Spore Wall Development Research Articles

Reactive oxygen species are required for spore wall formation in Physcomitrella patens.

A robust spore wall was a key requirement of terrestrialization by early plants. Sporopollenin in spore and pollen grain walls is thought to be polymerized and cross-linked to other macromolecular components partly through oxidative processes involving H2O2. Therefore, we investigated effects of scavengers of reactive oxygen species (ROS) on formation of spore walls in the moss, Physcomitrella patens. Exposure of sporophytes, containing spores in the process of forming walls, to ascorbate, dimethylthiourea or 4-hydroxy-TEMPO prevented normal wall development in a dose, chemical and stage-dependent manner. Mature spores, exposed while developing to a ROS scavenger, burst when mounted in water on a flat slide under a coverslip (a phenomenon we named "augmented osmolysis" since they did not burst in phosphate-buffered saline or in water on a depression slide). Additionally, walls of exposed spores were more susceptible to alkaline hydrolysis than those of control spores and some were characterized by discontinuities in the exine, anomalies in perine spine structure, abnormal intine and aperture and occasionally wall shedding. Our data support involvement of oxidative cross-linking in spore wall development, including sporopollenin polymerization or deposition, as well as a role for ROS in intine/aperture development.

Spore wall ultrastructure and development in a basal euphyllophyte: Psilophyton dawsonii from the Lower Devonian of Quebec (Canada).

Euphyllophytes, a clade including living ferns, horsetails, and seed plants, have a rich fossil record going back to the Early Devonian. The euphyllophyte spore wall has a complex structure, the evolutionary origins of which are incompletely understood. Psilophyton is the best-characterized basal euphyllophyte genus; thus, data on this genus can inform current hypotheses on spore wall structure and development, which propose a bilayered spore wall organization of combined spore and sporangial origin for the ancestral euphyllophyte. We employed cellulose acetate peel sectioning of permineralized Lower Devonian (Emsian) Psilophyton dawsonii sporangia, combined with electron microscopy, to document spore wall structure and development. The Psilophyton dawsonii spore wall is bilayered. The inner spore wall is homogeneous, probably of lamellar construction. The outer spore wall, loosely attached to the inner wall, covers distal and equatorial spore areas, and has a foveolate base layer upon which stacks of sporopollenin lumps accrete centrifugally, forming the scaffolding for the final apiculate ornamentation. This is the most complete account on spore wall structure, allowing developmental interpretations, in a basal euphyllophyte. The bipartite organization of the Psilophyton dawsonii spore wall reflects development as a result of two processes: an inner layer laid down by the spore cell and an outer layer of tapetal origin. Providing direct evidence on the spore wall of a basal euphyllophyte, these data confirm previous hypotheses and mark an empirically supported starting point for discussions of the evolution of spore wall development in euphyllophytes.

An ultrastructural study of spore wall development and septal pores in species of the Pachyphlodes (Pezizaceae, Pezizales) lineage, with a description of the new species Pachyphlodes annagardnerae

Pachyphlodes (Pezizaceae) is a genus of truffle-like fungi that is distributed across the Northern Hemisphere. These fungi form ectomycorrhizae primarily with trees in the Fagaceae family, and occasionally with other host plants. The genus Plicariella (= Scabropezia) is phylogenetically inferred as an ally of, or within, the Pachyphlodes lineage. Despite molecular phylogenetic analyses that show the close relationships of species in these two genera, morphological differences in ascomata shape and color, spore ornamentation, and ascus shape are profound. Here, we studied spore wall development to better understand affinities within the Pachyphlodes–Plicariella lineages. Electron microscopy studies indicate that the initial spore wall development is similar across six Pachyphlodes species and a Plicariella species, despite striking differences in mature spore ornamentation among species. Ultrastructural analyses reveal that differences in spore ornamentation among Pachyphlodes species are due to unique developmental events at the final stages of spore wall deposition. Septal pore ultrastructure in Pachyphlodes species is similar to other Pezizaceae that have been studied. Molecular analyses of the five species studied indicate that four of them have not been previously described. The new species Pachyphlodes annagardnerae is here described, and the ultrastructural features of species of Pachyphlodes, Plicariella, and other Pezizales are compared and discussed.

Callose is integral to the development of permanent tetrads in the liverwort Sphaerocarpos.

A striking feature of the liverwort Sphaerocarpos is that pairs of male and female spores remain united in permanent tetrads. To identify the nature of this phenomenon and to test the hypothesis that callose is involved, we examined spore wall development in Sphaerocarpos miche lii, with emphasis on the appearance, location and fate of callose vis-à-vis construction of the sculptoderm. All stages of sporogenesis were examined using differential interference contrast optics, and aniline blue fluorescence to locate callose. For precise localization, specimens were immunogold labeled with anti-callose antibody and observed in the transmission electron microscope. Callose plays a role in Sphaerocarpos spore wall development not described in any other plant, including other liverworts. A massive callose matrix forms outside of the sculptured sporocyte plasmalemma that predicts spore wall ornamentation. Consequently, layers of exine form across adjacent spores uniting them. Spore wall development occurs entirely within the callose and involves the production of six layers of prolamellae that give rise to single or stacked tripartite lamellae (TPL). Between spores, an anastomosing network of exine layers forms in lieu of intersporal septum development. As sporopollenin assembles on TPL, callose progressively disappears from the inside outward leaving layers of sporopollenin impregnated exine, the sculptoderm, overlying a thick fibrillar intine. This developmental mechanism provides a direct pathway from callose deposition to sculptured exine that does not involve the intermediary primexine found in pollen wall development. The resulting tetrad, encased in a single wall, provides a simple model for development of permanent dyads and tetrads in the earliest fossil plants.

Conservation of Male Sterility 2 function during spore and pollen wall development supports an evolutionarily early recruitment of a core component in the sporopollenin biosynthetic pathway

The early evolution of plants required the acquisition of a number of key adaptations to overcome physiological difficulties associated with survival on land. One of these was a tough sporopollenin wall that enclosed reproductive propagules and provided protection from desiccation and UV-B radiation. All land plants possess such walled spores (or their derived homologue, pollen). We took a reverse genetics approach, consisting of knock-out and complementation experiments to test the functional conservation of the sporopollenin-associated gene MALE STERILTY 2 (which is essential for pollen wall development in Arabidopsis thaliana) in the bryophyte Physcomitrella patens. Knock-outs of a putative moss homologue of the A. thaliana MS2 gene, which is highly expressed in the moss sporophyte, led to spores with highly defective walls comparable to that observed in the A. thaliana ms2 mutant, and extremely compromised germination. Conversely, the moss MS2 gene could not rescue the A. thaliana ms2 phenotype. The results presented here suggest that a core component of the biochemical and developmental pathway required for angiosperm pollen wall development was recruited early in land plant evolution but the continued increase in pollen wall complexity observed in angiosperms has been accompanied by divergence in MS2 gene function.

Evolutionary development of the plant and spore wall.

Many key innovations were required to enable plants to colonize terrestrial habitats successfully. One of these was the acquisition of a durable spore/pollen wall capable of withstanding the harsh desiccating and UV-B-rich environment encountered on land. The spores of 'lower' spore-bearing plants and the pollen of 'higher' seed plants are homologous. In recent years, researchers have begun to investigate the molecular genetics of pollen wall development in angiosperms (including the model organism Arabidopsis thaliana). However, research into the molecular genetics of spore wall development in more basal plants has thus far been extremely limited. This review summarizes the literature on spore/pollen wall development, including the molecular genetics associated with pollen wall development in angiosperms, in a preliminary attempt to identify possible candidate genes involved in spore wall development in more basal plants. Bioinformatic studies have suggested that genes implicated in pollen wall development in angiosperms are also present in moss and lycopsids, and may therefore be involved in spore wall development in basal plants. This suggests that the molecular genetics of spore/pollen development are highly conserved, despite the large morphological and functional differences between spores and pollen. The use of high-throughput sequencing strategies and/or microarray experiments at an appropriate stage of 'lower' land plant sporogenesis will allow the identification of candidate genes likely to be involved in the development of the spore wall by way of comparison with those genes known to be involved in pollen wall development. Additionally, by conducting gene knock-out and gene swap experiments between 'lower' land plant species, such as the moss model species Physcomitrella patens, and the angiosperm model species arabidopsis it will be possible to test the role of these candidate genes.

Ultrastructural observations on the ascomata and ascospores of the truffle Mattirolomyces terfezioides

The septal pore ultrastructure of non-ascogenous and reproductive hyphae, as well as development of the spore wall were examined in the truffle Mattirolomyces terfezioides (Mattir.) E. Fisch. (Ascomycota, Pezizales, Pezizaceae) and compared with other pezizalean taxa. The septal pore in ascogenous hyphae of M. terfezioides is a uni- or bi-convex band of the type found in the Pezizaceae. Septal pore ultrastructure in other hyphae included a uniconvex band with lamellate structure. Woronin bodies were globose to hexagonal, and infrequently ellipsoid in shape. Hexagonal Woronin bodies have not been previously reported in the Pezizaceae. Ascospore delimitation starts with two delimiting membranes and the spore-wall development is typical of the Pezizales. The heterogeneously staining spines of the secondary cell wall differ from the structures of Terfezia species. The ultrastructural characteristics of M. terfezioides show the general characteristics of the Pezizaceae, except that the hexagonal Woronin body form is new to this family. The spore delimitation, spine-cap structure, and Woronin body shape of M. terfezioides presented here are in agreement with molecular taxonomic results, which separate the genus from Terfezia and call into question the generic position of Mattirolomyces tiffanyae Healy.

ULTRASTRUCTURE OF DISPERSED ANDIN SITUSPECIMENS OF THE DEVONIAN SPORERHABDOSPORITES LANGII: EVIDENCE FOR THE EVOLUTIONARY RELATIONSHIPS OF PROGYMNOSPERMS

Abstract: The sporeRhabdosporites(Triletes)langii(Eisenack)Richardson, 1960is abundant and well preserved in Middle Devonian (Eifelian) ‘Middle Old Red Sandstone’ deposits from the Orcadian Basin, Scotland. Here it occurs as dispersed individual spores andin situin isolated sporangia. This paper reports on a detailed light microscope (LM), scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis of both dispersed andin situspores. The dispersed spores are pseudosaccate with a thick walled inner body enclosed within an outer layer that was originally attached only over the proximal face. The inner body has lamellate/laminate ultrastructure consisting of fine lamellae that are continuous around the spore and parallel stacked. Towards the outer part of the inner body these group to form thicker laminate structures that are also continuous and parallel stacked. The outer layer has spongy ultrastructure.In situspores preserved in the isolated sporangia are identical to the dispersed forms in terms of morphology, gross structure and wall ultrastructure. The sporangium wall is two‐layered. A thick coalified outer layer is cellular and represents the main sporangium wall. This layer is readily lost if oxidation is applied during processing. A thin inner layer is interpreted as a peritapetal membrane. This layer survives oxidation as a tightly adherent membranous covering of the spore mass. Ultrastructurally it consists of three layers, with the innermost layer composed of material similar to that comprising the outer layer of the spores. Based on the new LM, SEM and TEM information, consideration is given to spore wall formation. The inner body of the spores is interpreted as developing by centripetal accumulation of lamellae at the plasma membrane. The outer layer is interpreted as forming by accretion of sporopollenin units derived from a tapetum. The inner layer of the sporangium wall is considered to represent a peritapetal membrane formed from the remnants of this tapetum. The sporeR. langiiderives from aneurophytalean progymnosperms. In light of the new evidence on spore/sporangium characters, and hypotheses of spore wall development based on interpretation of these, the evolutionary relationships of the progymnosperms are considered in terms of their origins and relationship to the seed plants. It is concluded that there is a smooth evolutionary transition betweenApiculiretusispora‐type spores of certain basal euphyllophytes,Rhabdosporites‐type spores of aneurophytalean progymnosperms andGeminospora‐/Contagisporites‐type spores of heterosporous archaeopteridalean progymnosperms. Prepollen of basal seed plants (hydrasperman, medullosan and callistophytalean pteridosperms) are easily derived from the spores of either homosporous or heterosporous progymnosperms. The proposed evolutionary transition was sequential with increasing complexity of the spore/pollen wall probably reflecting increasing sophistication of reproductive strategy. The pollen wall of crown group seed plants appears to incorporate a completely new developmental mechanism: tectum and infratectum initiation within a glycocalyx‐like Microspore Surface Coat. It is unclear when this feature evolved, but it appears likely that it was not present in the most basal stem group seed plants.

Novel actin ring structure in sporulation of Zygosaccharomyces rouxii

Abstract The filamentous actin (F-actin) during sporulation of Zygosaccharomyces rouxii was visualized with rhodamine-phalloidin, and then the behavior was observed using confocal laser scanning microscopy. During spore formation, we found a novel actin ring structure that has not been reported in other yeasts and molds in sporulation. The ring surrounded each meiotic nucleus at the peripheral regions of spores. Three-dimensional observation suggested that the ring was not an artificial structure produced by spherical structure sectioning. The period and location of the ring's appearance suggest that the ring may have some relation to the spore membrane or wall development. In addition, this ring structure was more stable than other Factin structures against latrunculin A, an F-actin disrupting agent.

Mattirolomyces tiffanyae, a new truffle from Iowa, with ultrastructural evidence for its classification in the Pezizaceae

A new species of hypogeous Pezizales, Mattirolomyces tiffanyae, is described and illustrated. Its asci are typically three-spored, an unusually small number in the non-Tuber Pezizales. Ascus septal pore ultrastructure consists of a uni- or bi-convex band, which suggests an affinity with the Pezizaceae. Secondary spore-wall development is similar to that of Peziza, and several species of hypogeous Pezizaceae.

A model for the role of surfactants in the assembly of exine sculpture

The surface coatings of spores and pollen often show complex species or genus specific patterning and for which there is no obvious explanation in terms of evolutionary advantage. It has previously been suggested that these patterns are the product of a self-assembly process used by plants in spore and pollen wall development and are a consequence of the physical and chemical behaviour of the components and their interactions. Experiments have shown that comparable patterning and structure can be achieved by self-assembly of analogous components but the range of patterning obtainable was restricted. The introduction of specific surfactants into the reaction has now broadened the range of patterns that can be derived by this method and supports the view that surfactants must be important in the development of the naturally occurring patterns.

Ultrastructural Studies of Ascosporogenesis in Ascobolus stictoideus (Pezizales, Ascomycetes)

Ascosporogenesis in Ascobolus stictoideus is studied and compared with that found in other members of Pezizales. Early phases of ascus development involving spore delimiting membranes from the plasmalemma and spore formation within the epiplasm are much like those of other species of Pezizales, with the exception of the appearance of organelles, such as pigmented bodies and net bodies, which are not found in other taxa. Primary spore walls are very thick and electron translucent. Opaque globular bodies, amorphous dense material derived from net bodies, and dense membrane‐bound bodies associated with the endoplasmic reticulum appear in the epiplasm and later play a role in spore wall formation. Dense granular bodies containing small particles are transported from the epiplasm into the perisporic sac along with pigmented bodies. These deeply opaque bodies increase in size, appear to coalesce, and are deposited onto the epispore layer of the spore. The phylogenetic significance of spore wall development is discussed in light of recent molecular data.

Sporangia containing Scylaspora from the Lower Devonian of the Welsh Borderland

ABSTRACT. Bulk maceration of Early Devonian (Lochkovian) deposits from the Welsh Borderland has yielded eight specimens of a new type of sporangium characterized by its elongate shape and distinctive spores. The specimens have been examined using scanning electron, transmission electron and light microscopy. The elongate sporangia occur isolated and are fragmented to varying degrees. They contain trilete spores that possess a proximal surface with shallow murornate ornament and a distal surface that is laevigate. The spores belong to the dispersed spore genus Scylaspora, and this is the first report of such spores in situ. Ultrastructural studies demonstrate that the spore walls are bilayered with a lamellate inner layer overlain by an essentially homogeneous outer layer. There is little or no associated extra‐exosporal material. To date this is the earliest reported example of lamellate wall ultrastructure in trilete spores. Models of spore wall development are suggested in the light of evidence provided by spore wall ultrastructure. Detailed comparisons of the characters preserved in the fossils (morphological, anatomical and ultrastructural), with those in other fossil and extant plants, currently shed little light on the phylogenetic relationships of these specimens, primarily due to the paucity of comparable data. It is proposed that the plant is probably of vascular status, but in the absence of evidence for vascular tissue, it must be classified as rhyniophytoid.

Ultrastructure of laevigate hilate spores in sporangia and spore masses from the Upper Silurian and Lower Devonian of the Welsh Borderland

Spore masses and isolated sporangia, containing laevigate hilate cryptospores attributable to the dispersed taxonLaevolancis divellomedia sensu lato, have been recovered on bulk maceration of Upper Silurian (Pridoli) and Lower Devonian (Lochkovian) deposits from the Welsh Borderland. Detailed morphological, anatomical and ultrastructural analysis, using light microscope, scanning electron microscope and transmission electron microscope techniques, reveals subtle differences between the specimens and they can be grouped into five distinct types. The different groups are distinguished principally by using sporangia–spore mass characteristics, presence or absence of extra–exosporal material and nature of spore–wall ultrastructure. Of the groups, one has a uniformly homogeneous exospore and the other four groups have a bilayered exospore. In the former the spores lack extra–exosporal material and occur in a discoidal sporangium. Of the bilayered groups, two have exospores of homogeneous composition but with the two layers differing in electron density. They occur in discoidal sporangia and spore masses and are distinguished on the presence or absence of extra–exosporal material and differences in the widths of the two layers. Finally, two bilayered groups possess a lamellate inner layer, but vary in presumed sporangial shape. Elongate sporangia have spores with concentric continuous lamellae, lacking further ultrastructure. In contrast, spores from a discoidal spore mass have white–line–centred, presumably tripartite, lamellae which are laterally discontinuous, overlapping and irregularly spaced. These findings, which suggest that morphologically similar spores were produced by a number of plant taxa, have important implications regarding the assessment of early land–plant diversity. The affinities of hilate cryptospore–producing plants are unknown and problematic, particularly as no extant non–angiosperm plants produce dyads, other than through meiotic irregularity, and spore–sporangial characters have no exact counterpart in coeval plants. Studies of specimens within situhilate cryptospores suggest that they derive from rhyniophytoids, i.e. plants that resemble the simplest of vascular plants but lack evidence of vascular tissue, although hilate cryptospore–containing examples show no axial branching. It might be argued, based on evidence from spore wall ultrastructure, that some of the plants have more in common with lycopsids and filicopsids than bryophytes, a surprising finding bearing in mind the stratigraphic distribution of hilate cryptospores–dyads and inferences that the producers were bryophyte–like. Detailed studies of wall structure in the hilate cryptospores permit consideration of spore wall development. It is suggested that extra–exosporal material derives from a tapetum and is thus produced by the diploid sporophyte. The white–line–centred lamellae in a single specimen provide the earliest evidence for the presence of such structures in early land plant spores and provide further evidence that sporopollenin deposition on such structures is the most primitive mode of sporopollenin deposition among land plants.

Anatomy and ultrastructure of the sporophyte of Takakia ceratophylla (Bryophyta)

In this study, morphogenesis and structure of the sporophyte of Takakia ceratophylla are characterized beginning with the late embryo and culminating in the fully dehisced capsule. Information is presented on the development, ultrastructure, and anatomy of the three organographic regions of the sporophyte, namely capsule, seta, and foot. Diagnostic features that identify Takakia as a moss include the gradual elongation of seta, persistence of an apical calyptra, expansion of the capsule after cessation of seta elongation, existence of a columella, monoplastidic meiosis, spore ultrastructure (including a perine layer deposited late in spore wall (development), and the structure of the foot. Commonalities with the capsule of the Andreaeopsida include sporogenous tissue that overarchs a central columella, absence of stomata, and lack of a peristome and operculum. Peculiarities of the genus are seen in the internal structure of the capsule, the disintegration of the columella with spore maturation, and the dehiscence of the capsule along a single, spiralled, longitudinal suture line. Passive spore dispersal through longitudinal splitting of the capsule occurs in andreaeopsid mosses, liverworts, hornworts, and seedless vascular plants. The precise mechanism of dehiscence along a spiralled suture is unparalleled in extant archegoniates but finds counterparts in ancestral land plants such as the pteridophyte Tortilicaulis.

Ultrastructural Studies of Leucangium carthusianum (Hypogeous Pezizales)

Ascospores and septal structures of both vegetative and reproductive hyphae of Leucangium carthusianum were studied ultrastructurally. The apiculate ascospores contain a couple of large guttules and multiple nuclei (more than four). Early spore wall development is similar to that of most epigeous and hypogeous Pezizales studied. The mature spore wall has a very thick primary wall with a bricklike arrangement of wall fibers and a three layered epispore. Septal structures of vegetative hyphae are constructed of laminate pore plugs and globose, hexagonal, and cylindrical Woronin bodies, which are typically found in the Pezizales. In reproductive hyphae. septal pore plugs are dome shaped with central laminations. Cylindrical Woronin bodies are found around the septal pore in ascal bases. Using ultrastructural data, we find that L. carthusianum is closely related to the Morchellaceae and Helvellaceae. However, the placement of L. carthusianum in either family is not certain.

Spore ontogeny of Galiella rufa (Pezizales)

Galiella is one of the genera of the dark-colored apothecial Sarcosomataceae, tribe Galielleae, with cyanophilous spore markings. Ultrastructural studies show that spore wall development of Galiella rufa is similar to the subgenus Discina of Gyromitra in Helvellaceae and to the other Sarcosomataceae, especially Plectania nannfeldtii, which both have fine secondary wall spore ornaments. The multinucleate ascospores found in G. rufa may show relationship to the Morchellaceae and the Helvellaceae. Keywords: ascospore ontogeny, Galiella, Sarcosomataceae, ultrastructure.

Spore ontogeny in species of Phillipsia and Wynnea (Pezizales)

Some species of the genera Phillipsia and Wynnea have similar longitudinally ridged cyanophobic ascospore markings. Ultrastructural studies show that the cyanophobic spore markings are part of the primary wall. In contrast, the cyanophilous spore ornaments are formed by the secondary wall. The observation of spore wall development indicates that the sources of the spore wall components are the sporoplasm and the epiplasm. Based on the pinkish hymenial color of fresh young apothecia and the cyanophobic spore ridges found only in some members of the Sarcoscyphaceae, Wynnea belongs to the Sarcoscyphaceae. Keywords: Pezizales, Phillipsia, Sarcoscyphaceae, spore wall ontogeny, ultrastructure, Wynnea.

The tapetum inSchizaea pectinata (Schizaeaceae) and a comparison with the tapetum inPsilotum nudum (Psilotaceae)

A combination tapetum consisting of a cellular, parietal component and a plasmodial component occurs inSchizaea pectinata. A single, tapetal initial layer divides to form an outer parietal layer which maintains its cellular integrity until late in spore wall development. The inner tapetal layer differentiates into a plasmodium which disappears after the outer exospore has developed. In the final stages of spore wall development, granular material occurs in large masses and is dispersed as small granules throughout the sporangial loculus. No tapetal membrane develops. Comparisons are drawn with the combination tapetum found inPsilotum nudum.

Ultrastructure of Spore Ontogeny in Trichophaea brunnea (Pezizales)

Trichophaea brunnea is a small, pyrophylic, operculate discomycete with hyaline, uniguttulate ascospores and an excipulum fringed with short, brownish, tapering hairs. Most recent literature places the species in Sphaerosporella because of its smooth spherical spores, as opposed to ellipsoid spores of other species of Trichophaea. Ellipsoid-spored species of Trichophaea have both smooth and ornamented ascospores with variable guttulation and other apothecial characters. These differences raise questions as to the relationship of T. brunnea to other species of Trichophaea. In this article we examine the fine structure of spore wall development in T. brunnea to determine if it is comparable to that of ellipsoid-spored species. We found that while early stages of spore ontogeny are similar, there are differences in staining properties of the primary wall of three species compared, but wall development in T brunnea is much like that in the type T. abundans The pattern of deposition and staining properties of various wall layers support other conclusions that Sphaerosporella should not be recognized as a distinct genus.