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Simulated Self-assembly of Spore Exines
Annals of Botany 82 : 105–109, 1998
Simulated Self-assembly of Spore Exines A L A N R. H E M S L EY*, B R I A N V I N C E NT†, M A R G A R E T E. C O L L I N S O N‡, and P E T E R C. G R I F F I T H S§ * Department of Earth Sciences, Uniersity of Wales Cardiff, PO Box 914, Cardiff, CF1 3YE, Wales, UK, † School of Chemistry, Uniersity of Bristol, Cantock’s Close, Bristol BS8 1TS, UK, ‡ Geology Department, Royal Holloway Uniersity of London, Egham, Surrey TW20 0EX, UK and § Department of Chemistry, Uniersity of Wales Cardiff, PO Box 912, Cardiff, CF1 3TB, Wales, UK Received : 4 February 1998
Accepted 8 April 1998
The spores and pollen of higher plants have enormous value in taxonomic studies by contributing to our understanding of past and present biodiversity, and of environmental change. The critical features used in species identification are wall structure and surface pattern. However, ultrastructural and histo-developmental studies of spore and pollen walls have, so far, provided limited explanation of wall construction and surface pattern formation. The consistency of pattern form within any species suggests a high degree of genetic regulation, and yet few templates or other mechanisms of control have been demonstrated. Our experiments show that all layers and organizations within the spore wall of our test plant group (lycopodiopsid megaspores) can be simulated by the flocculation of mixed colloidal systems. This leads us to a possible explanation of the mode of genetic control over pattern formation. It also provides a feasible, largely self-assembling, mechanism of construction which has the potential to reflect the diversity of structure known to exist in all spore and pollen walls. # 1998 Annals of Botany Company Key words : Simulated self-assembly, spore exine development, sporopollenin, Lycopodiopsida, polyballs.
I N T R O D U C T I ON The diversity of pattern and ultrastructure shown by living spores and pollen is remarkable (e.g. Moore, Webb and Collinson, 1991 ; Tryon and Lugardon, 1991), as is that shown by the frequently well preserved spores and pollen of extinct plants (Traverse, 1988). Among living plants, pollen and spores from a particular species are often very consistent in terms of the patterning and ultrastructure they exhibit. They commonly share characters with those of related species or genera. This consistency and comparability has provided considerable scope for systematic work on living species, and on fossils where patterning and ultrastructure are also presumed to have been consistent in particular taxa. An understanding of how such patterning and ultrastructure is, and has been, generated is therefore of great importance if we are to be confident about presumed plant relationships based on spore or pollen morphology. In addition, it provides a valuable insight into the derivation of biological microstructure. This is especially significant with extinct species, where other evidence may be scant. Detailed studies of pollen and spore wall development have illustrated the role of the endoplasmic reticulum in the positioning and ultimate form of germinal apertures, but have so far failed to demonstrate clearly how surface patterning and internal ultrastructure is generated (Sheldon and Dickinson, 1986). Studies by Takahashi (1989), Takahashi and Skvarla (1991) and Paxson-Sowders, Owen and Makaroff (1997) have demonstrated a likely role for undulations of the plasma membrane in the initiation of pattern formation. However, it is difficult to see how these might translate into complex 0305-7364}98}07010505 $30.00}0
exine architecture, or surface pattern on thick walls or perispores. Lycopodiopsid megaspores (large female spores produced by clubmosses and their relatives, Fig. 1) provide many opportunities for the study of spore wall development. The walls are thick, up to 60 µm (Fig. 2), and the spores are large (200–1200 µm) with diverse surface patterning. In addition, such spores have a long and relatively continuous fossil record extending from the late Devonian. Ultrastructural studies of both living and extinct lycopodiopsid megaspores have been undertaken frequently (Taylor, 1990, 1991, 1993 ; Hemsley and Scott, 1991). Like other spore walls, those of the lycopodiopsid megaspores are constructed of sporopollenin, a biomacromolecule composed of aliphatic and aromatic moieties (van Bergen et al., 1995) with a remarkable resistance to degradation under anoxic conditions. This resistance contributes greatly to its longevity in the fossil record. However, sporopollenin has proven particularly difficult to analyse by conventional techniques, so we know little about either the detail of its chemical composition, or how it is formed. Previous investigations of lycopodiopsid megaspore walls have demonstrated that, in some groups, the walls contain layers consisting of domains of crystalline particles, commonly around 0±25 µm in diameter (Figs 2 and 3). This has been interpreted as a colloidal crystal (Hemsley, Collinson and Brain, 1992) and shows considerable similarity to other natural colloidal crystal aggregations (e.g. precious opal, Darragh, Gaskin and Sanders, 1976 ; iridoviridae, Devauchelle, Stoltz and Darcy-Tripier, 1985). Like these colloidal crystals, the specific layer within the lycopodiopsid
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F. 1. Megaspore of Selaginella myosurus exhibiting a robust reticulate ridge ornament (specimens held in the biological collection, University of Wales, Cardiff). Scale bar ¯ 500 µm. F. 2. The wall structure of Selaginella myosurus megaspore shows three different layers of ultrastructure : an inner layer of particles ; a middle layer of ordered, monodisperse particles ; and an outer layer (present within the ridges) of convoluted laminae. Scale bar ¯ 5 µm. F. 3. Detail of the middle (colloidal crystal) layer shown in Fig. 2, exhibiting ordering of component particles. Scale bar ¯ 1 µm. F. 4. The structure shown in Fig. 3 can be modelled (as shown here) by a monodisperse latex of polystyrene particles flocculated by carboxymethylcellulose. Scale bar ¯ 1 µm.
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T 1. A comparison of the components and their function from a generalized sporangium and from the synthetic system used here
Polymer forming latex Flocculant Hydrocarbon Supporting fluid Initiator
Plant sporangium
Model system
Sporopollenin Mucopolysaccharides Lipids (fatty acids) Water Enzyme ? UV ? O ?
Polystyrene Carboxymethylcellulose Cyclohexane Water Ammonium persulphate
megaspore wall is iridescent (Hemsley et al., 1994). These observations led to the hypothesis that this layer (at least) within the spore wall was of colloidal origin. The structures forming the remaining wall layers (irregular particles, rods, laminae), and indeed, those of other lycopodiopsid megaspores, could all theoretically be generated by the association of specific forms of colloidal unit. This hypothesis was supported by the transitional wall zones between, for example, layers consisting of regular particle arrays and layers consisting of laminae (Collinson, Hemsley and Taylor, 1993 ; Fig. 7). In addition, megaspores extracted from sporangia during development show features consistent with a colloidal mode of construction. A mechanism of depletion flocculation of a monodisperse sporopollenin colloid was proposed to account for the wall structures described above (Hemsley et al., 1994). A water-based monodisperse polystyrene latex (to simulate sporopollenin within the supporting sporangial fluid, Table 1), was flocculated using carboxymethylcellulose (representing the mucopolysaccharides ; Morbelli and Rowley, 1993). This gave rise to colloidal crystal forms similar to those within the megaspore wall layers (Fig. 4 ; Hemsley et al., 1996) and by a self-assembly process governed by the concentration and osmotic behaviour of the components. We are currently investigating the possibility of utilizing a polmer of pcoumaric acid for the production of polymer latices (which may be appropriate ; Wehling et al., 1989), but currently we view the volume of available literature on polystyrene colloid behaviour to outweigh any benefits to be obtained by using more ‘ natural ’ components. This study sought to expand the reproducible range of structures obtainable by
#
colloidal self-assembly, to demonstrate that the diversity of exine structure observable in clubmoss megaspores is consistent with their production by variations of a single mechanism, and to test the hypothesis that microspores are constructed by a similar mechanism. MATERIALS AND METHODS All these simulations used polystyrene in place of sporopollenin. Ammonium persulphate is used throughout as the polymeric reaction initiator. Sodium chloride is used to provide a quantitative increase in the electrolyte concentration of the solution, which consists mainly of doubledistilled water. Our initial experiments (Hemsley et al., 1996) were aimed at the production of a monodisperse colloid in an aqueous environment. The technique was based on that of Goodwin et al. (1973) utilizing the apparatus shown in Fig. 13. Our subsequent simulations have incorporated additional components (see Table 2) to reflect sporangial content and increase diversity of structure. The distilled water and sodium chloride were placed in the reaction vessel (with cyclohexane if used). The oil bath was raised to 70°C and the stirrer set to rotate at around 300 rpm. A slow stream of nitrogen was passed through the reaction vessel for 1 h to expel oxygen, which might otherwise initiate polymerization on addition of the styrene. This flow of nitrogen was maintained throughout the duration of the reaction. The styrene was added and left to integrate in solution for 0±5 h before addition of the initiator. The apparatus was then left for 24 h for the reaction to progress. Flocculations of subsequently cooled latex were achieved by
F. 5. The simpler wall structure of S. selaginoides consists of polydisperse particles which are partly fused (spores obtained from herbarium specimens held in the National Museum of Wales, Cardiff). Scale bar ¯ 4 µm. F. 6. The structure shown in Fig. 5 can be similarly modelled by a flocculation of polydisperse polystyrene latex, as shown here. Scale bar ¯ 5 µm. F. 7. Transitions between layers in lycopodiopsid megaspore walls indicate that the processes responsible for different ultrastructural types are closely related. Scale bar ¯ 2 µm. F. 8. Transitions from particles to strings and sheets can be reproduced using polystyrene}cyclohexane colloidal mixtures, as shown here. Scale bar ¯ 1±5 µm. F. 9. Sporopollenin particles and particle aggregates present in the developing megaspore wall of Selaginella laeigata (spores obtained from plants in the living collection at University of Wales, Cardiff). Scale bar ¯ 10 µm. F. 10. A droplet of cyclohexane acts as a surface upon which polystyrene latex particles and particle aggregates have flocculated, forming a pattern strikingly similar to that of many spores and some pollen. Scale bar ¯ 10 µm. F. 11. When broken, coated droplets (as in Fig. 10) illustrate a structure containing raspberry-like aggregates of polystyrene particles, compared to the living example in Fig. 9. Scale bar ¯ 3 µm. F. 12. Membranes with regular holes and foam-like arrangements may also be created from polystyrene, as shown here. Some resemble the structures found in certain fern megaspore walls, e.g. Salinia.
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the addition of the sodium salt of carboxymethylcellulose and}or air drying. Samples were later observed by scanning electron microscopy. R E S U L T S A N D D I S C U S S I ON Experiments using the simple components outlined above have created mixed, aqueous colloidal systems containing polydisperse particles, hydrocarbon droplets and particle aggregates. Flocculation of these mixtures demonstrates a wider range of pattern than previously reported (as dried down samples viewed by SEM, see Fig. 4) and has generated a diversity of relevant internal structures. The majority of those so far observed have analogues among either living or extinct lycopodiopsid megaspore walls (see Figs 5–8) but, significantly, some structures (obtained from solid residue adherent to the stirrer) resemble megaspore walls from other plant groups (Fig. 12), principally the ferns (for example Salinia). Specific concentrations of hydrocarbon and styrene monomer (Table 2) give rise to mixtures which, during latex production, produce raspberry-like aggregations of polystyrene particles. Sporopollenin ‘ raspberries ’ have been identified in developing megaspore exines of Selaginella (Fig. 9). Commonly these aggregate, with numerous free particles, on the surface of hydrocarbon droplets (mimicking sporopollenin aggregation around the spore body during wall development, Figs 10 and 11). The size distribution of the lipid droplets is dependent upon the degree of agitation (rpm) in the reaction vessel. Under the conditions used for latex production, the droplets are generally small (approx. 5 µm), but some reach 50 µm in diameter. Aggregations of particles and particle clusters upon the surface of these larger droplets result in structures comparable to many microspore, isospore and pollen walls in dimensions, and in some cases, in surface ornament. These results provide further support to the view that exine development within lycopodiopsid megaspores occurs principally by way of self-assembling colloids, and that a wide range of structures are potentially reproducible, based on variation in the initial components of such an assembly system. In addition, our experiments demonstrate an essential role for an oil or lipid component in the formation of surface patterning, either for the coalescence of preformed aggregates, or the restriction of individual particle aggregation around the developing wall.
Gas trap N2 N2 Reflux condenser
Thermometer Reaction vessel
Stirrer Oil bath
F. 13. Diagram of the apparatus used to prepare polystyrene latices. Adapted from Pieranski (1983).
Remarkably, these experiments suggest that microspore (small male spore) walls may be constructed from colloidal mixtures which differ little from those which give rise to the structure in megaspore walls, despite differences in ultimate wall thickness, patterning and ultrastructure. The processes by which particles are produced in the plants and in these experiments, and the particle composition, are clearly different (Table 1). However, the flocculation mechanisms are probably comparable (Lin et al., 1989). That we require similar types of component within the model to those found within the plant sporangia to produce similar structure is significant, and gives more support for our simulation. Within colloidal mixtures, there is clearly scope to construct an immense array of pattern and structural diversity which will, in many cases, be indistinguishable from that present in living and fossil spores. In addition, it is possible to quantify differences in ultimate structure in terms of differences in initial components, which may in turn be tested against comparative analyses of developing sporangial fluid content. This approach to an understanding of spore wall patterning underpins the growing consensus that spore and pollen wall development must, at least in part, be a selfassembly process resulting from physico-chemical properties of the sporangial contents (Scott, 1994). Wall development
T 2. The components and quantities used to produce the structures illustrated in Figs 4, 6, 8, 10 and 11. See methodology for details
Distilled water Styrene Cyclohexane Sodium chloride (10 % solution) Ammonium persulphate (3 % solution) Carboxymethylcellulose (in 20 ml distilled water)
Fig. 4
Fig. 6
670 ml 80 ml — 1±25 ml
335 ml 50 ml 335 ml 1±25 ml
Fig. 8 670 ml 50 ml 50 ml 1±25 ml
25 ml
25 ml
20 ml
5g
5g
—
Figs 10–11 670 ml 50 ml 100 ml 1±25 ml 20 ml —
Hemsley et al.—Simulated Self-assembly of Spore Exines by flocculation of mixed colloidal systems provides a simple mechanism by which polymerized sporopollenin monomer may aggregate forming elaborate structures. Within sporangia, mechanisms may be reliant, at least initially, upon controlled irregularities on the spore surface to induce pattern formation [probably cytoplasmic wall protein or charge distributions, perhaps associated with variation in plasma membrane topography (Paxson-Sowders et al., 1997)]. However, it is clear from our experiments that patterns and structures can arise in the absence of a physical template. Providing there is genetic control over the initial conditions and concentration of components within the colloidal system operating in the sporangium, the resulting spore wall ultrastructure and pattern can achieve consistency. The apparently high degree of genetic control involved in the production of patterning and ultrastructure would appear to be concerned not with the actual process of construction, but rather with the establishment of the precise conditions at the initiation of the self-assembly process (Hemsley, 1998). Simulations using inorganic components to produce structured aragonite shells around polystyrene particles (Walsh and Mann, 1995) have proven to be useful models in the understanding of pattern production mechanisms at the microscopic level. Such simulations not only reproduce the structures seen, but also reveal underlying processes which link the genetic basis of variation to actual morphology (in our case, colloidal aggregation). A C K N O W L E D G E M E N TS We thank P. D. Jenkins for his work in developing these models, and the Royal Society for a University Research Fellowship to ARH. We are indebted to J. Crawley, V. Williams, L. Axe, K. Habgood and A. Oldroyd for photographic and technical assistance. L I T E R A T U R E C I T ED Collinson ME, Hemsley AR, Taylor WA. 1993. Sporopollenin exhibiting colloidal organization in spore walls. Grana, Supplement 1 : 31–39. Darragh PJ, Gaskin AJ, Sanders JV. 1976. Opals. Scientific American 234 : 82–95. Devauchelle G, Stoltz DB, Darcy-Tripier F. 1985. Comparative ultrastructure of Iridoviridae. In : Willis DB, ed. Iridoiridae. Berlin : Springer Verlag, 1–21. Goodwin JW, Hearn J, Ho CC, Ottewill RH. 1973. The preparation and characterisation of polymer lattices formed in the absence of surface active agents. British Polymer Journal 5 : 347–362. Hemsley AR. 1998. Non-linear variation in simulated complex pattern development. Journal of Theoretical Biology (in press). Hemsley AR, Collinson ME, Brain APR. 1992. Colloidal crystal-like
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structure of sporopollenin in the megaspore walls of recent Selaginella and similar fossil spores. Botanical Journal of the Linnean Society 108 : 307–320. Hemsley AR, Collinson ME, Kovach WL, Vincent B, Williams T. 1994. The role of self-assembly in biological systems : evidence from iridescent colloidal sporopollenin in Selaginella megaspore walls. Philosophical Transactions of the Royal Society, London B 345 : 163–173. Hemsley AR, Jenkins PD, Collinson ME, Vincent B. 1996. Experimental modelling of exine self-assembly. Botanical Journal of the Linnean Society 121 : 177–187. Hemsley AR, Scott AC. 1991. Ultrastructure and relationships of the Upper Carboniferous spores from Thorpe Brickworks, West Yorkshire, UK. Reiew of Palaeobotany and Palynology 69 : 337–351. Lin MY, Lindsay HM, Weitz DA, Ball RC, Klein R, Meakin P. 1989. Universality in colloidal aggregation. Nature 339 : 360–362. Moore PD, Webb JA, Collinson ME. 1991. Pollen analysis. Oxford : Blackwell Scientific Publications. Morbelli MA, Rowley JR. 1993. Megaspore development in Selaginella. 1. ‘ Wicks ’, their presence, ultrastructure and presumed function. Sexual Plant Reproduction 6 : 98–107. Paxson-Sowders DM, Owen HA, Makaroff CA. 1997. A comparative ultrastructural analysis of exine pattern development in wildtype Arabidopsis and a mutant defective in pattern formation. Protoplasma 198 : 53–65. Pieranski P. 1983. Colloidal crystals. Contemporary Physics 24 : 25–73. Scott RJ. 1994. Pollen exine – the sporopollenin enigma and the physics of pattern. In : Scott RJ, Stead MA, eds. Molecular and cellular aspects of plant reproduction. Cambridge : Cambridge University Press, 49–81. Sheldon JM, Dickinson HG. 1986. Pollen wall formation in Lilium : The effect of chaotropic agents, and the organisation of the microtubular cytoskeleton during pattern development. Planta 168 : 11–23. Takahashi M. 1989. Pattern determination of the exine of Caesalpinia japonica (Leguminosae : Caesalpinioideae). American Journal of Botany 76 : 1615–1626. Takahashi M, Skvarla JJ. 1991. Exine pattern formation by plasma membrane in Bougainillea spectabilis Wild. (Nyctaginaceae). American Journal of Botany 78 : 1063–1069. Taylor WA. 1990. Comparative analysis of megaspore ultrastructure in Pennsylvanian lycophytes. Reiew of Palaeobotany and Palynology 62 : 65–78. Taylor WA. 1991. Megaspore wall ultrastructure in Selaginella. Pollen et Spores 31 : 251–288. Taylor WA. 1993. Megaspore wall structure in Isoetes. American Journal of Botany 80 : 165–171. Traverse A. 1988. Paleopalynology. Boston : Unwin Hyman. Tryon AF, Lugardon B. 1991. Spores of the Pteridophyta. New York : Springer-Verlag. Van Bergen PF, Collinson ME, Briggs DEG, de Leeuw JW, Scott AC, Evershed RP, Finch P. 1995. Resident biomacromolecules in the fossil record. Acta Botanica Neerlandica 44 : 319–342. Walsh D, Mann S. 1995. Fabrication of hollow porous shells of calcium carbonate from self-organizing media. Nature 377 : 320–323. Wehling K, Niester CH, Boon JJ, Willemse MTM, Wiermann R. 1989. p-Coumaric acid – a monomer in the sporopollenin skeleton. Planta 179 : 376–380.
Simulated Self-assembly of Spore Exines A L A N R. H E M S L EY*, B R I A N V I N C E NT†, M A R G A R E T E. C O L L I N S O N‡, and P E T E R C. G R I F F I T H S§ * Department of Earth Sciences, Uniersity of Wales Cardiff, PO Box 914, Cardiff, CF1 3YE, Wales, UK, † School of Chemistry, Uniersity of Bristol, Cantock’s Close, Bristol BS8 1TS, UK, ‡ Geology Department, Royal Holloway Uniersity of London, Egham, Surrey TW20 0EX, UK and § Department of Chemistry, Uniersity of Wales Cardiff, PO Box 912, Cardiff, CF1 3TB, Wales, UK Received : 4 February 1998
Accepted 8 April 1998
The spores and pollen of higher plants have enormous value in taxonomic studies by contributing to our understanding of past and present biodiversity, and of environmental change. The critical features used in species identification are wall structure and surface pattern. However, ultrastructural and histo-developmental studies of spore and pollen walls have, so far, provided limited explanation of wall construction and surface pattern formation. The consistency of pattern form within any species suggests a high degree of genetic regulation, and yet few templates or other mechanisms of control have been demonstrated. Our experiments show that all layers and organizations within the spore wall of our test plant group (lycopodiopsid megaspores) can be simulated by the flocculation of mixed colloidal systems. This leads us to a possible explanation of the mode of genetic control over pattern formation. It also provides a feasible, largely self-assembling, mechanism of construction which has the potential to reflect the diversity of structure known to exist in all spore and pollen walls. # 1998 Annals of Botany Company Key words : Simulated self-assembly, spore exine development, sporopollenin, Lycopodiopsida, polyballs.
I N T R O D U C T I ON The diversity of pattern and ultrastructure shown by living spores and pollen is remarkable (e.g. Moore, Webb and Collinson, 1991 ; Tryon and Lugardon, 1991), as is that shown by the frequently well preserved spores and pollen of extinct plants (Traverse, 1988). Among living plants, pollen and spores from a particular species are often very consistent in terms of the patterning and ultrastructure they exhibit. They commonly share characters with those of related species or genera. This consistency and comparability has provided considerable scope for systematic work on living species, and on fossils where patterning and ultrastructure are also presumed to have been consistent in particular taxa. An understanding of how such patterning and ultrastructure is, and has been, generated is therefore of great importance if we are to be confident about presumed plant relationships based on spore or pollen morphology. In addition, it provides a valuable insight into the derivation of biological microstructure. This is especially significant with extinct species, where other evidence may be scant. Detailed studies of pollen and spore wall development have illustrated the role of the endoplasmic reticulum in the positioning and ultimate form of germinal apertures, but have so far failed to demonstrate clearly how surface patterning and internal ultrastructure is generated (Sheldon and Dickinson, 1986). Studies by Takahashi (1989), Takahashi and Skvarla (1991) and Paxson-Sowders, Owen and Makaroff (1997) have demonstrated a likely role for undulations of the plasma membrane in the initiation of pattern formation. However, it is difficult to see how these might translate into complex 0305-7364}98}07010505 $30.00}0
exine architecture, or surface pattern on thick walls or perispores. Lycopodiopsid megaspores (large female spores produced by clubmosses and their relatives, Fig. 1) provide many opportunities for the study of spore wall development. The walls are thick, up to 60 µm (Fig. 2), and the spores are large (200–1200 µm) with diverse surface patterning. In addition, such spores have a long and relatively continuous fossil record extending from the late Devonian. Ultrastructural studies of both living and extinct lycopodiopsid megaspores have been undertaken frequently (Taylor, 1990, 1991, 1993 ; Hemsley and Scott, 1991). Like other spore walls, those of the lycopodiopsid megaspores are constructed of sporopollenin, a biomacromolecule composed of aliphatic and aromatic moieties (van Bergen et al., 1995) with a remarkable resistance to degradation under anoxic conditions. This resistance contributes greatly to its longevity in the fossil record. However, sporopollenin has proven particularly difficult to analyse by conventional techniques, so we know little about either the detail of its chemical composition, or how it is formed. Previous investigations of lycopodiopsid megaspore walls have demonstrated that, in some groups, the walls contain layers consisting of domains of crystalline particles, commonly around 0±25 µm in diameter (Figs 2 and 3). This has been interpreted as a colloidal crystal (Hemsley, Collinson and Brain, 1992) and shows considerable similarity to other natural colloidal crystal aggregations (e.g. precious opal, Darragh, Gaskin and Sanders, 1976 ; iridoviridae, Devauchelle, Stoltz and Darcy-Tripier, 1985). Like these colloidal crystals, the specific layer within the lycopodiopsid
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F. 1. Megaspore of Selaginella myosurus exhibiting a robust reticulate ridge ornament (specimens held in the biological collection, University of Wales, Cardiff). Scale bar ¯ 500 µm. F. 2. The wall structure of Selaginella myosurus megaspore shows three different layers of ultrastructure : an inner layer of particles ; a middle layer of ordered, monodisperse particles ; and an outer layer (present within the ridges) of convoluted laminae. Scale bar ¯ 5 µm. F. 3. Detail of the middle (colloidal crystal) layer shown in Fig. 2, exhibiting ordering of component particles. Scale bar ¯ 1 µm. F. 4. The structure shown in Fig. 3 can be modelled (as shown here) by a monodisperse latex of polystyrene particles flocculated by carboxymethylcellulose. Scale bar ¯ 1 µm.
Hemsley et al.—Simulated Self-assembly of Spore Exines
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T 1. A comparison of the components and their function from a generalized sporangium and from the synthetic system used here
Polymer forming latex Flocculant Hydrocarbon Supporting fluid Initiator
Plant sporangium
Model system
Sporopollenin Mucopolysaccharides Lipids (fatty acids) Water Enzyme ? UV ? O ?
Polystyrene Carboxymethylcellulose Cyclohexane Water Ammonium persulphate
megaspore wall is iridescent (Hemsley et al., 1994). These observations led to the hypothesis that this layer (at least) within the spore wall was of colloidal origin. The structures forming the remaining wall layers (irregular particles, rods, laminae), and indeed, those of other lycopodiopsid megaspores, could all theoretically be generated by the association of specific forms of colloidal unit. This hypothesis was supported by the transitional wall zones between, for example, layers consisting of regular particle arrays and layers consisting of laminae (Collinson, Hemsley and Taylor, 1993 ; Fig. 7). In addition, megaspores extracted from sporangia during development show features consistent with a colloidal mode of construction. A mechanism of depletion flocculation of a monodisperse sporopollenin colloid was proposed to account for the wall structures described above (Hemsley et al., 1994). A water-based monodisperse polystyrene latex (to simulate sporopollenin within the supporting sporangial fluid, Table 1), was flocculated using carboxymethylcellulose (representing the mucopolysaccharides ; Morbelli and Rowley, 1993). This gave rise to colloidal crystal forms similar to those within the megaspore wall layers (Fig. 4 ; Hemsley et al., 1996) and by a self-assembly process governed by the concentration and osmotic behaviour of the components. We are currently investigating the possibility of utilizing a polmer of pcoumaric acid for the production of polymer latices (which may be appropriate ; Wehling et al., 1989), but currently we view the volume of available literature on polystyrene colloid behaviour to outweigh any benefits to be obtained by using more ‘ natural ’ components. This study sought to expand the reproducible range of structures obtainable by
#
colloidal self-assembly, to demonstrate that the diversity of exine structure observable in clubmoss megaspores is consistent with their production by variations of a single mechanism, and to test the hypothesis that microspores are constructed by a similar mechanism. MATERIALS AND METHODS All these simulations used polystyrene in place of sporopollenin. Ammonium persulphate is used throughout as the polymeric reaction initiator. Sodium chloride is used to provide a quantitative increase in the electrolyte concentration of the solution, which consists mainly of doubledistilled water. Our initial experiments (Hemsley et al., 1996) were aimed at the production of a monodisperse colloid in an aqueous environment. The technique was based on that of Goodwin et al. (1973) utilizing the apparatus shown in Fig. 13. Our subsequent simulations have incorporated additional components (see Table 2) to reflect sporangial content and increase diversity of structure. The distilled water and sodium chloride were placed in the reaction vessel (with cyclohexane if used). The oil bath was raised to 70°C and the stirrer set to rotate at around 300 rpm. A slow stream of nitrogen was passed through the reaction vessel for 1 h to expel oxygen, which might otherwise initiate polymerization on addition of the styrene. This flow of nitrogen was maintained throughout the duration of the reaction. The styrene was added and left to integrate in solution for 0±5 h before addition of the initiator. The apparatus was then left for 24 h for the reaction to progress. Flocculations of subsequently cooled latex were achieved by
F. 5. The simpler wall structure of S. selaginoides consists of polydisperse particles which are partly fused (spores obtained from herbarium specimens held in the National Museum of Wales, Cardiff). Scale bar ¯ 4 µm. F. 6. The structure shown in Fig. 5 can be similarly modelled by a flocculation of polydisperse polystyrene latex, as shown here. Scale bar ¯ 5 µm. F. 7. Transitions between layers in lycopodiopsid megaspore walls indicate that the processes responsible for different ultrastructural types are closely related. Scale bar ¯ 2 µm. F. 8. Transitions from particles to strings and sheets can be reproduced using polystyrene}cyclohexane colloidal mixtures, as shown here. Scale bar ¯ 1±5 µm. F. 9. Sporopollenin particles and particle aggregates present in the developing megaspore wall of Selaginella laeigata (spores obtained from plants in the living collection at University of Wales, Cardiff). Scale bar ¯ 10 µm. F. 10. A droplet of cyclohexane acts as a surface upon which polystyrene latex particles and particle aggregates have flocculated, forming a pattern strikingly similar to that of many spores and some pollen. Scale bar ¯ 10 µm. F. 11. When broken, coated droplets (as in Fig. 10) illustrate a structure containing raspberry-like aggregates of polystyrene particles, compared to the living example in Fig. 9. Scale bar ¯ 3 µm. F. 12. Membranes with regular holes and foam-like arrangements may also be created from polystyrene, as shown here. Some resemble the structures found in certain fern megaspore walls, e.g. Salinia.
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Hemsley et al.—Simulated Self-assembly of Spore Exines
the addition of the sodium salt of carboxymethylcellulose and}or air drying. Samples were later observed by scanning electron microscopy. R E S U L T S A N D D I S C U S S I ON Experiments using the simple components outlined above have created mixed, aqueous colloidal systems containing polydisperse particles, hydrocarbon droplets and particle aggregates. Flocculation of these mixtures demonstrates a wider range of pattern than previously reported (as dried down samples viewed by SEM, see Fig. 4) and has generated a diversity of relevant internal structures. The majority of those so far observed have analogues among either living or extinct lycopodiopsid megaspore walls (see Figs 5–8) but, significantly, some structures (obtained from solid residue adherent to the stirrer) resemble megaspore walls from other plant groups (Fig. 12), principally the ferns (for example Salinia). Specific concentrations of hydrocarbon and styrene monomer (Table 2) give rise to mixtures which, during latex production, produce raspberry-like aggregations of polystyrene particles. Sporopollenin ‘ raspberries ’ have been identified in developing megaspore exines of Selaginella (Fig. 9). Commonly these aggregate, with numerous free particles, on the surface of hydrocarbon droplets (mimicking sporopollenin aggregation around the spore body during wall development, Figs 10 and 11). The size distribution of the lipid droplets is dependent upon the degree of agitation (rpm) in the reaction vessel. Under the conditions used for latex production, the droplets are generally small (approx. 5 µm), but some reach 50 µm in diameter. Aggregations of particles and particle clusters upon the surface of these larger droplets result in structures comparable to many microspore, isospore and pollen walls in dimensions, and in some cases, in surface ornament. These results provide further support to the view that exine development within lycopodiopsid megaspores occurs principally by way of self-assembling colloids, and that a wide range of structures are potentially reproducible, based on variation in the initial components of such an assembly system. In addition, our experiments demonstrate an essential role for an oil or lipid component in the formation of surface patterning, either for the coalescence of preformed aggregates, or the restriction of individual particle aggregation around the developing wall.
Gas trap N2 N2 Reflux condenser
Thermometer Reaction vessel
Stirrer Oil bath
F. 13. Diagram of the apparatus used to prepare polystyrene latices. Adapted from Pieranski (1983).
Remarkably, these experiments suggest that microspore (small male spore) walls may be constructed from colloidal mixtures which differ little from those which give rise to the structure in megaspore walls, despite differences in ultimate wall thickness, patterning and ultrastructure. The processes by which particles are produced in the plants and in these experiments, and the particle composition, are clearly different (Table 1). However, the flocculation mechanisms are probably comparable (Lin et al., 1989). That we require similar types of component within the model to those found within the plant sporangia to produce similar structure is significant, and gives more support for our simulation. Within colloidal mixtures, there is clearly scope to construct an immense array of pattern and structural diversity which will, in many cases, be indistinguishable from that present in living and fossil spores. In addition, it is possible to quantify differences in ultimate structure in terms of differences in initial components, which may in turn be tested against comparative analyses of developing sporangial fluid content. This approach to an understanding of spore wall patterning underpins the growing consensus that spore and pollen wall development must, at least in part, be a selfassembly process resulting from physico-chemical properties of the sporangial contents (Scott, 1994). Wall development
T 2. The components and quantities used to produce the structures illustrated in Figs 4, 6, 8, 10 and 11. See methodology for details
Distilled water Styrene Cyclohexane Sodium chloride (10 % solution) Ammonium persulphate (3 % solution) Carboxymethylcellulose (in 20 ml distilled water)
Fig. 4
Fig. 6
670 ml 80 ml — 1±25 ml
335 ml 50 ml 335 ml 1±25 ml
Fig. 8 670 ml 50 ml 50 ml 1±25 ml
25 ml
25 ml
20 ml
5g
5g
—
Figs 10–11 670 ml 50 ml 100 ml 1±25 ml 20 ml —
Hemsley et al.—Simulated Self-assembly of Spore Exines by flocculation of mixed colloidal systems provides a simple mechanism by which polymerized sporopollenin monomer may aggregate forming elaborate structures. Within sporangia, mechanisms may be reliant, at least initially, upon controlled irregularities on the spore surface to induce pattern formation [probably cytoplasmic wall protein or charge distributions, perhaps associated with variation in plasma membrane topography (Paxson-Sowders et al., 1997)]. However, it is clear from our experiments that patterns and structures can arise in the absence of a physical template. Providing there is genetic control over the initial conditions and concentration of components within the colloidal system operating in the sporangium, the resulting spore wall ultrastructure and pattern can achieve consistency. The apparently high degree of genetic control involved in the production of patterning and ultrastructure would appear to be concerned not with the actual process of construction, but rather with the establishment of the precise conditions at the initiation of the self-assembly process (Hemsley, 1998). Simulations using inorganic components to produce structured aragonite shells around polystyrene particles (Walsh and Mann, 1995) have proven to be useful models in the understanding of pattern production mechanisms at the microscopic level. Such simulations not only reproduce the structures seen, but also reveal underlying processes which link the genetic basis of variation to actual morphology (in our case, colloidal aggregation). A C K N O W L E D G E M E N TS We thank P. D. Jenkins for his work in developing these models, and the Royal Society for a University Research Fellowship to ARH. We are indebted to J. Crawley, V. Williams, L. Axe, K. Habgood and A. Oldroyd for photographic and technical assistance. L I T E R A T U R E C I T ED Collinson ME, Hemsley AR, Taylor WA. 1993. Sporopollenin exhibiting colloidal organization in spore walls. Grana, Supplement 1 : 31–39. Darragh PJ, Gaskin AJ, Sanders JV. 1976. Opals. Scientific American 234 : 82–95. Devauchelle G, Stoltz DB, Darcy-Tripier F. 1985. Comparative ultrastructure of Iridoviridae. In : Willis DB, ed. Iridoiridae. Berlin : Springer Verlag, 1–21. Goodwin JW, Hearn J, Ho CC, Ottewill RH. 1973. The preparation and characterisation of polymer lattices formed in the absence of surface active agents. British Polymer Journal 5 : 347–362. Hemsley AR. 1998. Non-linear variation in simulated complex pattern development. Journal of Theoretical Biology (in press). Hemsley AR, Collinson ME, Brain APR. 1992. Colloidal crystal-like
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structure of sporopollenin in the megaspore walls of recent Selaginella and similar fossil spores. Botanical Journal of the Linnean Society 108 : 307–320. Hemsley AR, Collinson ME, Kovach WL, Vincent B, Williams T. 1994. The role of self-assembly in biological systems : evidence from iridescent colloidal sporopollenin in Selaginella megaspore walls. Philosophical Transactions of the Royal Society, London B 345 : 163–173. Hemsley AR, Jenkins PD, Collinson ME, Vincent B. 1996. Experimental modelling of exine self-assembly. Botanical Journal of the Linnean Society 121 : 177–187. Hemsley AR, Scott AC. 1991. Ultrastructure and relationships of the Upper Carboniferous spores from Thorpe Brickworks, West Yorkshire, UK. Reiew of Palaeobotany and Palynology 69 : 337–351. Lin MY, Lindsay HM, Weitz DA, Ball RC, Klein R, Meakin P. 1989. Universality in colloidal aggregation. Nature 339 : 360–362. Moore PD, Webb JA, Collinson ME. 1991. Pollen analysis. Oxford : Blackwell Scientific Publications. Morbelli MA, Rowley JR. 1993. Megaspore development in Selaginella. 1. ‘ Wicks ’, their presence, ultrastructure and presumed function. Sexual Plant Reproduction 6 : 98–107. Paxson-Sowders DM, Owen HA, Makaroff CA. 1997. A comparative ultrastructural analysis of exine pattern development in wildtype Arabidopsis and a mutant defective in pattern formation. Protoplasma 198 : 53–65. Pieranski P. 1983. Colloidal crystals. Contemporary Physics 24 : 25–73. Scott RJ. 1994. Pollen exine – the sporopollenin enigma and the physics of pattern. In : Scott RJ, Stead MA, eds. Molecular and cellular aspects of plant reproduction. Cambridge : Cambridge University Press, 49–81. Sheldon JM, Dickinson HG. 1986. Pollen wall formation in Lilium : The effect of chaotropic agents, and the organisation of the microtubular cytoskeleton during pattern development. Planta 168 : 11–23. Takahashi M. 1989. Pattern determination of the exine of Caesalpinia japonica (Leguminosae : Caesalpinioideae). American Journal of Botany 76 : 1615–1626. Takahashi M, Skvarla JJ. 1991. Exine pattern formation by plasma membrane in Bougainillea spectabilis Wild. (Nyctaginaceae). American Journal of Botany 78 : 1063–1069. Taylor WA. 1990. Comparative analysis of megaspore ultrastructure in Pennsylvanian lycophytes. Reiew of Palaeobotany and Palynology 62 : 65–78. Taylor WA. 1991. Megaspore wall ultrastructure in Selaginella. Pollen et Spores 31 : 251–288. Taylor WA. 1993. Megaspore wall structure in Isoetes. American Journal of Botany 80 : 165–171. Traverse A. 1988. Paleopalynology. Boston : Unwin Hyman. Tryon AF, Lugardon B. 1991. Spores of the Pteridophyta. New York : Springer-Verlag. Van Bergen PF, Collinson ME, Briggs DEG, de Leeuw JW, Scott AC, Evershed RP, Finch P. 1995. Resident biomacromolecules in the fossil record. Acta Botanica Neerlandica 44 : 319–342. Walsh D, Mann S. 1995. Fabrication of hollow porous shells of calcium carbonate from self-organizing media. Nature 377 : 320–323. Wehling K, Niester CH, Boon JJ, Willemse MTM, Wiermann R. 1989. p-Coumaric acid – a monomer in the sporopollenin skeleton. Planta 179 : 376–380.