Pollen walls in angiosperms typically consist of an outer exine composed of sporopollenin and an inner intine composed of cellulose and pectin (Fig. 4) (Paxson-Sowders et al. 1997; Morant et al. 2007). Models of development have been proposed based on observations on numerous species, including Lilium and arabidopsis (e.g. Suzuki et al. 2008). Similar processes have been described in both these species.Fig. 4
Diagram of arabidopsis pollen wall structure. The inner intine and the various components of the outer exine are indicated. Note the pollen coat (tryphine and pollenkitt) filling the cavities of the exine sculpture. Taken from Suzuki et al. (2008).
Once again, prior to meiosis, the pollen mother cell is surrounded by a callose special cell wall (Blackmore et al. 2007). Immediately after meiosis, four microspores derived from the pollen mother cell form a tetrad. A callose special wall surrounds the microspores (Blackmore et al. 2007). A cellulose primexine then forms between the plasma membrane and callose wall of each microspore.
Both the callose wall and primexine are deposited at the surface of the microspore through processes mediated by the plasma membrane (Blackmore et al. 2007). A section of the primexine is then adapted to form column-like structures called the probaculae upon which sporopollenin, secreted by the microspore, will eventually accumulate and polymerize. Sporopollenin deposition and accumulation extend the probaculae, which form the baculae and the tectum (Heslop-Harrison 1963, 1968).
The callose wall then degrades and the developing baculae and tectum are exposed to the fluid of the locule and receive sporopollenin secreted by the tapetum. Wall formation is complete when the nexine and intine layers are formed and the primexine recedes and disappears (Suzuki et al. 2008). The mature pollen grain is then coated by tryphine and pollenkitt, which are synthesized by the tapetum (Dickinson and Lewis 1973; Blackmore et al. 2007).
Spore walls have been investigated in a number of pteridophyte species representing all of the major pteridophyte groups (reviewed in Lugardon 1990; Tryon and Lugardon 1991).
Spore wall development is well understood in the homosporous lycopsid Lycopodium clavatum (Uehara and Kurita 1991). Shortly after meiosis, the plasma membrane of the sporogenous cell folds into a pattern that later becomes the reticulate spore sculpture. Small WLCL form on the plasma membrane and accumulate in a centripetal fashion, forming the greater part of the exospore. After the main lamellate part of the exospores is formed, an inner granular layer, possibly derived from the spore cytoplasm, is deposited. In some Lycopodium there are no extra-exosporal layers (Uehara and Kurita 1991) whereas in others a thin extra-exosporal layer is deposited after the completion of the exospore (Tryon and Lugardon 1991).
Spore structure and development in heterosporous lycopsids differ between microspores and megaspores. In the clubmoss selaginella, microspores possess an exospore consisting of two layers (Fig. 3). The thin inner layer is the first to develop and comprises imbricate lamellae that are formed on WLCL in a centripetal direction (Tryon and Lugardon 1991). The outer layer starts to form only once the inner layer is complete. Some selaginella species may also develop a thin perispore or a paraexospore. In the microspores of the heterosporous lycopsid Isoetes japonica, a large gap is developed between the two exospore layers (Uehara et al. 1991). The outer exospore layer is regarded as a paraexospore as it begins to form before the inner exospore, consists of similar sporopollenin, and is completed at the same time as the inner exospore.Fig. 3
Proposed model of spore wall development in selaginella microspores. The thin inner exine layer forms first and comprises lamellae formed centripetally on WLCL. The outer exine starts to form once the inner layer is complete. Note the presence of callose at early developmental stages around the spore mother cell.
Selaginella megaspore walls contain two layers of similar thickness (Morbelli 1995). The inner and outer layers consist of lamellae and poorly segregated components, respectively. The inner layer does not thicken during exospore development and a dense basal layer is formed by the lamellae. In contrast, the outer layer increases significantly in thickness due to self-assembly (Hemsley et al. 1994, 2000; Gabarayeva 2000). During the final stages of sporogenesis, the endospore forms between the plasma membrane and the exospore. In Isoetes, the megaspore wall is similar to that of selaginella in terms of development and structure, consisting of two layers, with the formation of the outer layer commencing prior to that of the inner layer. Substantial quantities of silica are deposited within and on top of the outer layer before the exospore is completed. Finally, the endospore is laid down between the plasma membrane and the exospore.
The exospore in homosporous ferns develops centrifugally and is once again bilayered. The inner layer acts as a substructure and consists of varying numbers of fused sheets (extensive interconnected laminae) that form by sporopollenin accumulation on WLCL. The homogeneous outer layer is considerably thicker and contains thin radial fissures and small cavities. An extra-exosporal layer (perispore) forms once the exospore is complete and is deposited from the decaying tapetum. Spore wall development in heterosporous ferns is similar to that observed in homosporous ferns, and is also similar in both microspores and megaspores.
In sphenopsids the spore walls appear to be highly derived (Lugardon 1990), and observations of Equisetum arvense have shown that four layers are present in the form of an exospore, an endospore, a middle layer and pseudoelators (Uehara and Kurita 1989). The exospores comprise inner and outer exospores. The broad and homogeneous inner exospore forms first by way of plate-like structures accumulating on the plasma membrane. The outer exospore is then formed by the deposition of granular material on the inner exospore and is similarly wide and homogeneous. Once exospore formation is complete, the middle layer forms in the gap between the exospore and the plasma membrane. The pseudoelators are the next structure to form and consist of two layers. The inner layer comprises longitudinal microfibrils during the early stages of development but eventually becomes homogeneous. The outer layer is also homogeneous and is formed by granules that are released from vesicles in the plasmodial cytoplasm. The pseudoelators are connected to the spore, by way of the middle layer, at the aperture. The endospore is the final component of the wall to form on the inside of the exospores (Taylor 1986; Uehara and Kurita 1989).
Spore wall development has been studied in all three of the traditional bryophyte groups (reviewed in Brown and Lemmon 1988, 1990). In the majority of liverworts, immediately after meiosis, a polysaccharide wall (the spore special wall) is laid down outside the plasma membrane (Brown and Lemmon 1985). In many liverworts, this spore special wall seems to function as a primexine in which the pattern of exospore ornamentation is established (Brown and Lemmon 1993).
However, in some liverworts exospore ornamentation appears to be determined by exospore precursors produced by the diploid sporocyte prior to meiosis and formation of the haploid spores (Brown et al. 1986). The exospore develops centripetally (Brown and Lemmon 1993) based on WLCL formed outside the spore cytoplasm. At completion, the entire exospore comprises sporopollenin deposited on WLCL.
At maturity, the lamellate structure thus formed is clearly discernible and is highly characteristic of the liverwort exospore. Liverworts lack a tapetum and there is therefore no input from this source. The innermost layer of fibrillar intine is the final wall layer to be formed (Brown and Lemmon 1993).
Studies of spore wall development in hornworts are limited. As with liverworts, a spore special wall is formed after meiosis and functions as a primexine in which the exospore is set down. It was initially thought that the exospore formed in the absence of WLCL, but Taylor and Renzaglia have recently demonstrated their presence (W.A. Taylor, University of Wisconsin-Eau Claire, USA, pers. comm., 2011).
Recent analyses of Phaeomegaceros fimbriatus have shown that the mature spore wall has a thin perine-like outer layer, but this represents the remnants of the spore mother cell wall rather than extra-exosporal material derived from a tapetum (Villarreal and Renzaglia 2006).
Three types of spore wall have been recognized in mosses: Bryopsida type, Andreaeidae type and Sphagnidae type (Brown and Lemmon 1990). All three of these types appear to form in the absence of a spore special wall. Bryopsida-type spore walls are homogeneous except for an inconspicuous foundation layer (Fig. 2). This foundation layer forms first via sporopollenin accumulation on WLCL.
Subsequently, the homogeneous exospore layer is laid down outside the foundation layer in a centrifugal manner. This layer is probably mainly extrasporal in origin. Sometimes additional homogeneous material is also deposited inside the foundation layer. This layer is almost certainly derived from the spore. Following the accumulation of homogeneous material, the spores are coated by an additional extra-exosporal layer, referred to as the perine or perispore, which is derived from the tapetum. Finally, the intine forms.Fig. 2
Proposed model of spore wall development in physcomitrella. The exine foundation layer is laid down first by way of sporopollenin accumulation on WLCL. The rest of the exine layer is deposited outside the foundation layer centrifugally. Note the appearance of callose in the inner exine, which is confined to the expanded aperture region at the proximal pole.
Spore wall development in the Andreaeidae type is unique among mosses in that they have a spongy exospore that appears to form in the absence of WLCL (Brown and Lemmon 1984). By studying Andreaea rothii, Brown and Lemmon (1984) demonstrated that the exospore is instead initiated as discrete homogeneous globules within the coarsely fibrillar network of the spore mother cell.
These globules accumulate and form an irregular layer with numerous interstitial spaces. The sequence of spore wall layer development is essentially the same as that of other mosses and the mature wall consists of an inner intine, a spongy exospore and an outer perine (Brown and Lemmon 1984).
Sphagnidae-type moss spore walls are more complex than those of the other mosses and consist of five layers (Brown et al. 1982). Unlike other mosses, the exospore of Sphagnidae type comprises two layers: an inner lamellate layer (A-layer) and a thick homogeneous outer layer (B-layer). In addition to the exospore, there is an intine, a unique translucent layer and the outermost perine.
The A-layer is the first to form and does so by sporopollenin accumulation on WLCL, and develops evenly around the young spore immediately after meiosis. The homogeneous B-layer is deposited outside the A-layer. Overlying the exospore is a translucent layer that consists of unconsolidated exospore lamellae in a medium of unknown composition. The tapetally derived perine is deposited on top of this unique layer. The study of spore wall development in Sphagnum lescurii by Brown et al. (1982) suggests that the ontogeny of the wall layers is not strictly centripetal.
The basic mechanisms involved in the formation of the spore wall, and the deposition of sporopollenin in the exospore/exine, have been illuminated by numerous ultrastructural studies performed on extant and fossil species across the plant kingdom (Paxson-Sowders et al. 2001). Blackmore and Barnes (1987) proposed a number of sporopollenin deposition processes apparent in the spore wall. Firstly, they recognized the role of white-line-centred lamellae (WLCL) in this process.
The accumulation of sporopollenin on an array of WLCL is regarded as being the most primitive method of sporopollenin deposition and has been identified in a number of algal groups and most, if not all, embryophytes (Wellman 2004). These lamellae materialize at the plasma membrane with sporopollenin polymerizing out onto either side of the white line. They accumulate in a variety of ways to form the spore/pollen wall (Blackmore and Barnes 1987; Blackmore et al. 2000; Wellman 2004).
Another mode of exospore/exine formation involves the deposition of sporopollenin from the surrounding cells of the tapetum. Transmission electron microscopy has shown that the tapetal cells possess a highly active secretory system containing lipophilic globules, which are thought to contain the precursors of sporopollenin and are deposited onto the surface either directly contributing to the exospore/exine or forming extra-exosporal layers (Piffanelli et al. 1998).
‘ Blackmore et al. (2000) suggested that a tapetal contribution to the spore wall can take place in a variety of ways, including the addition to the layers formed by the WLCL or directly onto WLCL. Studies of pollen wall formation in angiosperms highlight the role that tapetal cells play in supplying nutrients and lipid components to developing microsporocytes and microspores (Scott et al. 1991; Ariizumi et al. 2004; de Azevedo Souza et al. 2009). Interestingly, the most basal extant land plants (liverworts) lack a tapetum, which is acquired in mosses and vascular plants.
An alternative deposition process involves centripetal accumulation of sporopollenin onto previously formed layers. Blackmore et al. (2000) noted that exospore formation may be achieved by sporopollenin accumulation below a pre-existing layer, either by WLCL accumulation or by the deposition of granular or unstructured sporopollenin. A further mode of deposition is observed in seed plants where sporopollenin accumulates within a pre-patterned cell surface glycocalyx referred to as the primexine (Blackmore and Barnes 1987; Blackmore et al. 2000; Wellman 2004), which is essentially an exine precursor.
The spore/pollen walls of embryophytes have multiple layers and components that are laid down in a regulated manner during spore/pollen development. Layers containing the macromolecule sporopollenin are the component enabling the resistance of the spore/pollen wall to numerous environmental factors that make life on land challenging. Sporopollenin is highly resistant to physical, chemical and biological degradation procedures.
Consequently, its precise chemical composition, structure and biosynthetic route have not yet been ascertained (Meuter-Gerhards et al. 1999). Traditional convention asserts that sporopollenin is a polymer of carotenoid esters (Cronk 2009). However, modern purification, degradation and analytical techniques have shown that it is comprised of polyhydroxylated unbranched aliphatic units with small quantities of oxygenated aromatic rings and phenylpropanoids (Ahlers et al. 1999; Domínguez et al. 1999).
The colonization of land by plants in the Palaeozoic was a highly significant event in Earth’s history, both from an evolutionary point of view and because it fundamentally changed the ecology and environment of the planet (Beerling 2007). Land plants evolved to form crucial components of all modern terrestrial ecosystems through evolutionary adaptations involving changes in anatomy, physiology and life cycle (Waters 2003; Menand et al. 2007; Cronk 2009). Key adaptations include rooting structures, conducting tissues, cuticle, stomata, and sex organs such as gametangia and spores/pollen.
Development of a durable spore wall is essential for terrestrialization as it enables the spore to withstand physical abrasion, desiccation and UV-B radiation (Wellman 2004). As part of their life cycle, sexually reproducing embryophytes manufacture either spores, or their more derived homologues pollen. The major component of the spore/pollen wall proposed to be of primary importance in enabling resistance to the conditions described above is the highly resistant biopolymer sporopollenin (Ito et al. 2007; Cronk 2009).
It seems reasonable to hypothesize that colonization of the land by plants was not possible prior to the evolution of the sporopollenin spore wall, and this adaptation is considered to be a synapomorphy of the embryophytes. Additionally, spore walls are not present in the hypothesized embryophyte antecedents, the green algae (Wellman 2004). However, the production of sporopollenin is highly likely to be pre-adaptive as it is present in a number of different algal groups such as the charophyceans, which have been proposed as the sister group to the embryophytes. In certain charophyceans, sporopollenin occurs, but is located in an inner layer of the zygote wall (Graham 1993).
Phylogenetic studies and fossil evidence have shown that the most basal living land plants are the paraphyletic ‘bryophytes’ (Kenrick and Crane 1997; Qui et al. 2006) . They comprise the liverworts, mosses and hornworts, and their phylogenetic position should allow us to further elaborate the evolutionary changes that facilitated the conquest of land by plants (Rensing et al. 2008). The moss Physcomitrella patens is the first ‘bryophyte’ genome to be sequenced. This genome, through comparisons with angiosperm genomes, is proving to be a valuable tool in experimental studies that attempt to reconstruct genome evolution during the colonization of land (Reski and Cove 2004; Quatrano et al. 2007; Rensing et al. 2008).
Phylogenetic tree for land plant evolution derived from analysis by Qui et al. (2006). The bryophytes are a paraphyletic group comprising three separate lineages. Together with the vascular plants (which include the angiosperms), bryophytes form the embryophytes, which have a sister group relationship to the green algae.
In this review, we first outline the nature of spore/pollen wall development in the major plant groups, before considering emerging understanding of the molecular genetics of pollen wall development. The latter includes identification of genes involved in sporopollenin biosynthesis and exospore formation, callose wall formation and tetrad separation. We also report results from BLAST searches of the basal land plant physcomitrella and the clubmoss Selaginella moellendorfii using genes implicated in pollen wall development in arabidopsis.
Pollen grains are produced by seed plants (angiosperms and gymnosperms), and spores by ferns, lycopods, horsetails, mosses and fungi. They are produced in generally very large numbers during the reproduction process, and are dispersed by wind, water and animals.
As pollen and spores are made up of an extremely resistant organic material called sporopollenin, they preserve well and thus the pollen and spore associations in sediments give us a very detailed overview of the complex vegetation and climate history of the past.
In addition, pollen and spores of individual plant species that have only existed during short periods of geological history, or that have produced very characteristic associations related to particular time periods only, can be used to date continental or shallow marine sedimentary deposits (“biostratigraphy”).
The study of pollen grains and spores, in addition to that of other organic-walled microfossils such as dinoflagellate cysts, is generally referred to as palynology.
Above: modern bisaccate Pinus pollen, middle: fern spore from the Upper Jurassic, below: pollen from the Upper Triassic
