gels were used. Amplification of DNA by the Polymerase Chain Reaction was done using Taq polymerase (Gibco-BRL) and a DNA thermocycler (Perkin-Elmer). All frequently used buffers and reagents were made and used as described in Sambrook et al. (1989). 2.4 Ruthenium Red Assay: Samples were placed in micro-titer plates and shaken in 250 ul of 0.01% Ruthenium Red for varying lengths of time on an orbital shaker with sufficient speed as to keep the seeds suspended in the liquid. Seeds were then rinsed in distilled water before viewing with the dissection microscope for phenotypic analysis. Alternatively, seeds were not rinsed prior to phenotypic analysis when determining seed phenotypes in large populations (segregation analysis, double mutant analysis and mapping). This method still succeeded in distinguishing between parental phenotypes but was much less time consuming 2.5 Scanning Electron Microscopy (SEM): Tissue samples were generally prepared for SEM by fixing for 3-10 hrs in 3% gluteraldehyde in 0.05 M phosphate buffer. They were then rinsed thoroughly with 0.05 14 M phosphate buffer post-fixed in Os04 for 2 hrs, rinsed several times with water and, dehydrated through an ethanol series (30, 50, 70, 95, 100%x2) for 45min at each step. Dehydrated tissue was critical point dried using liquid CO2, sputter coated with gold (Nanotech SEM rep2 Sputter Coater) and examined under the SEM (Cambridge model 250T Scanning Electron Microscope [Leica], with a 20 kv accelerating voltage). Mature dry seeds were sputter coated with gold and examined directly without fixation or dehydration. 2.6 Sample Preparation for Light Microscopy: Young seeds (less than 18 days after pollination (dap) were fixed in 3% gluteraldehyde in 0.05 M phosphate buffer for at least 3 hrs and then post-fixed in Os04 for 2 hrs. They were then washed with water and dehydrated through an ethanol series as described for the SEM protocol. Seeds were then embedded in Spurr's Resin (REF) by successive treatment in a graded series of solutions of Spurr's Resin in ethanol (25%, 50%, 75% and 100%), each for 1 hr, and finally embedded in fresh resin and baked overnight at 60°C. For mature dry seeds, a microwave and vacuum chamber were used to enhance penetration of the fixative. Also, seeds were incubated in 75% and 100% Spurr's resin solutions for a minimum of 12 hrs before embedding and baking at 60°C. Samples were sectioned on a Reichert microtome with glass knives, floated onto a drop of water on a glass slide, fixed to the slide by heating to approximately 60°C and stained with Toluidine Blue in 1% sodium borate for approximately 30 sec at 60°C. Samples were then rinsed with tap water and dried on a hot plate before they were 15 observed. Sections were mounted in TE buffer with 50% glycerol under cover slips and sealed with nail polish. 2.7 Analysis of Seed Coat Development: Flowers were marked at the time of self-pollination (defined as the time at which the stamens reach the same length as the gynoecium thereby allowing pollination) at 11 am each day for six days with one of six different colors of non-toxic water paints. This procedure was repeated two additional times for a total of 18 days. All marked flowers/siliques were then harvested and fixed on the 18th day. Siliques older than 7 days were dissected and the seeds removed for improved fixation. Samples were prepared either for SEM or light microscopy as described above. 2.8 Generation of F2 Population for Genetic Mapping: Since pty-1 exists in the WS ecotype, and the majority of polymorphisms which are known to occur in Co-dominant Amplified Polymorphic Sequence (CAPS) markers exist between Col and Ler ecotypes, F2 plants from both Ws x Col and Ws x Ler crosses were planted (Jarvis, et. al., 1994) Approximately 100 F2 plants from each cross were planted. The the seed coat phenotype of each line was determined by using the Ruthenium Red assay as described previously. DNA from mutant plants was used as a Polymerase Chain Reaction (PCR) template. The PCR product was then digested with a specific restriction endonuclease to determine the genotype of those plants at each of the available CAPS marker loci (as described below). 16 2.9 Preparation of Plant Genomic DNA for Mapping: Genomic DNA was prepared from leaf tissue of each F2 line as described by Edward (1991): 2-3 young to mature leaves were macerated for 5 sec in a microfuge tube using a plastic microfuge tube pestle. 400 ul PCR buffer (200 mM Tris-HCI pH 7.5, 250 mM NaCl., 25 mM EDTA and 0.5% SDS) was added and the solution was centrifuged for 1 min. 300 ul of the supernatant was transferred to a new tube containing 300 ul isopropanol and centrifuged for 5 min. The pellet was washed with 100 ul 70% ethanol, air dried and resuspended in 100 ul of water for storage at4°C. DNA concentrations were determined by spectrophotometry using a Beckman Du-64 Spectrophotometer (UV radiation at 260 nm). 2.10 Polymerase Chain Reaction Amplification of DNA for mapping: Approximately 100 ng of genomic DNA from mutant plants was used in each PCR reaction as a template to amplify each specific marker. Table 1 shows the sequences and map positions of the full set of 18 oligonucleotide primer pairs which were used to amplify DNA at each marker locus (Konieczny and Ausubel, 1993), as well as two additional CAPS markers, EG7F2 and g2368, which were used to confirm the position of the pty locus. All primers were between 19 and 22 bp and yielded PCR products which, when digested with a specific restriction endonucleases and separated on agarose gels, produced a polymorphic banding pattern distinguishing either the WS and Col or WS and Ler ecotypes. Primer pairs were obtained from Research Genetics (Huntsville, AL) and were positioned in the genome at 40 map unit intervals. Also 17 shown in Table 1 is the cross (either WS x Ler or WS x Col) from which progeny were analyzed, as well as the restriction endonuclease used to detect genetic polymorphisms between ecotypes. A comprehensive list of all available CAPS markers, primer sequences and restriction fragments length polymorphisms can be found at http://genome-www.stanford.edu/Arabidopsis/maps/CAPS.html. Since sequences of primer pairs are different for each of these markers, the PCR conditions for each marker were also modified. Table 2 shows the different reaction conditions, amplification programs and annealing temperatures which were found to be the most effective for each marker. 18 Table 1. Summary of all CAPS markers used in genetic mapping of pty. Indicated are the marker name, its position on the Lister-Dean Rl map (Jarvis, et. al. 1994) of the Arabidopsis genome, Restriction Fragment Length Polymorphisms (RFLPs), oligotnucleotide primers used to amplify DNA at each marker, and the cross of either Col or Ler ecotypes to WS used for each marker depending on the parental combination showing polymorphism for the marker. Marker Map Position on Rl map RFLPs Primer Sequences Cross chr. #1 PW4 1.13 BsaAl: Col=Ws= (0.706, 0.311, 0.047); Ler, 1 (0.753, 0.311) 5'-GTTTGAAAGTGTAGATGTAACGAC-3' 5' GGTTGTG I I I I GCTAGCATC-3' WS x Ler NCC1 10.48 Rsal: Col, (0.87, 0.05); Ler= Ws, (0.92, 0.05) 5'-GTCCTATCTCTACGATGTGGATG-3' 5'-AAGTTATAAG G C ATTAG AATC ATAATC-3' WS x Col GAPB 59.1 Ddel: Col=Ws (0.605, 0.284, 0.225, 0.174); Ler, (0.35, 0.284, 0.255, 0.225, 0.174) 5'-TCTGATCAGTTGCAGCTATG-3' 5'-GGCACTATGTTCAGTGCTG-3' WS x Ler ADH 115.41 Xbal: Col=Ws,(1.291); Ler(1.097, 0.262); Bfal: 5'-GCGTGACCATCAAGACTAAT-3' 5'-AAAAATGGCAACACTTTGAC-3' WS x Ler chr. #2 m246 11.03 Maelll: Col,(1.354); Ler=Ws, (1.122, 0.232) 5'-TGAAGAGCTATCCGAGATGG-3' 5'-GCTTGAACTCCTCCTCCTTC-3' WS x Col GPA1 48.9 Af I III: Col=Ws, (0.705, 0.680, 0.209); Ler, (1.385, 0.209) 5'-GGGATTTGATGAAGGAGAAC-3' 5'-ATTCCTTGGTCTCCATCATC-3' WS x Ler m249 73.19 ScrFI: Col=(0.316); Ler=Ws, (0.216, 0.100) ScrFI: Col=No-0, 0 (0.316); Ler=C24=CV=R=Ws, 1 (0.216, 0.100) WS x Col chr. #3 GAPC 8.41 EcoRV: Col=(0.735, 0.713); Ler=Ws (0.713, 0.39, 0.34) 5'-CTGTTATCGTTAGGATTCGG-3' 5'-ACGGAAAGACATTCCAGTC-3' WS x Col GAPA 43.77 Ddel: Col, 5 (0.42, 0.178, 0.1, 0.033, 0.019, 0.01); Ler=Ws, (0.24, 0.19, 0.178, 0.1, 0.033, 5'-CACCGTGATCTAAGGAGAGCAAG-3' 5' •TGTGCTCAACCAAACTTAGCC-3' WS x Col GL1 48.45 Taql: Col, (0.298, 0.1, 0.074, 0.047); Ler, (0.372, 0.1, 0.047); 5'-ATATTGAGTACTGCCTTTAG-3' 5'-CCATGATCCGAAGAGACTAT-3' WS x Col BGL1 75.24 Rsal: Col, (0.785, 0.34, 0.105); Ler=Ws, (0.785, 0.485) 5'-TCTTCTCGGTCTATTCTTCG-3' 5'-TTATCACCATAACGTCTCCC-3' WS x Col 19 chr. #4 GA1 17.72 BsaBI: Col=Ws, (0.707, 0.527); Ler, (1.196) 5'-AAGCTTCGAACTCAAGGTTC-3' 5'-CCGGAGAATCGTACGGTAC-3' WS x Ler AG 63.16 Xbal: Col, (1.366); Ler=Ws, (1.073, 0.293) 5'-CAACAGGTTTCTTCTTCTTCTC-3'5'-'CAAACACCATTTAATCTTGACA-3' WS x Col PG11 75.16 Bfal: Col=Ws,(0.644, 0.296, 0.263, 0.090); Ler, (0.644, 0.353, 0.296) 5'-CGCAACTAACCACACATTAC-3'5'-AGTGAAATTCACCAGCATG-3' WS x Ler DHS1 108.54 Ddel: Col,(1.491, 0.129, 0.048); Ler=Ws,(1.62, 0.048); 5'-CAAGTGACCTGAAGAGTATCG-3' 5'-AGAGAGAATGAGAAATGGAGG-3' . WS x Col chr. #5 ASM 18.35 Bell: Col, (1.042, 0.686); Ler=Ws, (0.686, 0.553, 0.489) 5'-CTTACTCCTGTTCTTGCTTAC-3' 5'-CCTCTAGCCTGAATAACAGAAC-3' WS x Col DFR 89.51 BsaAl: CoNWs, (0.609, 0.534); Ler, (0.609, 0.318, 0.216) 5-AGATCCTGAGGTGAG Mill C-3' 5'-TGTTACATGGCTTCATACCA-3' WS x Ler LFY3 116.88 0.126, 0.078, 0.035); Ler=Ws, (0.855, 0.236, 0.126, 0.078, 0.035) 5-TAACTTATCGGGCTTCTGC-3' 5'-GACGGCGTCTAGAAGATTC-3' WS x Col EG7F2 1 cM distal of LFY Xbal: Col, (1.2); Ler=WS, (0.7, 0.5); 5'-GATCTGTGTAGGACTACGAGAC-3' 5'-GCATAGAAI I I GACGATAACGAGC-3' WS x Col g2368 125.12 Hindlll: Col=WS (1.4); Ler, (1.35, 0.05) 5'-AAGCTTTTGAATAGGACAGCATTG-3' 5' CGTTTTCATTGGTCCACTGCATGG-3' WS x Ler 20 Table 2. Summary of PCR conditions used for different CAPS markers. Each row of the grid indicates Reaction conditions and Thermocycler Program steps (1-6) for the marker(s) indicated in that section. Markers Annealing T Reaction conditions Thermocycler Program PW4 55°C 2 ul 10x Buffer 1. 94°C 3 min NCC1 2 ul 2mM dNTP 2. 94°C 1 min ADH " 1 ul 50mM MgCI2 3. 55°C 2 min m249 " 9 ul Water 4. 72°C 1.5 min BGL1 2 x 2.5 ul 2uM primer 5. (2, 3, 4) x 30 AG " 100 ng template DNA 6. 72°C 7 min DFR " 0.1 ul Taq polymerase GA1 " 10 ul 10x Buffer 1. 94°C 5 min 10 ul 2mM dNTP 2. 94°C 0.5 min GAPA II 3 ul 50mM MgCI2 3. Ta 0.5 min 50 ul Water 4. 72°C 45sec 2 x 10 ul 2uM primer 5. (2, 3, 4) x 30 0.1 ul Taq polymerase 6. 72°C 7 min 100 ng template DNA GAPC 52°C 2 ul 10x Buffer 1. 94°C 5 min LFY3 52°C 2 ul 2mM dNTP 2. 94°C 1 min PG11 55°C 0.75 ul 50mM MgCI2 3. Ta 1 min EG7F2 65°C 17 ul Water 4. 72°C 1 min g2368 52.5°C 2 x 1 ul 2 uM primer 5. (2, 3, 4) x 30 0.1 ul Taq polymerase 6. 72°C 5 min 100 ng template DNA 2.11 Preparation of DNA Probes for Southern Analysis: All probes were amplified and labelled with 32P by PCR where a single reaction consisted of 50 ng of DNA template, 2 ul 10x Buffer (Gibco-BRL), 2 ul dCTP, dGTP, 21 dTTP (2 mM), 1 ul MgCI2 (50 mM), 9 ul H20, 0.1 ul Taq DNA Polymerase (Gibco-BRL), 1 ul each primer (100 ng/ul) and 5 ul of dAT32P (50 uCi). All probes were amplified with the following PCR program: 94°C, 4 min; (94°C, 30sec; 50°C, 15sec; 72°C, 80 sec) x 30, 72°C;4 min. Probes were purified in QIAquick columns according to the manufacturer's protocol, boiled for 5 min and then added to the pre-hybridization buffer. The 800 bp NPT2 probe was amplified from the pBIN19 plasmid vector using the-following primers: (A/P7"2-forward: 5' TCAGAAGAACTCGTCAAG 3';/VP7"2-reverse: 5' GATGGATTGCACGCAGGT 3'). The 500 bp Right Border probe was amplified from genomic DNA of another T-DNA insertional mutant, Bell (Reiser et. al., 1995). This was done using the following primers: (RB2-forward: 5'GGTTAAACTGAAGGCGGG3'; RB2-reverse: 5' CTCGGTGGTGATAACTCC 3). The 600bp AmpR probe was amplified from the plasmid vector pBR322 and consisted of the coding portion of the gene imparting ampicillin resistance in bacteria (beta lactamase). The following primers were used: (AmpR-forward: 5' GCTCACCCAGAAACGCTG 3'; AmpR-reverse: 5'CCAGTGCTGCAATGATAC 3'). 2.11 DNA extraction and Southern Blotting: Genomic DNA from both pty-1 and Col-2 plants was extracted using the standard CTAB protocol (Dean et. al., 1991). 5 ug of DNA were then digested with Hind\\\ and EcoRI restriction endonucleases, fragments separated on a 1% agarose gel and transferred to a Hybond-N+ (Amersham) membrane according to the protocol of the manufacturer. The membrane was then incubated for 2 hrs in 10 ml pre-hybridization 22 buffer (0.5 M Sodium phosphate, 7% SDS) in a glass hybridization tube. ^P-labelled probes were then added and allowed to hybridize for at least 12 hours. The membrane was removed, washed twice at low stringency (1X SSC, 0.1% SDS) and exposed to Kodak XAR-5 film. 2.13 Sequencing: Right border and AmpR probes were both sequenced to ensure they were identical with the published sequence of the T-DNA (Feldmann, 1991). An automated 377 DNA Sequencer (Perkin-Elmer, Applied Bio-Systems) was used for all sequencing reactions. Sequencing was carried out at the Nucleic Acid Protein Services (NAPS) branch of the Biotechnology Department at UBC. One reaction consisted of 4 ul premix, 90ng template (purified PCR product), 3.2 pmol primer and distilled water to a volume of 20 ul. The PCR programme used for amplification of the template was as follows: (96°C for 30 sec, 50°C for 15 sec, 60°C for 4 min) x 25. 23 3. Results 3.1 Phenotypic Analysis of pty-1: 3.1.1 Extrusion of mucilage in Ruthenium Red stain: Morphological and genetic studies were carried out in order to characterize the pty-1 mutant, and to provide information regarding the possible function of the PTY gene. It was already known, based on the original screen of the T-DNA lines using a Ruthenium Red staining assay (see Materials and Methods), that pty-1 extruded mucilage differently than wild type (WS ecotype) seeds. Therefore, this aspect of the pty-1 phenotype was described in detail. Staining of wild type seed with aqueous Ruthenium Red produced bright red staining after 20 minutes (Fig. 4d). After two hours, the halo had darkened in color somewhat, but had changed little (Fig. 4e) Upon staining overnight, epidermal cells and the mucilage halo stained very darkly. (Fig 4,f). The difference between wild type and pty-1 seeds was apparent after 20 minutes of staining. In pty-1 seeds, mucilage was extruded in discreet patches in approximately 50% of the mutant seed, and not in the other 50%. The extruded mucilage generally stained pink in both the mutant and the wild type seeds, although there was some variation in coloring among individual mutant seeds (Fig. 4a). After 2 hours of staining 80% of the mutant seeds extruded some mucilage. Of those 80%, some cells of the seed coat associated with mucilage patches are seen to be staining a darkish red.(Fig. 4b). When seeds were left staining and shaking overnight, 90% of pty-1 seeds extruded at least some mucilage, but only 1% succeed in extruding all of it. The remaining 10% 24 Figure 4: Appearance of pty-1 and wild type (WS ecotype) seeds after staining with Ruthenium Red for different time intervals. (a) pty-1 after 20 min. Note small patches of pink-staining mucilage as indicated with arrow. (37x) (b) pty-1 after 2 hrs, shows patches of mucilage staining more darkly than at 20 min.(37x) (c) pty-1 stained overnight shows red-staining seed coat and darkly staining patches of mucilage. (37x) (d) WS after 20 min shows darkly staining mucilage halo. (50x) (e) WS after 2 hrs (28x) (f) WS overnight shows very darkly staining mucilage (28x) have no patches and their epidermal cells stain red. Patches vary in size as does the extent of staining of the cells in the outer layer of the seed coat. Some seeds stained to a lesser extent and some were similar to wild type in appearance. Many were seen to stain to varying extents between these two extremes (Fig. 4c). 25 In order to further characterize the pty-1 phenotype, the development of the seed coat epidermal cells of pty-1 was characterized using scanning and light microscopy. 3.1.2 Development of the seed coat epidermis in wild type and in pty-1 seeds: Scanning electron microscopy and light microscopy of stained sections were used to characterize the development of the seed coat epidermis in pty-1 relative to that of wild type to determine the earliest observable defects in the cell type. This was done for seeds between 0 and 17 dap. Figure 5 summarizes the normal development of the wild type (WS ecotype) Arabidopsis seed coat epidermal cells. Events were similar to those described previously for the Columbia ecotype. The earliest event involves growth to produce cells 4 times the size of ovular cells (Fig 5, a,e). This is followed by an accumulation of amyloplasts 5 days after pollination (dap) (Fig 5 b,f,). At 7dap, the vacuole and cytoplasm shrink away from the outer tangential wall toward the center of the cell forming a columella-shaped structure (Fig. 5g) The cell produces mucilage at this time which accumulates between the plasma membrane and the primary cell wall (Fig. 5 c). Mucilage production continues with the deposition of a secondary cell wall outside the plasma membrane. The secondary cell wall gradually displaces the cytoplasm and forms the mature columella (Fig. 5d,h). 26 27 Fig 5. Developing cells of the wild type seed coat. Developing seeds (WS ecotype) were harvested at different times following pollination (days after pollination =dap), sectioned, stained with Toluidine Blue and observed by light microscopy (a-d) or observed by scanning electron microscopy (e-h). (a) . 2 dap. Arrow indicates outer layer of the outer integument of the ovule. (900x) (b) 5 dap. Arrow indicates amyloplasts (a). (600x) (c) 7 dap. Arrow indicates mucilage in epidermal cells (m). (280x) (d) 14 dap. Arrow indicates columella. (85x) (e) 2 dap. Shown are cells of the epidermis of the ovule. (f) 5 dap. Epidermal cells with thickened outer tangential cell wall and granular objects believed to be amyloplasts (arrow ). (g) 7 dap. Arrow indicates columella. (h) 14 dap. Magnification bars: (e)=25pm; (f)=100xpm; (g), (h)=40pm Using stained sections and SEM to analyze seed coat development in pty-1 no detectable differences were found between the mutant and wild type until 17dap (data not shown). At 17 dap, however, the extrusion of mucilage seemed to occur differently in the mutant than in the wild type. When wild type seed at 17dap were exposed to an aqueous solution, the primary wall of every cell ruptured and mucilage extruded (Fig. 6a). In contrast, in 60% oi pty-1 seed at 17dap, the primary cell walls of epidermal cells separated from the seed as an intact sheet, but did not rupture (compare Fig. 6 a, b). The extrusion of mucilage was thereby prevented, and mucilage remained trapped between the outer primary cell wall and the epidermis. These data indicate that the mutant is capable of making mucilage in every cell of its epidermis, even though it is not extruded in all cells. 28 ! Figure 6. Wild type (WS ecotype) and pty-1 developing seeds at 17 days after pollination sectioned, stained with Toluidine Blue and observed by light microscopy. (a) Wild type seed. Note the absence of mucilage and primary cell walls. Only the intact columella remains of the epidermal cells. Arrows indicate columellae (c), and regions previously occupied by mucilage deposits (md). (100x, insert = 200x) (b) pty seed. Note the presence of mucilage contained by the outer primary cell wall of the epidermal layer. Arrows indicate the outer primary cell wall (cw), mucilage (m) and columellae (c). (100x, insert = 240x) 3.1.3 Analysis of mature pty and wild type seeds: The mutant phenotype was most evident in mature dry seeds which had been allowed to dry either on the senesced parent plant or in storage for at least two weeks and then stained with Ruthenium Red. The oldest developing seed examined was only 7 days. Therefore, mature dry seeds were sectioned, stained with Toluidine Blue and observed using light microscopy. Under these conditions, both the mutant and the wild type seed had a similar phenotype in that the outer primary cell wall of the epidermal cells was ruptured and mucilage had escaped into the extracellular environment (Fig. 7). Remnants of outer primary cell wall material are seen to remain attached to the peak of the columella in both samples. The empty spaces between columella represents the space previously occupied by deposits of mucilage. 29 The results for sectioned mature dry seed presented in Fig. 7 seem to contradict the results for whole seed (Fig. 4) and for sectioned developing seed (17dap) (Fig 6). The experiments conducted on whole seeds and developing seeds suggest that pty-1 is defective in extrusion while experiments conducted on mature dry seeds seems to suggest that pty-1 extrusion is the same as wild type. The simplest explanation for this apparent paradox is that the mature dry seed could only be properly fixed and embedded by using vacuum infiltration of the dry seed coat both in glutaraldehyde and in osmium as well as long incubation times in resin solutions. Since seeds at 17dap were not treated so harshly, this may explain the difference between these two treatments. Fig 7. Comparison of wild type (WS ecotype) and pty-1 mature seed sections stained with Toluidine Blue and observed by light microscopy. (a) Wild type seed coat showing remnant primary cell wall material (cwm) and lack of mucilage.Arrows also indicates spaces previously occupied by mucilage deposits (md) before extrusion. (900x) (b) pty-1 seed coat showing no significant differences from the wild type sample. (900x) 30 Fig. 8 Scanning electron micrographs of wild type (a,c) and pty-1 (b,d) mature dry seed when dry-mounted (a, b) or fixed in aqueous glutaraldehyde to allow the potential extrusion of mucilage (c, d). (a) Wild type seeds dry mounted. Morphology of epidermal cells shows normal tangential cell walls (cw) and normal columellae (c) . (b) pty-1 seeds dry-mounted. Morphology of epidermal cells shows normal tangential cell walls (cw) and normal columellae (c). (c) Wild type seeds fixed in aqueous glutaraldehyde. Mucilage (m) has been extruded from all epidermal cells. (d) pty-1, seeds fixed in aqueous glutaraldehyde. Outer primary cell walls of individual epidermal cells act as an intact sheet to prevent the extrusion of mucilage. Magnification Bars: (b)=60pm; (a), (d)=100u.m; (c)=70um SEM data was collected for mature seed in aqueous fixative as described above and . for unfixed dry seed which was dry-mounted (no chemical fixation), sputter coated and viewed directly (Fig 8). The results indicate that unfixed, dry mutant seed appear virtually identical to wild type seed treated in the same way (Fig.8 compare a and b). Mature seed of the mutant which had been fixed in aqueous glutaraldehyde (allowing the extrusion of mucilage) before critical point drying and sputter coating displays a smoother seed coat with reduced columellar morphology where the hexagonal outer 31 tangential cell walls are invisible (Fig. 8d). This is similar to the behavior of the mutant at 17 dap when viewed as a stained section (Fig. 6b). Conversely, in the wild type, the outer tangential cell wall of each cell can be seen as an independent unit (Fig. 8c). 3.2 Genetic Analysis: 3.2.1 Inheritance of pty-1: Phenotypic analysis shows the pty-1 mutant to be defective in mucilage extrusion, although the specific nature of the defect remained unclear. To examine the inheritance of this mutation, pty-1 was crossed to wild type and the phenotypes in the F1 and F2 generations were characterized by immersing the seed in 0.01% Ruthenium Red. F1 seed was found to be wild type, suggesting that the mutation causing the pty-1 phenotype is recessive. In the F2 population, approximately one quarter of the plants had a pty-1 phenotype (223 wild type: 84 mutant). According to chi square analysis: I (O-E)2 = (84 - 76.75)2 + (223 - 23Q.25)2 = 0.48 E 76.75 230.25 where 0= number of observed individuals in a class, E= number of expected individuals in a class. So Oi=84, 02=223, Ei=76.75, E2=230.25, and the Null Hypothesis (Ho) is that the pty phenotype is due to a mutation at a single locus. The Chi Square value obtained is 0.48 and p > 0.1. We therefore accept the Null Hypothesis that the pty-1 phenotype is due to a mutation at a single locus. 3.2.2 Complementation tests: Complementation tests were performed to determine if pty-1 is allelic to known seed coat mutants. The pty-1 mutant was crossed previously to mum1-5, gl2 and ttg 32 mutants and F2 seed generated (Western and Haughn, unpublished data). The F1 phenotype of these crosses was determined by staining seed from each cross with Ruthenium red and examining the mucilage halo. Seeds from all crosses appeared wild type suggesting that the pty-1 mutant is defective at a novel locus: PTY. 3.2.3 Double mutant analysis: Seeds of several known seed coat mutants fail to extrude mucilage following exposure to an aqueous solution including mum1, (Fig. 9a; Western and Haughn, unpublished data). Figure 9. mum1 phenotype and ptyx mum1 double mutant seed phenotype after staining in Ruthenium Red (a) mum1 seed showing red staining but no extrusion of mucilage. (75x) (b) pty-1, mum1 double mutant seed showing lack of staining and lack of extrusion of mucilage. (75x) A possible interaction between PTY and the MUM1 gene product was tested by making a double mutant. Crosses were made, and the mucilage phenotype of F3 seed from individual F2 plants was determined by staining their seeds with Ruthenium Red as previously described. For this analysis, however, seeds were left in stain overnight 33 rather than for two hours. A novel phenotype was observed for F3 progeny of 9 F2 plants (Fig. 9b). Seeds with this novel phenotype failed to stain red and therefore appear pale when compared to stained wild type seed. No significant differences were observed when comparing dry, unstained seeds of this class with dry, unstained wild type seeds (data not shown). This indicates that this novel phenotype is not due to a alteration in pigmentation or testa coloration, but rather to the reduced staining of pectins in the epidermal cells with Ruthenium Red. Table 3 indicates all the phenotypes observed in the seed of F2 plants of the pty-1 x mum1 cross. Table 3: Numbers of different phenotypes seen in F2 seed of pty x mum1 cross and the numbers of each phenotype expected if the novel non-staining seed phenotype represents the double mutant class. Phenotypes pty-1 x mum1 obs. exp. wild type 94 107 pty-1 44 36 mum1 40 36 Non-staining 9 12 Chi-square 4.5(<7.8) The frequency of non-staining seed lines (approximately 1/16 of the F2 population) suggested that they are double mutants. This hypothesis was supported by chi-square analysis using the values in Table 3. This data from the pty-1 x mum1 cross was within the allowed Chi-Square value of 7.8 for 3df (p>0.1): (Q-E)2 = (9- 12)2 + (40 - 36)2 + (44 - 36)2 + (94- 107)2 = 4.5 E 12 36 36 107 34 Where Oi=9, O2=40, 03=44, 04=94, Ei=12, E2=36, E3=36, E4=107. We therefore accept the Null Hypothesis that these phenotypes are segregating in a 9:3:3:1 ratio. Test crosses later confirmed that plants with this novel phenotype were homozygous for mutations at both the pty-1 and mum1 loci (data not shown). Analysis of mature pty-1, mum1 double mutant seed by sectioning and by SEM was completed. These data indicate that the putative double mutants make mucilage (Fig. 10b), but like the mum1 mutant it is not extruded (Compare fig. 10a with b). In addition, the double mutants make a normal outer tangential cell wall as well as a normal columella (Fig. 10c) as is the case in both parental mutants (for the pty-1 parent, see Fig. 5g and 8b; data not shown for mum1). Figure 10. mum1 and pty-1, mum1 double mutant seed sections stained with Toluidine Blue and dry-mounted SEM of pty-1, mum1 double mutant. (a) mum1, mature dry seed stained with Toluidine Blue showing lack of extrusion of mucilage. (650x) (b) pry, mum1 Double mutant, mature dry seed stained with Toluidine Blue showing lack of extrusion of mucilage. (51 Ox) (c) pry, mum1 Double mutant, dry-mounted SEM showing normally shaped cell walls and columella. Magnification Bar: (c)=160u.m 35 3.2.4 Allelism test with scm2: During the course of this study a series of mutants (scm1-4) defective in seed coat mucilage were isolated and made available by Sattler and Marks (unpublished results). Complementation tests were completed between these mutants and all the mum mutants including pty. Table 4 summarizes the results and indicates allelism between mum2 and scm4 and also between scm2 and pty-1. Table 4. Results of Complementation Tests between mum and scm mutants. "c"=complements, "nc" = does not complement semi scm2 scm3 scm4 mum1 c c c c mum2 c c nc mum3 c c c c mum3 c c c mum5 c c c c pty-1 nc c c Since pty-1 and scm2 were shown to be alleles at the same locus, the scm2 allele will now be referred to as pty-2. Examination of the mature seed coat epidermis of pty-2 by light microscopy (Fig. 11a,d) and by SEM analysis (Fig 11 b, c) show that the epidermal cells appear to have low amounts of mucilage and abnormally shaped cell walls and columella. When stained with Ruthenium Red, pty-2 whole seeds stain more lightly than either mum1 or pty-1, and fails to extrude any mucilage (Fig. 11 a). These data suggest that pty-2 is a stronger allele than pfy-7, and that the PTV gene may affect the morphogenesis of this particular cell type including mucilage synthesis and formation of the columella. 36 Figure 11. pty-2 seed phenotype. (a) Ruthenium Red stained seeds showing lack of red staining which distinguishes them from wild type and mum1 seeds. (50x) (b) Dry-mounted SEM showing abnormally shaped cells and lack of columella. (c) SEM of seed fixed in aqueous glutaraldehyde showing lack of extrusion of mucilage. (d) section stained with Toluidine Blue. Arrows showing reduced mucilage (rm), abnormal columella (c) and abnormal outer tangential cell wall (tew). (680x) Magnification bars: (b), (c)=84u.m If pty-1 is weaker than pty-2, the phenotype of the pty-1'Ipty-2 heteroallelic mutant can be predicted to be less severe than the pty-2 phenotype, but more severe than pty-1. Data from the analysis of the pty-1/pty-2 heteroallelic mutant phenotype support this hypothesis. Unlike pty-2, the heteroallelic mutant appears to make a significant amount of mucilage (compare Fig. 11d with Fig. 12b), as well as a normal outer cell wall and columella (compare Fig. 11 b,c,d with Fig. 12 b,c,d). Unlike pty-1, the heteroallelic mutant fails to extrude mucilage in distinct patches (compare Fig. 4c with Fig. 12 a). In addition, the outer tangential cell wall of the heteroallelic mutant fails to lift off of the epidermal cells in the presence of aqueous fixative unlike the pty-1 mutant (compare figures 12 c and 8 d) These are essentially intermediate phenotypes as compared to those of the original parents, pty-1 and pty-2. 37 Figure 12. Phenotype of pty-11pty-2 heteroallelic mutant seed. (a) Ruthenium Red stained seeds showing lack of red color which distinguishes them from wild type and mum1 seeds. (65x) (b) Seeds sectioned and stained with Toluidine Blue showing the presence of mucilage. Note that the mucilage is not extruded. (350x) (c) SEM of seeds fixed in aqueous glutaraldehyde showing normal cell shape and columellae. (d) SEM of dry-mounted seeds showing normal cell shape and columellae. Magnification bars: (c), (d)=120um 3.3 Molecular Analysis: 3.3.1 Co-segregation of pty-1 with NPT2: The pty-1 mutant was isolated from a T-DNA transformed population. Therefore, the pty-1 mutation may be due to an insertion of a T-DNA fragment into the wild type PTV gene. Since this T-DNA fragment contains an NPT2 gene, it is expected to impart kanamycin resistance in plants. Cosegregation of kanamycin resistance and the pty-1 phenotype can therefore be used to test for linkage. For a cross oi pty-1 to wild type plants, all F1 progeny, and approximately three quarters of the F2 progeny were resistant to kanamycin (161 out of 223), suggesting that KanR was segregating 3 resistant: 1 sensitive as expected of a single Mendelian trait. This hypothesis was supported by Chi-square analysis. 38 X (0-E)2 = (62.0-55.8)2 + (161-167.2)2 = 0.9 E 55.8 167.2 Where Oi=62, 02=161, Ei=55.8, E2=167.2, chi-square = 0.9 and p >0.1. We therefore accept Ho that there is only one copy of the NPT2 gene. If the pty-1 mutation is due to a T-DNA insertion, all pty-1 mutants should be homozygous for the NPT2 gene. At least 9 F3 progeny from each of 15 pty mutants from the F2 generation were tested for kanamycin resistance. 100% of the 9 F3 progeny tested for each pty-1 mutant were found to be resitstant to kanamycin. The probability of such an event happeneing by chance assuming no linkage between pty-7and the T-DNA is [(0.75 chance 1 F3 progeny is KanR)9]15 = 1.4 x 10"17, essentially zero. In addition approximately 1/3 of the kanamycin resistant F2 progeny were pty-1 mutants (115 wild type to 48 pty-1). Y (O-E)2 = (48-54)2 + (115-108)2 = 1.1 E 54 108 Where 0-i=48, 02=115, Ei=54, E2=108 and Ho states that pty-1and NTP2 are linked. The obtained Chi-square value is 1.1 and p > 0.1. We therefore accept Ho that pty-1 and NPT2 are linked. These data strongly support the hypothesis that pty-1 and the T-DNA insert are linked. 39 3.3.2 Southern Analysis: Cloning of a gene into which the T-DNA has been inserted requires the isolation of genomic DNA flanking the insert. Since T-DNA often inters the genome as a concatamer and is associated with rearrangements it is prudent to determine the structure of the T-DNA prior to cloning. Analysis by Southern blotting was used to address this issue, while simultaneously confirming the presence of a single copy of the T-DNA. Hindlll Hindlll Hindlll Hindlll a. | I 1 S 4.4 7.2 2.3 2 EcoRI EcoRI pBR322 NPT1 NPT2 pBR322 NOS Left Border Right Border Figure 13. Structure of the T-DNA used to transform the population from which pty-1 was derived (c). The positions of the Hind\\\ (a) and EcoRI (b) restrictions endonuclease sites are shown. The numbers indicate the size of fragments. The bars above the T-DNA (c) indicate the position of the probes used. Gray shaded areas indicate flanking Arabidopsis genomic DNA for pty-1 as deduced from this study. Wild type and pty-1 genomic DNAs were digested with Hind\\\ and EcoRI and probed with three different probes corresponding to sequences found within the T-DNA: the NPT2 gene, the right border region (NOS) and the beta lactamase gene in pBR322 (Fig. 13). 40 A summary of the observed and expected band sizes for Southern blots of Hind\\\ and EcoRI digests is shown in Table 5. Expected bands of probes are based on the original T-DNA construct. The NPT2 probe hybridized to Hind\\\ fragments of approximately 7 kb and to EcoRI fragments of 7 kb (Fig. 14c) as expected (Fig. 13 and Table 5). The pBR322 probe hybridized to Hind\\\ fragments of 4 and 7 kb (Fig. 14a), as expected (Fig 13 and Table 5). The same probe hybridized to EcoRI fragments of both 7 kb (Fig. 14a), as expected (Fig. 13 and Table 5), and also to an 11 kb fragment, indicating the presence of an EcoRI restriction site approximately 5.5 kb outside the right Restriction Digest Hind\\\ EcoRI Probe expected observed expected observed Right Border 4kb Hkb pBR322 4kb 4kb 7kb 7kb 7kb 7kb 11kb NPT2 7kb "/kb 7kb 7kb Table 5: Observed and Expected band sizes for Southern blots of Hind\\\ and EcoRI digests. border region of the T-DNA (Fig. 13b). Finally, the right border probe hybridized to an 11 kb EcoRI fragment (Fig. 14b), as expected (Fig. 13 and Table 5), and also to a smaller 4 kb fragment (Fig. 14b), indicating the presence of a Hind\\\ restriction site approximately 2 kb outside the right border region (Fig. 13a). 41 a pBR322 probe pty-1 WS b. Right Border probe pty-1 WS Hindlll EcoRI Hindlll EcoRI Hindlll EcoRI Hindlll EcoRI c. NPT2 probe pty-1 WS Hindlll EcoRI Hindlll EcoRI Figure 14. Southern blots showing fragments oi pty-1 genomic DNA which hybridize to three T-DNA probes: pBR322 (a), the Right border region (b) and NPT2 (c). No hybridization was observed for wild type control genomic DNA. (a) The pBR322 probe hybridized to 7 kb and 4 kb bands of genomic DNA digested with Hind\\\ and 11 kb and 7 kb bands when digested with EcoRI. (b) The Right Border probe hybridized to a 4 kb fragment of genomic DNA digested with Hind\\\ and to an 11 kb fragment of genomic DNA digested with EcoRI. (c) The NPT2 probe hybridized to a 7 kb band of genomic DNA digested with Hind\\\ and a 7 kb band when digested with EcoRI. 42 3.3.3 Genetic mapping of pty-1: Mapping was carried out using Co-dominant Amplified Polymorphic Sequences (CAPS). These markers have been characterized to the extent that they detect polymorphisms only between Columbia and Landsberg ecotypes. Since pty-1 has the WS genetic background, the pty-1 allele of any CAPS marker could resemble either the , Columbia or Landsberg allele. Therefore, DNA was extracted from 100 F2 lines from both pty-1x Ler and pty-1x Col crosses. The seed coat phenotype of these F2 lines was determined and 18 pty-1 mutants selected from each cross. PCR amplification of genomic DNA of the mutant lines was done using specific DNA primers corresponding to known CAPS markers. Each marker represents a specific location in the Arabidopsis genome (Table 2). Amplification was followed by restriction digests with specific enzymes to reveal polymorphisms between the two ecotypes Columbia and Landsberg. If pty-1 were unlinked to a particular marker where WS corresponded to the. Columbia genotype (C), a 1:2:1 ratio of C:C/L:L in the F2 would be expected for any given CAPS marker. Linkage of pty-1 to a particular marker would be indicated by the presence of the Col allele at a frequency significantly higher than 50%. Given the small samples sizes used in this study, recombination frequencies of over 30% were considered unlinked. A summary of the data for all markers is shown in Table 6. The data indicate linkage between pty-1 and three CAPS markers all found on the distal end of chromosome 5: g2368, Lfy3 and EG7F2 (See "b" in Table 6). 43 Table 6. Summary of genotypes for various CAPS marker loci of F2 pty-1 mutants from pty-1 (WS) x Col and pty-1 (WS) x Ler crosses, "c" indicates a plant homozygous for a Columbia allele, "I" indicates a plant homozygous for the Landsberg allele and "h" indicates a heterozygote. Markers showing linkage to pty-1 are shown . Marker cross controls aSelected lines of F2 pty mutants Map distance co ler ws F1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 NCC1 m249 GL1 AG DFR bLFY3 bEG7F2 Ws x Col Ws x Col Ws x Col Ws x Col Ws x Col Ws x Col Ws x Col I h c I I h c I c I I c I c h c I I h c I I h c c ccl h h h I I hclchh hhch hlh Ichhh cl chhc he hhhc clhc llhlhh hhhchlhh chlhchcch Ihl Ihlllllcllclhll Ihllhlllllcllclhll unlinked unlinked unlinked unlinked unlinked b19.4% b19.4% controls ABCDEFGH I JKLMNOPQR PW4 ADH GPA1 GA1 bg2368 Ws x Ler Ws x Ler Ws x Ler Ws x Ler Ws x Ler c I c c I c h c I c I c h c I c h Illll cl c I I I c I hhh llch Ihlhlhhil chile hi hhchhih clhhh chh hccccchi cchcc ccl hets? d34.3% unlinked unlinked b21% controls Marker cross co ler ws F1 CF2 pty mutants chosen randomly PG11 GAPC ASA1 BGL1 Ws x Col Ws x Col Ws x Col Ws x Col I c I c I h c I I h h h h h h I I I I I I h h h h h c c hhhh I Icccc cccccl I I I Ihhhhh d30% (low #s) unlinked unlinked unlinked a Since CAPS markers reveal more polymorphisms between Col and Ler ecotypes than with WS, and since pty-1 only exists in the WS ecotype, F2 mutants were analyzed from WS x Col and WS x Ler crosses. Columns 1-18 indicate F2 mutants from a Ws x Col cross, and columns A-R indicate F2 mutants from a Ws x Ler cross b Indicates markers which show strong linkage to the pty locus. c These data are derived from pty-1 mutants from the F2 population of the cross pty-1 (WS) x Col. However, since the lineage for each F2 plant was not recorded, correspondance to lines 1-18 cannot be given. d Indicates markers which show weak linkage to the pty locus (% Recomb>30%). 44 a Marker GA1 pos. 17, Chr. 4. % recomb. = 17 Lalleles/ 36 chromosomes =47.2% CHCHHHC HHLH C LLHHLC b. Marker EG7F2 pos. 117 on Chr. 5 % recomb. = 7/36 = 19.4% LHLLLLHLLLCLLCLHLL c. Marker Lfy3 pos. 117 on chr. 5 % recomb. = 7/36 = 19.4% L H L L H LLLLLCLLCLHLL d. Marker g2368 pos. 125 on chr. 5 % recomb. = 8/28 = 28.6% Fig. 15. CAPS banding patterns of 4 different markers for individual pty-1 F2 plants. All lanes shown are results for pty F2 mutants selected from either a pty-1 x Col or a pty-1x Ler cross . A preponderance of the parental pty-1 genotype (WS ecotype) indicates linkage to a particular marker. The WS is similar to Columbia (c), or Landsberg (L), depending upon the cross, as indicated below, c = plant homozygous for Columbia allele. L = plant homozygous for Landsberg allele. H = plant heterozygous for Columbia and Landsberg alleles. Map positions from Lister and Dean Rl map (Jarvis, et. al., 1994). (a) Marker: GA1. WS genotype = "c". Results indicate that pty-1 is not linked to the marker GA1 since there is no significant preponderance of the "c" genotype in the F2 progeny tests. (b) Marker EG7F2. WS genotype = "L". Results indicate linkage of pty-1 to EG7F2 since there is significant preponderance of the "L" genotype. (c) Marker Lfy3. WS genotype = "L". This gel indicates linkage of pty-1 to Lfy3 since there is significant preponderance of the "L" genotype. (d) Marker g2368. WS genotype = "c". This gel indicates linkage of pty-1 to g2368 since there is significant preponderance of the "c" genotype. Data for markers EG7F2, Lfy3 and g2368 is shown in figure 15 b, c, d. The data indicate that pty-1 is located on the distal end of chromosome 5, within approximately 20 map units of the CAPS markers g2368, Lfy3 and EG7F2 (Fig. 16). Lfy3 (117) Pty? t I Distal End of Chromosome 5. (135) DFR EG7F2 g2368 (90) (116) (125) Figure 16. Proposed map location of pty-1 in relation to four CAPS markers on chromosome 5. 46 4. Discussion 4.7 The possible functions of the PTY gene The pty-2 allele appears to be the stronger of the two alleles of the PTY locus identified to date, pty-2 seeds show an abnormal columella and reduced amounts of mucilage. Three mutants described previously, ttg, gl2 and mum4 are known to have similar phenotypes (Western, thesis 1998,). The TTG and GL2 genes are cloned and encode putative transcription factors involved in regulating epidermal cell differentiation (Koorneef, 1991 and Rerie et. al. 1994). Therefore, the PTV gene may also encode a transcription factor regulating seed-coat epidermis differentiation. Alternatively, the PTY gene could encode a seed coat epidermis specific component of the cytoskeleton. For example, since polar secretion depends upon the cytoskeleton for vesicle transport and for cytoplasmic rearrangements (Fowler, 1997), a cytoskeletal defect could be responsible for reduced amounts of mucilage and abnormally shaped columellae. These defects could, in turn, be responsible for reduced extrusion by decreasing the amount of pressure exerted by intracellular components upon the outer tangential cell wall. 4.2 The pty-1 phenotype: Phenotypic analyses suggest that the pty-1 mutant is defective in extruding seed mucilage. Rather than extruding mucilage over the entire surface of the seed, the pty-1 mutant extrudes seed mucilage only in patches when placed in an aqueous solution of Ruthenium Red (Fig. 4c). In addition, mucilage in pty-1 is seen to be contained by the 47 outer cell wall in seeds sectioned later in development (Fig, 6b). Given the results and hypotheses obtained from data on the stronger pty-2 allele, the simplest explanation for the pty-1 phenotype is that a small reduction in the amount of mucilage made leads to a decreased hydrostatic pressure in the seed coat upon imbibition causing defects in extrusion. However, the possibilities that the pty-1 extrusion defects are due to differences in pty-1 mucilage composition or cell wall structure cannot be ruled out since this hypothesis was not directly tested. It is possible that the patchiness of pty-1 is due to a threshold effect. For example, since pty-1 is a weak allele, perhaps reduced function at that locus is sufficient ' to prevent extrusion only in some cells. This could depend upon natural, but slight variations in mucilage quantities between adjacent cells where some are above, and some are below the minimum or "threshold" quantity of mucilage which allows extrusion. This hypothesis would also explain the variability of the phenotype in terms of the different sizes of patches seen on different seeds. 4.3 PTY and MUM1 both affect the extrusion of mucilage: pty-1 x mum1 double mutants were shown to make mucilage, but they fail to extrude it, like the mum1 parent (Fig. 10b. Unlike either parent (especially mum1), the double mutant fails to stain in Ruthenium Red (Fig 9b) even though Toluidine blue staining indicates that mucilage is present (Fig. 10) The simplest explanation for these data is that the double mutant is both deficient in the chemical composition if its mucilage (imparted by the mum1 parent) and also deficient in the amount of mucilage made (imparted by the pty-1 parent). 48 The mum1 phenotype, therefore, may be due to a change in composition of mucilage and the pty-1 phenotype may be to a reduction in the amount of mucilage in epidermal cells, as indicated by the stronger pty-2 phenotype. In addition, since the pty-2 mutant phenotype suggests a more general upstream role for the PTV gene, it may also be possible that both chemistry and amount of mucilage could be affected by a mutation at this gene. This hypothesis would also be consistent with the observed double mutant phenotype. 4.4 The T-DNA insert in pty-1 is intact: Cloning of the PTV gene may provide insight into the biochemical role of the PTY gene in wild type plants. The T-DNA seems to be intact in structure and linked to the PTY gene. Therefore, isolation of DNA from the PTY locus should be possible. The restriction sites which are present in the flanking genomic DNA are shown in Figure 13 a, b. With this information, it should be possible to clone the pty-1 gene either by plasmid rescue (Gallois et. al., 1992) or by Inverse PCR (iPCR) (Dean, et. al., 1991). Both of these techniques were attempted more than once with no success but should be repeated. Positional cloning (Shih et. al., 1991) may be an option given that the approximate map position of PTY is already known. 4.5 pty-1 maps to distal chromosome 5: The map position of pty-1 was determined as an alternative method to cloning the gene given the difficulties encountered with plasmid rescue and iPCR. Linkage 49 was shown to exist between the pty-1 mutation and three CAPS markers on distal chromosome 5 (Lfy3, EG7F2 and g2368). The position oi pty-1 was originally tested and confirmed to be approximately 20cM away from Lfy_3. The g2368 marker was chosen to establish whether pty-1 was proximal or distal of Lfy3. Suprisingly, the recombination frequency between pty-1 and g2368 was also found to be approximately 20cM. An explanation for this problem is that g2368 is not where it is thought to be on the Rl map relative to Lfy3. This region is not as well characterized as other regions of the genome and discrepancies in the placement of markers on the map is possible. In addition, error inherent to using relatively small sample sizes may have produced these conflicting results. Since pty-1 was shown to be unlinked to DFR (the CAPS marker proximal to Lfy3 on chromosome 5) but linked to both Lfy3 and EG7F2, I proposed that pty-1 is located distal to g2368 on chromosome 5 (Fig. 16). This location assumes that the recombination frequencies between pty, g2368, and Lfy3 were underestimated by approximately 7%, and that that the recombination frequency between pty and g2368 was overestimated by approximately 7%. The conflicting results obtained for the markers shown to be linked to pty-1 on chromosome 5 can be resolved. Once the Arabidopsis sequencing project is complete and additional markers define the proposed map location of PTY, it should be relatively simple to map PTY more accurately. 50 Conclusions: This study of the PTY locus has shown that the pty-1 and pty-2 alleles are involved in controlling the morphogenesis of mucilage-producing cells of the seed coat epidermis, pty-1 is a T-DNA tagged allele. The T-DNA insert is intact and does not seem to have rearranged upon insertion into the genome. PTY maps to distal chromosome 5 in the vicinity of the CAPS marker Lfy3. Future directions: The discovery that pty-1 is allelic to pty-2 dictates that the next step in this project is to further characterize the pty-2/pty-2 mutant. Although mature dry seed has been sectioned and studied by SEM, analysis by these same means should be undertaken to characterize this mutant throughout its development (0-18daf). In addition, analysis of mucilage extracts using gas chromatography and closer analysis of the cell wall by transmission electron microscopy may be necessary to further specify the nature of the phenotypic defect. An examination of other tissues of the plant (trichomes, root hairs, flowers) should also be undertaken to determine if the effects of the pty-2 mutation are pleiotropic. This would provide better data on the nature and timing of the phenotypic defect as a result of a mutation at this locus which was apparently more severe than that of the pty-1 allele. Since double mutant analysis yielded interesting results when pty-1 was crossed with mum1, a cross should also be done with pty-2. In fact, attempts could be made at constructing double mutants with mum1, 2 and 4 as well as other mutants known to affect morphogenesis of epidermal cells such as ttg and gl2. 51 Finally, cloning and sequencing of the pty-2 allele may be less problematic than with pfy-1 and should be attempted using the same means. However, since the T-DNA orientation and map position in pty-1 have already been studied, it may be more expedient to continue to focus on pty-1 for future cloning attempts. 52 Bibliography Boeswinkel, D. and Bouman, F (1984). The seed: structure. In: Embryology of Angiosperms. Spreinger-Verlag, New York, 567-610. Boesewinkel, F. and Bouman, F. (1995). The seed: structure and function. In: Seed Development and Germination. Ed. Kigel, J. and Galili, G., Marcel Deker, Inc., New York, 1-24 Bowman, J., Smyth, D. and Meyerowitz, E. (1989). Genes directing flower development in Arabidopsis. Plant Cell. 1(1):37-52. Bystedt, P. and Vennigerholz, F. (1991). 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Fowler, J. and Quatrano, R. (1997). Plant Cell Morphogenesis: Plasma Membrane interactions with the cytoskeleton and Cell Wall. Annual Review in Cell Developmental Biology 13,697-743. Gallois, P., Lindsey, K. Malone, R. Kreis, m and Jones, M. (1992). Gene rescue in plants by direct gene transfer of total genomic DNA into protoplasts. Nucleic Acids Research 20(15):3977-82. Goodwin, T. and Mercer, E. (1972). Introduction to Plant Biochemistry. Pergamon Oxford Gould, J. and Northcote, D. (1986). Cell-Cell Recognition of Host Surfaces by Pathogens. Biochemical Journal 233, 395-405. Grant, g., NcNab, D., Rees, D. and Skerrett, R. (1969). Seed Mucilages as Examples of Polysaccharide Denaturation. Chemical communications 785, 805-806. Gregory, M. and Baas, P. (1989). A Survey of Mucilage Cells in Vegetative Organs of the Dicotyledons: a Review. Israel Journal of Botany 38, 125- 174. Hirst, E., Rees, D. and Richardson, N. (1965). Seed Polysaccharides and their Role in Germination. 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(1991). A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana. Mol Gen Genet.229(1):57-66. Leon-KI oosterziel, van de Bunt, G., Zeevaart, J. and Koorneef, M. (1996). Mutants with Reduced Seeed Dormancy. Plant Physiology 110,233-240 Mauseth, J. (1988). Plant Anatomy. California: Benjamin/Cummings. Moore, P., Swords, K., Lynch, M. and Staehelin, A. (1991). Spatial Organization of the Assembly Pathways of glycoproteins and complex Polysaccharides in the Golgi Apparatus of Plants. The Journal of Cell biology 112(4), 589-602). Morton, J. (1990). Mucilaginous Plants and their Uses in Medicine. Journal of Ethnopharmacology 29, 245-266. Northcote, D. (1969). Fine structure of cytoplasm in relation to synthesis and secretion in plant cells. Proceedings of the Royal Society of Botany 173, 21-30. Northcote, D. and Gould, J. (1989). Cell-cell recognition of host surfaces by pathogens. The adsorption of maize (Zea mays) root mucilage by surfaces of pathogenic fungi. Biochemistry Journal 233(2):395-405. Reiser, L., Modrusan, Z., Margossian, L., Samach, A., Ohad, N., Haughn, G. and Fischer, R. (1995). The BELLI gene encodes a homeodomain protein involved in pattern formation in the Arabidopsis ovule primordium. Cell. 1 ;83(5):735-42. Rerie, W. G., Feldmann, K.A. and Marks, M.D. (1994). The GLABRA2 gene encodes a homeo domain protein requred for normal trichome development in Arabidopsis. Genes and Development 8, 1388-1399. Sambrook, J., Fritsch, E. and Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual. 2nd. Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Shih, M., Heinrich, P. and Goodman, H. (1991). Cloning and chromosomal mapping of nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate-dehydrogenase from Arabidopsis thaliana. Gene 104(2): 133-8. 55 

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