- Top of page
- Materials and Methods
Primula plants have heteromorphic flowers and a sporophytically controlled, diallelic incompatibility system that prevents or reduces both self-fertilization and intramorph fertilization. The two forms of flower – pin and thrum – develop on separate plants. Thrum plants have flowers with anthers visible at the mouth of the corolla tube, and the stigma approximately half way down the corolla tube on a short style and with short stigmatic papillae. Pin plants have flowers with the stigma presented at the mouth of the corolla tube, long stigmatic papillae, and anthers approximately half way down inside the corolla tube. The visible differences in flower morphology are shown in Fig. 1. Heteromorphy is common in Primula, with 91% of all species showing this phenomenon (Richards, 1993), but it is not limited to Primula, and has been documented in a number of species (reviewed in Ganders, 1979; Barrett et al., 2000) with examples found in 28 different families (Barrett et al., 2000). A number of these examples have attracted specific attention, including Linum (for example Dulberger, 1973) and Turnera (for example Truyens et al., 2005), but the genus which has been most widely studied is Primula.
Figure 1. Pin and thrum flowers of Primula vulgaris var. Blue Jeans. Longitudinal section of pin flower (a) and thrum flower (b). The length of the style and the positions of the stigma and anthers are shown. The lengths of the upper and lower corolla tubes are also indicated. Top view of the pin (c) and thrum flowers (d) showing the diameter of the flower, the extent of the petal and the diameter of the flower mouth. All size bars are 5 mm.
Download figure to PowerPoint
Observations on the significance of the reciprocal arrangement of reproductive organs in promoting out-crossing in Primula were documented in the middle of the 19th century (Darwin, 1862; Hildebrand, 1863). Subsequent to this, other associated differences between the two different floral morphs were reported in different species of Primula. These differences include: (1) pollen size – the pollen produced by pin plants is smaller than that produced by thrum plants (Darwin, 1862; Ornduff, 1979; Heslop-Harrison et al., 1981); (2) pollen number – anthers from pin plants produce twice as much pollen as anthers from thrum plants (Piper & Charlesworth, 1986); (3) style cell length and stigmatic papillae length – the presence of longer cells in the style and longer stigmatic papillae in pin flowers as compared to thrum flowers has been observed (Ganders, 1979; Heslop-Harrison et al., 1981). It has been proposed that stigmatic papillae length is a correlate of style cell length (Richards, 1993), and a mutation in P. sinensis was found that reduces style cell length as well as shortening the length of the stigmatic papillae (Mather, 1950). Other studies on pin and thrum styles from P. sinensis (Stirling, 1932) revealed that, in addition to the presence of longer cells in pin styles, there were also more cells in the pin style than in the thrum style. The differences in style length, pollen size and anther position between pin and thrum flowers are controlled by genes designated G, P and A, respectively, which are tightly linked and collectively known as the S locus.
In combination, these differences in flower form promote intermorph crosses (Darwin, 1862). Pin plants are homozygous for the s allele (ss), and thrum plants heterozygous with a dominant S allele (Ss). Before the genetic basis for this observation had been established, the significance of this breeding system was recognised and documented by Darwin (Darwin, 1862), who observed that pin-thrum and thrum-pin crosses yielded equal numbers of pin and thrum progeny. This ratio is what would be expected from crosses between ss and Ss parents. Although illegitimate within-morph pollinations can be achieved, the seed set in such cases is dramatically reduced, and homozygous true-breeding thrum plants are rarely obtained from such forced crosses between thrum plants (Wedderburn & Richards, 1990), indicating the lethality of this genotype and suggesting the presence of an additional gene at the S locus that prevents SS homozygosity (Kurian & Richards, 1997). Other genes linked to the S locus, but not directly involved in the control of heteromorphy, include Hose in Hose (Ernst, 1931; Webster & Grant, 1990), sepaloid (Webster & Gilmartin, 2003), Staminoid Carpels, a possible new allele of Hose in Hose (Webster & Gilmartin, 2003) and Oak Leaf (Webster, 2005). In addition, the further genes magenta (mg) and maroon (ma) involved in the control of red and purple flower colour, have been localised some distance at either side of the S locus (Kurian, 1986; Richards, 1997).
As a prelude to the molecular genetic investigation of heteromorphic flower development in Primula it is important to fully define the developmental events that coordinate the formation of pin and thrum flowers. Knowledge of the processes and insight into the mechanisms, such as cell elongation and cell division, that result in the differential development and placement of structures within the two forms of flowers, is required to inform the molecular analysis of genes implicated in the control of this phenomenon. Furthermore, a definition of the timing of distinct events that lead to differential formation of pin and thrum flower forms will contribute to an interpretation of the coordination in gene expression once candidate genes at the S locus have been identified.
We have previously defined the early stages of development of both wild-type and mutant forms of P. vulgaris (Webster & Gilmartin, 2003) and found that Primula flowers are homomorphic during their early development. Here we have investigated pin and thrum flowers to reveal the temporal development of heteromorphy in P. vulgaris. We have analysed cell morphology in pin and thrum flowers in order to investigate the basis of differences in style and anther positions between them. A previously undocumented heteromorphic feature was discovered by comparing pin and thrum flowers of the same size and with the normal complement of five petals. The thrum corolla tube mouth is demonstrably wider than the pin corolla tube mouth.
These analyses not only underpin ongoing investigations identifying and characterizing genes located at the Primula S locus, but will contribute to their subsequent functional analysis. Once such genes are identified, they will provide appropriate tools for an investigation and interpretation of the evolutionary events which resulted in the establishment of heterostyly in Primula.
- Top of page
- Materials and Methods
The presence of pin and thrum flowers in Primula and the phenomenon of heteromorphic flower development has reputedly been known since Clusius, 1583 (van Dijk, 1943, quoted in Ornduff, 1993), and has been of scientific interest since Darwin, 1877. However, there is as yet no insight into the identity of the three genes G, P and A, or understanding of the cellular processes controlled by these genes that contribute to the differential development of pin and thrum flowers. This analysis provides new insight into the temporal development of heteromorphic flowers and in addition we revealed a previously unreported characteristic of heteromorphic flowers in P. vulgaris.
Definition of the upper and lower corolla tube
Previous ontogenetic studies on other species has revealed that, where stamens are attached to the corolla tube, the two parts of the tube above and below the anthers can be formed by two spatially and temporally separate processes (Erbar, 1991). In Vinca for example, the upper corolla tube is formed by post-genital fusion while the lower corolla tube forms by congenital fusion (Boke, 1948). This division of the corolla tube into two parts, the upper corolla tube as the part above the point of anther attachment and the lower corolla tube as the part below anther attachment, has also been employed by Sporne (Sporne, 1974). Studies in Lactuca sativa and other members of the Compositae with epipetalous stamens have revealed that both parts of the petal develop by intercalary growth (Sporn, 1974). The lower part of the corolla tube develops later than the upper part of the corolla tube and, as the lower corolla tube develops, the stamens are carried up to their final epipetalous position. Erbar also referred to the lower corolla tube as the stamen-corolla tube (Erbar, 1991). Expression analysis of the Primula vulgaris MADS box gene PvPlena (Cook, 2002), which is the presumed homologue of the Antirrhinum and Arabidopsis C function genes Plena (Carpenter & Coen, 1990; Bradley et al., 1993) and Agamous (Bowman et al. 1989; Yanofsky et al., 1990), revealed that this gene is expressed in the lower corolla tube, but not in the upper corolla tube above the anthers (Cook, 2002). This observation is in agreement with Erbar's definition of the stamen-corolla tube (Erbar, 1991).
As shown in Fig. 2, development of the upper corolla tube in Primula flowers proceeds ahead of expansion of the lower corolla tube in both pin and thrum flowers. However, when elongation of the lower corolla tube initiates, this growth is observed earlier in thrum than in pin flowers (Fig. 2e,f). Observation of a series of developing flowers from eight thrum plants (not shown) revealed that in all cases, growth of the lower corolla tube was evident by the time whorl 2 had reached approximately 10 mm in length. In contrast, observation of a series of developing flowers from 19 pin plants (not shown) revealed only two that exhibited any elongation of the lower corolla tube by the time that whorl 2 had reached 10 mm. This observation reveals some flexibility in the temporal aspect of growth, but it is the differential physical growth of the corolla tube above and below the attachment point of the stamens that results in different anther positions in the two morphs. Differential physical growth of the corolla tube above and below the anthers is therefore an integral feature of heteromorphy in Primula.
The temporal development of heteromorphic characteristics sheds light on the functioning of S locus genes
Despite considerable interest in the S locus for very many years (Ernst, 1936; Lewis & Jones, 1993; Richards, 1997), there have been no previous observations on the timing of events that lead to the final architecture of the mature Primula flower, and our data provides the first visual analysis of the developmental timing of heteromorphy. We have shown that observable differences between pin and thrum flowers are first seen in buds of 5 mm containing a corolla of 2 mm (Fig. 2a,b), when in pin flowers, the stigma is slightly elevated above the top of the anthers (Fig. 2a) and in the corresponding thrum flower the tip of the stigma remains below the top the anthers (Fig. 2b). The observation that pin style elongation is the first discernible step in heteromorphic development suggests that the visible effects mediated by the G locus may be the first to be implemented. Pin plants are homozygous for the recessive g allele, and therefore an initial key event in Primula heteromorphy may be the action of the dominant G allele in thrum flowers in inhibiting style elongation. Elevation of the thrum anthers as observed by growth of the lower corolla tube is not discernible until the flowers are at least 11 mm long and contain a corolla of 7 mm (Fig. 2e,f) indicating that since the visible effect of A activity is seen later than the visible effect of G activity it is possible that the timing of A activity may occur later than G activity. Pin plants are homozygous for the recessive a allele and do not show an elevation of anthers from the base of the corolla until much later than thrum flowers of the same size (Fig. 2e–j). Taken together, these observations suggest that A plays a dominant role in promoting anther elevation by growth of the lower corolla tube, while G plays a dominant role by suppression of growth of the style.
These observations provide the first indication of a differential timing of developmental events during heteromorphy and indicate that temporal control of S locus gene expression may be an integral component of pin and thrum flower development.
Heteromorphic features achieved by G and A are determined by different mechanisms
The mechanistic distinction where G suppresses style elongation and A promotes anther elevation raises the possibility that they may achieve their respective functions by distinct and separate mechanisms. From our observations of pin and thrum flowers (Fig. 1) and those of others (Darwin, 1877; Heslop-Harrison et al., 1981) it is apparent that the final length of the style in pin flowers is approximately twice the length of the style in thrum plants. Similarly, the proportions of upper and lower corolla tube lengths are approximately 1 : 1 in pin flowers and 1 : 3 in thrum flowers (Figs 1, 2). The developmental difference could in both cases be achieved either by increased cell elongation, or by increased cell division, or by a combination of both. Studies on cell size and shape in the styles (Heslop-Harrison et al., 1981) and corollas of pin and thrum flowers (this study) have indicated that the predominant mechanisms underpinning these developmental events are different. Previous observations on style cell length in Primula vulgaris (Heslop-Harrison et al., 1981) have shown that pin style cells were longer than the corresponding cells in thrum styles. Stylar cell length differences between pins and thrums can also be found in other Primula species (Richards, 1997; Al Wadi & Richards, 1993). The predominant determinant of different style lengths in pin and thrum plants is differential cell elongation, although this is not the sole determinant, as pin style cells are not twice the length of thrum style cells (data not shown, Heslop-Harrison et al., 1981).
The dominant G allele in thrum individuals must therefore play a role in the suppression of style cell elongation, reducing the average thrum style cell length. In contrast, anther height in pin and thrum flowers is not controlled primarily by differential cell elongation, but is mediated initially by different profiles of cell division above and below the points of anther attachment in the corolla tube of pin and thrum flowers. Despite clear differences in the position of the anthers in 10 mm pin and thrum flowers, both of which contain petals that are 7 mm in length (Fig. 2e,f), the lower corolla tube cell lengths are not significantly different (Fig. 4a). The finding that these differences only become significant as the flower approaches maturity suggests that the elevation of anthers in thrum flowers (Fig. 2e,f) must include an increased cell division below the point of anther attachment. Conversely, the corolla tube cell size above and below the point of anther attachment in pin flowers is essentially similar (Figs 3, 4) and is consistent with the position of the anthers in the middle of the corolla tube. Given this observation it is surprising that the mean lower corolla cell lengths in pin and thrum flowers are not significantly different (Fig. 4). A comparison of all cell sizes provides a possible explanation for this finding (Fig. 5). Although the mean cell lengths show small and consistent differences, the variability in cell size within an individual flower causes the spread of the samples. When all cell sizes are ordered and plotted against each other (Fig. 5c) a consistent, but small difference can be seen in the lengths of the lower corolla tube cells. These combined analyses indicate that the elevated anther position in thrum plants is mediated by an increase in cell division in the thrum corolla cells below the point of anther attachment.
Corolla tube mouth diameter, a new heteromorphic character
This analysis was undertaken in duplicate using both wild-type primrose hybrid flowers and a horticultural variety P. vulgaris cv. Blue Jeans. In both examples, the observed greater diameter of the flower mouth in thrum as compared to pin flowers indicated that the presence of this previously undocumented characteristic correlated with floral morph. The selection of equal numbers of pin and thrum flowers of similar sizes and with the normal complement of five petals enabled the elimination of differences that could be attributed to different flower sizes or to the presence of extra petals. The above difference is more pronounced when expressed as a measure of flower diameter to mouth diameter.
An examination of corolla tube cells from above the anthers in thrum and pin flowers using both light microscopy (not shown) and scanning electron microscopy (Fig. 3) showed the corolla tube cells above the anthers to be broader in mature thrum flowers than in mature pin flowers. This observation indicates that the increased width of cells in the thrum upper corolla tube leads to an increased diameter of the flower mouth in thrum as compared to pin flowers. However, quantification of this observation provides a further indication that the increased flower mouth diameter of thrum plants is due entirely to the increased cell widths in the upper corolla.
Given a wild primrose pin flower mouth diameter of 2.0 mm (Fig. 6b), the circumference of the opening can be calculated to be 6.3 mm. Similarly, the circumference of a 3.3 mm diameter opening in thrum flowers is 10.4 mm. Given a mean width of 21 µm in pin upper corolla cells, 300 cells would be needed to create this opening. A similar opening created from approximately 300 cells of 33 µm in diameter, the width of thrum upper corolla cells, would generate a mouth of circumference 9.9 mm – remarkably close to the experimental value of 10.4 mm. Similarly for the horticultural variety P. vulgaris Blue Jeans (Fig. 6c), the circumference of the pin flower mouth can be calculated as 9.1 mm, and for the corresponding thrum flower as 12.6 mm (Fig. 6c). The respective numbers of cells of 21 µm and 33 µm diameter (Fig. 6b) required to form openings of this size is approximately 430 and 380, respectively. Again these are remarkably similar numbers suggesting that flower mouth diameter in flowers with the normal complement of five petals is primarily dictated by the width, and not the number, of cells in the upper corolla. It is considered that this phenomenon may be a direct consequence of the presence of a dominant A allele at the S locus. This new difference between pin and thrum flowers may have previously gone unnoticed due to the varying sizes of flowers in bloom on a plant at any one time, with the earliest flowers to bloom always being larger than the later ones. We propose that this new heteromorphic feature is probably under the control of the A component of the S locus.
These studies have demonstrated the spatial and temporal functions of the dominant alleles of the S locus-linked genes A and G, and our analyses of cell size and shape have identified a new aspect of the Primula heteromorphic phenotype. Isolation and characterisation of the genes located at the Primula S locus, including G and A, will be required to provide a molecular explanation for these cellular and developmental observations. We are currently engaged in identifying DNA sequences and genes that are linked to the Primula S locus (Manfield et al., 2005), with the objective of establishing an integrated physical and genetic map to facilitate the identification and characterisation of genes that control heteromorphy in Primula.