Honours Thesis:
An Investigation of Self-Incompatibility in Turnera Species Using Light and Fluorescence Microscopy, and an in vitro Pollen Germination System.
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by: Sebastian Molnar Abstract. Homostyly can arise through recombination in a putative distyly supergene. A controlled crossing program was used to test the recombinant hypothesis through identifying compatible and incompatible pollinations in the progeny of a self-compatible mutant homostyle (Turnera). Analysis using the G-statistic revealed heterogeneity between pollinations. The homostyle progeny exhibited self-compatibility as expected, as well as compatibility with shorts, and incompatibility with longs. Unexpected results were obtained with long styled plants. Longs are expected to be self-incompatible, and yet selfed longs were not significantly different in seed production from longs crossed with shorts. This would suggest that pollen from such longs contain incompatibility features of pollen from shorts. Further testing, with immunodetection to determine the presence or absence of self-incompatibility proteins, is required to resolve this enigma. An in vitro pollen germination system was developed for species of Turnera. A two-phase system was found to generate a higher germination frequency than a single-phase system. Components of the pollen germination medium (PGM) were optimized using the two-phase system. The optimized PGM consisted of 30% sucrose, 0.5 mM boric acid, and 10 mM CaCl2 (pH 4.2, not adjusted). The addition of polygalacturonase to the PGM resulted in a significantly lower germination frequency, as well as a lower frequency of intact pollen tubes, than the controls. It is expected that a polygalacturonase involved in self-incompatibility would be morph-specific in its inhibition of pollen tube growth. The addition of style-specific polygalacturonases to the PGM, however, remains untested.
Below, I have provided only the introduction to my thesis and the references that I used. I did not included anything else as it would require vast amounts of time to modify all the figures, tables, and diagrams I created. I did include some photos that I took to help illustrate what I actually did.
I. Introduction I.1: Self-incompatibility systems Reproductive systems play important roles in the evolution of species. The plant kingdom contains a variety of reproductive systems, with hermaphroditism being the most common (Dellaporta and Calderon-Urrea, 1993). The presence of male and female sex organs within the same flower, or on the same individual plant, can increase the chances of self-pollination. Deleterious alleles may accumulate through selfing and can result in a decrease in the fitness of selfed progeny relative to outcrossed progeny (Weller and Ornduff, 1991), although inbreeding depression can be alleviated by an increase in polyploid level (e.g. Husband and Schemske, 1997). Plants have, therefore, evolved a number of mechanisms to promote outcrossing, such as dichogamy, dioecy, and self-incompatibility (Barrett, 1992; Bateman, 1952; Dellaporta and Calderon-Urrea, 1993). Self-incompatibility is a genetic mechanism that tends to prevent zygote formation in a selfed plant which produces viable gametes. There are two major types of self-incompatibility (SI): homomorphic SI, and heterostylous SI. Homomorphic SI is characterized by the occurrence of multiple alleles at an ‘S’ locus (Brewbaker, 1957; Haring et al., 1990), and is itself composed of two distinct incompatibility systems, which will be discussed only briefly here. Gametophytic homomorphic SI occurs when pollen is rejected if it has an allele at the S locus that is also carried by the pollen recipient. In contrast, the sporophytic homomorphic SI system inhibits fertilization when either S allele of the pollen parent is found in the pollen recipient. Species with the gametophytic SI system (e.g. Nicotiana spp.) typically have binucleate pollen grains and pollen tube growth is inhibited in the style, whereas species with the sporophytic SI system (e.g. Brassica spp.) have trinucleate pollen and inhibition occurs at the stigma (Brewbaker, 1957). Some exceptions to this generalization can be found in the Poaceae: pollen tube growth is inhibited in the style and yet grass species have trinucleate pollen (Brewbaker, 1957). Sequence comparisons of S-locus genes from different families reveal that homomorphic SI systems probably evolved independently on separate occasions (Haring et al., 1990). In the Solanaceae, S-glycoproteins show sequence homology to fungal RNases (reviewed in Haring et al., 1990; and in Matton et al., 1994). In the Brassicaceae, the S locus genes encode an extracellular S-locus glycoprotein and a serine/threonine kinase (Stein et al., 1991; Goring and Rothstein, 1992). The S-locus genes in the Papaveraceae encode a glycoprotein that is unrelated to the S locus products in either the Brassicaceae or the Solanaceae, and may be involved in calcium signaling that is associated with the actin cytoskeleton (Foote et al., 1994; Franklin-Tong, 1999; Geitmann et al., 2000). Continuing genetic and molecular studies will likely reveal further differences in the self-incompatibility mechanisms of other plant families. The second major type of self-incompatibility is heterostyly. Unlike homomorphic SI, heterostylous self-incompatibility is a diallelic genetic polymorphism associated with floral morphs exhibiting reciprocal herkogamy (Barrett, 1992). Distylous species, as in the Primulaceae and Turneraceae, have two floral morphs being either long styles with short stamens, or short styles with long stamens. Tristylous species, as in the Lythraceae and Pontederiaceae, have three floral morphs: long-, mid-, or short-styles with two sets of anther whorls at heights separate from that of the stigmas. There may also be floral-morph associated differences in pollen size, pollen production, and exine sculpturing, as well as in size and shape of the stigmas and papillae (reviewed in Dulberger, 1992). As with the homomorphic SI systems, heterostyly probably evolved independently on separate occasions, as it occurs in at least 25 different families (Charlesworth, 1982; Lloyd and Webb, 1992a; Vuilleumier, 1965). It also seems that the mechanism of self-incompatibility can differ between morphs in the same species (Athanasiou and Shore, 1997; Lloyd and Webb, 1992a). Heterostylous species are animal pollinated, rather than wind pollinated. ‘Legitimate’ pollination occurs when pollen is transferred to a stigma from an anther at the same relative height. In distyly, pollen from a long-styled morph transferred to the stigma of a short styled morph, and pollen from a short-styled morph transferred to the stigma of a long styled morph, are legitimate pollinations. ‘Illegitimate’ pollinations occur when pollen is transferred from an anther at a different relative height to the recipient stigma (i.e. selfing or intra-morph pollination). As suggested by Darwin in 1877, the floral polymorphism appears to enhance fecundity through increasing legitimate pollinations, while the self-incompatibility response functions to promote outcrossing (Ganders, 1974; Barrett and Glover, 1985; reviewed in Lloyd and Webb, 1992a, b). The genetics of heterostyly has been described in some species (reviewed in Lewis and Jones, 1992). The distyly S-locus is a supergene consisting of at least three (and possibly six) tightly linked genes which control the style length (G), pollen size (P), and anther height (A), as well as the pollen and stylar incompatibility (Lewis and Jones, 1992). Despite a few exceptions, long-styled plants are, in general, homozygous recessive, while short-styled plants are dominant, for both distylous and tristylous species (Lewis and Jones, 1992). Heterostyly can break down through recombination and give rise to homostyly (Barrett, 1992; Lewis and Jones, 1992; Shore and Barrett, 1985). Homostyly is the occurrence of anthers and stigmas at the same relative heights. The recombinant hypothesis predicts that homostylous plants can accept pollen from shorts, as well as fertilize longs, while rejecting pollen from longs and are unable to fertilize shorts. Homostylous plants are also self-compatible. This is due to the presence of the pollen incompatibility features of one floral type (e.g. pollen from short-styled plants), with the stylar incompatibility features of the other type (e.g. long-styles). The recombinant hypothesis can be tested through inter- and intra-morph pollinations directly with homostylous plants, or with their descendents. Residual effects of the incompatibility response are expected in pollinations of progeny from homostyles that are crossed with long and short styled plants. As such, this is one component that the present study will focus on. Pollen tubes produce callose, a b-1,3-glucose polymer, which functions by forming plugs that section off the pollen tube cytoplasm as they grow down a style (Stanley, 1971). Callose plugs can be detected under fluorescence microscopy after staining with aniline blue (Martin, 1958). Therefore, compatibility and incompatibility responses can be detected in styles by observing the presence of callose plugs with fluorescence microscopy. The second component of this paper, in a related aspect of plant reproduction and self-incompatibility, deals with pollen germination and pollen tube growth. I.2: The Pollen Grain and Pollen Tube Growth Pollination occurs when pollen is transferred (via wind, water, or animal) to the stigma. If the conditions are appropriate, the pollen grain undergoes hydration and then it germinates to develop a pollen tube, which grows down the style in the transmitting tissue towards the ovules (Mascarenhas, 1989). The pollen tube is guided to the filiform apparatus at the micropylar end of the ovule, through which the pollen tube penetrates into one of two synergid cells in the embryo sac (Higashiyama et al., 1998; Wilhelmi and Preuss, 1996). Recently, Higashiyama et al. (2000) have demonstrated that the pollen tube ruptures and explosively discharges its contents through the tip, which is followed by the degeneration of the synergid. The mechanism by which the immotile sperm cells are transported to carry out double fertilization is uncertain (Higashiyama et al., 2000), however, they may be transported by myosin along actin filaments (Zhang and Russell, 1999). One sperm cell fuses with the egg cell, and the second sperm cell fuses with the polar nuclei, to complete double fertilization (Russell, 1993). The pollen grain is the male gametophyte (i.e. it contains the sperm cells). Pollen grains derived from meiotic divisions of pollen mother cells (PMCs) in the anther (Mascarenhas, 1989). There are two types of angiosperm pollen grain cytology: binucleate, and trinucleate. Most angiosperms produce binucleate pollen grains (Brewbaker, 1967). Binucleate pollen grains are those that have only the generative cell and the vegetative cell upon release from the anther. The generative cell later undergoes mitosis to form two sperm cells during pollen tube growth. In contrast, the trinucleate pollen grain is one that has the vegetative cell and two sperm cells upon release from the anther. The Turneraceae, a Neotropical plant family, have pollen of the binucleate type (Brewbaker, 1967). Distylous species of Turnera also exhibit pollen size dimorphism, with pollen from short-styled plants having a larger size than pollen from long-styled plants. Pollen development and biochemistry has been studied extensively (e.g. Brewbaker, 1971; Stanley, 1971; reviewed in Mascarenhas, 1975). Although the mature pollen grain contains much of what it requires (e.g. organelles, RNA and proteins) for its germination and early tube growth, both transcription and translation are activated upon hydration, and continue during germination and tube growth (reviewed in Mascarenhas, 1971 and 1975). Pollen is released from the anther in a dehydrated state, and then it hydrates when it is deposited on a stigma. The pollen grain wall and apertures act as “harmomegathic” devices that allow for volumetric-changes upon dehydration and re-hydration (Blackmore and Barnes, 1986; Thanikaimoni, 1986). The apertures also function in germination for pollen tube emergence (Thanikaimoni, 1986). Pollen tube growth occurs by the addition of cell wall components (e.g. pectin), that are transported by vesicles, to the tip region (Franklin-Tong, 1999; Stepka et al., 2000). Understanding mechanisms in pollen tube growth may provide insight into other tip-growing cells. Tip growth is a characteristic feature in root hairs (Schiefelbien et al., 1993) and fungal hyphae (Heath, 1995), axon guidance (Palanivelu and Preuss, 2000), and ameboid movement (Heath and Steinberg, 1999). Although there are differences in these systems (Heath and Steinberg, 1999; Palanivelu and Preuss, 2000), there are also a number of similarities in the mechanisms involved in tip growth. For example, the presence of a tip-high calcium gradient has been implicated in the regulation of tip growth (Felle and Hepler, 1997; Franklin-Tong, 1999; Jackson and Heath, 1993). The apical calcium gradient may be involved in regulating vesicle secretion (Franklin-Tong, 1999). Calcium ions also cross-link de-esterified pectins and provide rigidity to the pollen tube wall (Franklin-Tong, 1999; Morris et al., 1982; Powell et al., 1982). Pectin is a major component of plant cell walls and has been shown to play important roles in a number of developmental stages (e.g. Rhee and Somerville, 1998). Pectinases (or polygalacturonases), which hydolyse a-1,4-glycosidic bonds in pectic substances, occur in plants (Torki et al., 2000), bacteria (Chanliand and Gidley, 1999), and fungi (Blanco et al., 1999). Pectinases are involved in pollen grain development (Allen and Lonsdale, 1993; Rhee and Somerville, 1998), fruit ripening and organ abscission, and have been used in the food industry (Blanco et al., 1999). Pectin is a major component of pollen tube walls, and therefore may provide style-specific polygalacturonases with a role in the self-incompatibility response of certain species (e.g. Turnera spp.). This idea can be tested with in vitro pollen germination systems. I.3: Objectives The present study is divided into two major components. The first part deals with an investigation into the inheritance patterns of a self-compatible mutant homostyle. This homostyle arose as a spontaneous somatic mutation from an otherwise short-styled plant, and has been crossed to a number of long- and short-styled plants. The occurrence of self-compatibility in long- and homo-styled progeny from these crosses will be used to test the recombinant hypothesis for the origin of the homostyle. In the second part of this study, an in vitro system for pollen germination is developed for species of Turnera. A stylar polygalacturonase may be involved in the self-incompatibility response. Therefore, it will be determined whether the addition of polygalacturonase to the pollen germination system inhibits pollen tube growth.
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