Genome-wide analysis of the GDSL esterase/lipase family genes in Physcomitrium patens and the involvement of GELP31 in spore germination.

In plants, the ability to produce hydrophobic substances that would provide protection from dehydration was required for the transition to land. This genome-wide investigation outlines the evolution of GDSL-type esterase/lipase (GELP) proteins in the moss Physcomitrium patens and suggests possible functions of some genes. GELP proteins play roles in the formation of hydrophobic polymers such as cutin and suberin that protect against dehydration and pathogen attack. GELP proteins are also implicated in processes such as pollen development and seed metabolism and germination. The P. patens GELP gene family comprises 48 genes and 14 pseudogenes. Phylogenetic analysis of all P. patens GELP sequences along with vascular plant GELP proteins with reported functions revealed that the P. patens genes clustered within previously identified A, B and C clades. A duplication model predicting the expansion of the GELP gene family within the P. patens lineage was constructed. Expression analysis combined with phylogenetic analysis suggested candidate genes for functions such as defence against pathogens, cutin metabolism, spore development and spore germination. The presence of relatively fewer GELP genes in P. patens may reduce the occurrence of functional redundancy that complicates the characterization of vascular plant GELP genes. Knockout lines of GELP31, which is highly expressed in sporophytes, were constructed. Gelp31 spores contained amorphous oil bodies and germinated late, suggesting (a) role(s) of GELP31 in lipid metabolism in spore development or germination. Future knockout studies of other candidate GELP genes will further elucidate the relationship between expansion of the family and the ability to withstand the harsh land environment.

Sporogenesis in Physcomitrium patens: Intergenerational collaboration and the development of the spore wall and aperture.

Although the evolution of spores was critical to the diversification of plants on land, sporogenesis is incompletely characterized for model plants such as Physcomitrium patens. In this study, the complete process of P. patens sporogenesis is detailed from capsule expansion to mature spore formation, with emphasis on the construction of the complex spore wall and proximal aperture. Both diploid (sporophytic) and haploid (spores) cells contribute to the development and maturation of spores. During capsule expansion, the diploid cells of the capsule, including spore mother cells (SMCs), inner capsule wall layer (spore sac), and columella, contribute a locular fibrillar matrix that contains the machinery and nutrients for spore ontogeny. Nascent spores are enclosed in a second matrix that is surrounded by a thin SMC wall and suspended in the locular material. As they expand and separate, a band of exine is produced external to a thin foundation layer of tripartite lamellae. Dense globules assemble evenly throughout the locule, and these are incorporated progressively onto the spore surface to form the perine external to the exine. On the distal spore surface, the intine forms internally, while the spiny perine ornamentation is assembled. The exine is at least partially extrasporal in origin, while the perine is derived exclusively from outside the spore. Across the proximal surface of the polar spores, an aperture begins formation at the onset of spore development and consists of an expanded intine, an annulus, and a central pad with radiating fibers. This complex aperture is elastic and enables the proximal spore surface to cycle between being compressed (concave) and expanded (rounded). In addition to providing a site for water intake and germination, the elastic aperture is likely involved in desiccation tolerance. Based on the current phylogenies, the ancestral plant spore contained an aperture, exine, intine, and perine. The reductive evolution of liverwort and hornwort spores entailed the loss of perine in both groups and the aperture in liverworts. This research serves as the foundation for comparisons with other plant groups and for future studies of the developmental genetics and evolution of spores across plants.

An optimized transformation protocol for Anthoceros agrestis and three more hornwort species.

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high-quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimized transformation protocol for A. agrestis, which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussii, and Phaeoceros carolinianus. The new transformation method is less laborious, faster, and results in the generation of greatly increased numbers of transformants compared with the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localization signal peptides for hornworts providing new tools to better understand the hornwort cell biology.

Cell wall polymers in the Phaeoceros placenta reflect developmental and functional differences across generations.

The placenta of hornworts is unique among bryophytes in the restriction of transfer cells that are characterized by elaborate wall labyrinths to the gametophyte generation. During development, cells around the periphery of the sporophyte foot elongate, forming smooth-walled haustorial cells that interdigitate with gametophyte cells. Using immunogold labeling with 22 antibodies to diverse cell wall polymers, we examined compositional differences in the developmentally and morphologically distinct cell walls of gametophyte transfer cells and sporophyte haustorial cells in the placenta of Phaeoceros. As detected by Calcofluor White fluorescence, cellulose forms the cell wall scaffolding in cells on both sides of the placenta. Homogalacturonan (HG) and rhamnogalacturonan I (RG-I) pectins are abundant in both cell types, and haustrorial cells are further enriched in methyl-esterified HGs. The abundance of pectins in placental cell walls is consistent with the postulated roles of these polymers in cell wall porosity and in maintaining an acidic apoplastic pH favorable to solute transport. Xyloglucan hemicellulose, but not mannans or glucuronoxylans, are present in cell walls at the interface between the two generations with a lower density in gametophytic wall ingrowths. Arabinogalactan proteins (AGPs) are diverse along the plasmalemma of placental cells and are absent in surrounding cells in both generations. AGPs in placental cell walls may play a role in calcium binding and release associated with signal transduction as has been speculated for these glycoproteins in other plants. Callose is restricted to thin areas in cell walls of gametophyte transfer cells. In contrast to studies of transfer cells in other systems, no reaction to the JIM12 antibody against extensin was observed in Phaeoceros.

HAG1 and SWI3A/B control of male germ line development in P. patens suggests conservation of epigenetic reproductive control across land plants.

KEY MESSAGE: Bryophytes as models to study the male germ line: loss-of-function mutants of epigenetic regulators HAG1 and SWI3a/b demonstrate conserved function in sexual reproduction. With the water-to-land transition, land plants evolved a peculiar haplodiplontic life cycle in which both the haploid gametophyte and the diploid sporophyte are multicellular. The switch between these phases was coined alternation of generations. Several key regulators that control the bauplan of either generation are already known. Analyses of such regulators in flowering plants are difficult due to the highly reduced gametophytic generation, and the fact that loss of function of such genes often is embryo lethal in homozygous plants. Here we set out to determine gene function and conservation via studies in bryophytes. Bryophytes are sister to vascular plants and hence allow evolutionary inferences. Moreover, embryo lethal mutants can be grown and vegetatively propagated due to the dominance of the bryophyte gametophytic generation. We determined candidates by selecting single copy orthologs that are involved in transcriptional control, and of which flowering plant mutants show defects during sexual reproduction, with a focus on the under-studied male germ line. We selected two orthologs, SWI3a/b and HAG1, and analyzed loss-of-function mutants in the moss P. patens. In both mutants, due to lack of fertile spermatozoids, fertilization and hence the switch to the diploid generation do not occur. Pphag1 additionally shows arrested male and impaired female gametangia development. We analyzed HAG1 in the dioecious liverwort M. polymorpha and found that in Mphag1 the development of gametangiophores is impaired. Taken together, we find that involvement of both regulators in sexual reproduction is conserved since the earliest divergence of land plants.

The placenta of Physcomitrium patens: transfer cell wall polymers compared across the three bryophyte groups.

Following similar studies of cell wall constituents in the placenta of Phaeoceros and Marchantia, we conducted immunogold labeling TEM studies of Physcomitrium patens to determine the composition of cell wall polymers in transfer cells on both sides of the placenta. 16 monoclonal antibodies were used to localize cell wall epitopes in the basal walls and wall ingrowths in this moss. In general, placental transfer cell walls of P. patens contain fewer pectins and far fewer AGPs than those of the hornwort and liverwort. P. patens also lacks the differential labeling that is pronounced between generations in the other bryophytes. In contrast, transfer cell walls on either side of the placenta of P. patens are relatively similar in composition with slight variation in HG pectins. Compositional similarities between wall ingrowths and primary cell walls in P. patens suggest that wall ingrowths may simply be extensions of the primary cell wall. Considerable variability in occurrence, abundance, and types of polymers among the three bryophytes and between the two generations suggests that similarity in function and morphology of cell walls does not require a common cell wall composition. We propose that the specific developmental and life history traits of these plants may provide even more important clues in understanding the basis for these differences. This study significantly builds on our knowledge of cell wall composition in bryophytes in general and transfer cells across plants.

The hornworts: morphology, evolution and development.

Extant land plants consist of two deeply divergent groups, tracheophytes and bryophytes, which shared a common ancestor some 500 million years ago. While information about vascular plants and the two of the three lineages of bryophytes, the mosses and liverworts, is steadily accumulating, the biology of hornworts remains poorly explored. Yet, as the sister group to liverworts and mosses, hornworts are critical in understanding the evolution of key land plant traits. Until recently, there was no hornwort model species amenable to systematic experimental investigation, which hampered detailed insight into the molecular biology and genetics of this unique group of land plants. The emerging hornwort model species, Anthoceros agrestis, is instrumental in our efforts to better understand not only hornwort biology but also fundamental questions of land plant evolution. To this end, here we provide an overview of hornwort biology and current research on the model plant A. agrestis to highlight its potential in answering key questions of land plant biology and evolution.

Differential localization of cell wall polymers across generations in the placenta of Marchantia polymorpha.

To further knowledge on cell wall composition in early land plants, we localized cell wall constituents in placental cells of the liverwort Marchantia polymorpha L. using monoclonal antibodies (MAbs) in the transmission electron microscope and histochemical staining. The placenta of M. polymorpha is similar to the majority of bryophytes in that both generations contain transfer cells with extensive wall ingrowths. Although the four major cell wall polymers, i.e., cellulose, pectins, hemicelluloses, and arabinogalactan proteins, are present, there are variations in the richness and specificity across generations. An abundance of homogalacturonan pectins in all placental cell walls is consistent with maintaining cell wall permeability and an acidic apoplastic pH necessary for solute transport. Although similar in ultrastructure, transfer cell walls on the sporophyte side in M. polymorpha are enriched with xyloglucans and diverse AGPs not detected on the gametophyte side of the placenta. Gametophyte wall ingrowths are more uniform in polymer composition. Lastly, extensins and callose are not components of transfer cell walls of M. polymorpha, which deviates from studies on transfer cells in other plants. The difference in polymer localizations in transfer cell walls between generations is consistent with directional movement from gametophyte to sporophyte in this liverwort.

With Over 60 Independent Losses, Stomata Are Expendable in Mosses.

Because stomata in bryophytes are uniquely located on sporangia, the physiological and evolutionary constraints placed on bryophyte stomata are fundamentally different from those on leaves of tracheophytes. Although losses of stomata have been documented in mosses, the extent to which this evolutionary process occurred remains relatively unexplored. We initiated this study by plotting the known occurrences of stomata loss and numbers per capsule on the most recent moss phylogeny. From this, we identified 40 families and 74 genera that lack stomata, of which at least 63 are independent losses. No trends in stomata losses or numbers are evident in any direction across moss diversity. Extant taxa in early divergent moss lineages either lack stomata or produce pseudostomata that do not form pores. The earliest land plant macrofossils from 400 ma exhibit similar sporangial morphologies and stomatal distribution to extant mosses, suggesting that the earliest mosses may have possessed and lost stomata as is common in the group. To understand why stomata are expendable in mosses, we conducted comparative anatomical studies on a range of mosses with and without stomata. We compared the anatomy of stomate and astomate taxa and the development of intercellular spaces, including substomatal cavities, across mosses. Two types of intercellular spaces that develop differently are seen in peristomate mosses, those associated with stomata and those that surround the spore sac. Capsule architecture in astomate mosses ranges from solid in the taxa in early divergent lineages to containing an internal space that is directly connected to the conducing tissue and is involved in capsule expansion and the nourishment, hydration and development of spores. This anatomy reveals there are different architectural arrangements of tissues within moss capsules that are equally effective in accomplishing the essential processes of sporogenesis and spore dispersal. Stomata are not foundational to these processes.

Characterisation of evolutionarily conserved key players affecting eukaryotic flagellar motility and fertility using a moss model.

Defects in flagella/cilia are often associated with infertility and disease. Motile male gametes (sperm cells) are an ancestral eukaryotic trait that has been lost in several lineages like flowering plants. Here, we made use of a phenotypic male fertility difference between two moss (Physcomitrella patens) ecotypes to explore spermatozoid function. We compare genetic and epigenetic variation as well as expression profiles between the Gransden and Reute ecotype to identify a set of candidate genes associated with moss male infertility. We generated a loss-of-function mutant of a coiled-coil domain containing 39 (ccdc39) gene that is part of the flagellar hydin network. Defects in mammal and algal homologues of this gene coincide with a loss of fertility, demonstrating the evolutionary conservation of flagellar function related to male fertility across kingdoms. The Ppccdc39 mutant resembles the Gransden phenotype in terms of male fertility. Potentially, several somatic (epi-)mutations occurred during prolonged vegetative propagation of Gransden, causing regulatory differences of for example the homeodomain transcription factor BELL1. Probably these somatic changes are causative for the observed male fertility defect. We propose that moss spermatozoids might be employed as an easily accessible system to study male infertility of humans and animals in terms of flagellar structure and movement.

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.

Contrasting pectin polymers in guard cell walls of Arabidopsis and the hornwort Phaeoceros reflect physiological differences.

BACKGROUND AND AIMS: In seed plants, stomata regulate CO2 acquisition and water relations via transpiration, while minimizing water loss. Walls of guard cells are strong yet flexible because they open and close the pore by changing shape over the substomatal cavity. Pectins are necessary for wall flexibility and proper stomata functioning. This study investigates the differences in pectin composition in guard cells of two taxa that represent key lineages of plants with stomata: Arabidopsis, an angiosperm with diurnal stomatal activity, and Phaeoceros, a bryophyte that lacks active stomatal movement. METHODS: Using immunolocalization techniques in transmission electron microscopy, this study describes and compares the localization of pectin molecule epitopes essential to stomata function in guard cell walls of Arabidopsis and Phaeoceros. KEY RESULTS: In Arabidopsis, unesterified homogalacturonans very strongly localize throughout guard cell walls and are interspersed with arabinan pectins, while methyl-esterified homogalacturonans are restricted to the exterior of the wall, the ledges and the junction with adjacent epidermal cells. In contrast, arabinans are absent in Phaeoceros, and both unesterified and methyl-esterified homogalacturonans localize throughout guard cell walls. CONCLUSIONS: Arabinans and unesterified homogalacturonans are required for wall flexibility, which is consistent with active regulation of pore opening in Arabidopsis stomata. In contrast, the lack of arabinans and high levels of methyl-esterified homogalacturonans in guard cell walls of Phaeoceros are congruent with the inability of hornwort stomata to open and close with environmental change. Comparisons across groups demonstrate that variations in guard cell wall composition reflect different physiological activity of stomata in land plants.

Hornwort stomata do not respond actively to exogenous and environmental cues.

Backgrounds and Aims: Because stomata in bryophytes occur on sporangia, they are subject to different developmental and evolutionary constraints from those on leaves of tracheophytes. No conclusive experimental evidence exists on the responses of hornwort stomata to exogenous stimulation. Methods: Responses of hornwort stomata to abscisic acid (ABA), desiccation, darkness and plasmolysis were compared with those in tracheophyte leaves. Potassium ion concentrations in the guard cells and adjacent cells were analysed by X-ray microanalysis, and the ontogeny of the sporophytic intercellular spaces was compared with those of tracheophytes by cryo-scanning electron microscopy. Key Results: The apertures in hornwort stomata open early in development and thereafter remain open. In hornworts, the experimental treatments, based on measurements of >9000 stomata, produced only a slight reduction in aperture dimensions after desiccation and plasmolysis, and no changes following ABA treatments and darkness. In tracheophytes, all these treatments resulted in complete stomatal closure. Potassium concentrations are similar in hornwort guard cells and epidermal cells under all treatments at all times. The small changes in hornwort stomatal dimensions in response to desiccation and plasmolysis are probably mechanical and/or stress responses of all the epidermal and spongy chlorophyllose cells, affecting the guard cells. In contrast to their nascent gas-filled counterparts across tracheophytes, sporophytic intercellular spaces in hornworts are initially liquid filled. Conclusions: Our experiments demonstrate a lack of physiological regulation of opening and closing of stomata in hornworts compared with tracheophytes, and support accumulating developmental and structural evidence that stomata in hornworts are primarily involved in sporophyte desiccation and spore discharge rather than the regulation of photosynthesis-related gaseous exchange. Our results run counter to the notion of the early acquisition of active control of stomatal movements in bryophytes as proposed from previous experiments on mosses.

The Ceratopteris (fern) developing motile gamete walls contain diverse polysaccharides, but not pectin.

MAIN CONCLUSION: Unlike most plant cell walls, the five consecutive walls laid down during spermatogenesis in the model fern Ceratopteris contain sparse cellulose, lack pectin and are enriched with callose and hemicelluloses. Seed-free plants like bryophytes and pteridophytes produce swimming male gametes for sexual reproduction. During spermatogenesis, unique walls are formed that are essential to the appropriate development and maturation of the motile gametes. Other than the detection of callose and general wall polysaccharides in scattered groups, little is known about the sequence of wall formation and the composition of these walls during sperm cell differentiation in plants that produce swimming sperm. Using histochemistry and immunogold localizations, we examined the distribution of callose, cellulose, mannan and xylan-containing hemicelluloses, and homogalacturonan (HG) pectins in the special walls deposited during spermatogenesis in Ceratopteris. Five walls are produced in sequence and each has a unique fate. The first wall (W1) contains callose and sparse xylan-containing hemicelluloses. Wall two (W2) is thin and composed of cellulose crosslinked by xylan-containing hemicelluloses. The third wall (W3) is thick and composed entirely of callose, and the fourth wall (W4) is built of cellulose heavily crosslinked by galactoxyloglucan hemicelluloses. Wall five (W5) is an arabinogalactan protein (AGP)-rich matrix in which the gamete changes shape and multiple flagella elongate. We detected no esterified or unesterified HG pectins in any of the walls laid down during spermatogenesis. To consider evolutionary modifications in cell walls associated with motile gametes, comparisons are presented with male gametophyte and spermatogenous cell walls across plant groups.

Hornwort Stomata: Architecture and Fate Shared with 400-Million-Year-Old Fossil Plants without Leaves.

As one of the earliest plant groups to evolve stomata, hornworts are key to understanding the origin and function of stomata. Hornwort stomata are large and scattered on sporangia that grow from their bases and release spores at their tips. We present data from development and immunocytochemistry that identify a role for hornwort stomata that is correlated with sporangial and spore maturation. We measured guard cells across the genera with stomata to assess developmental changes in size and to analyze any correlation with genome size. Stomata form at the base of the sporophyte in the green region, where they develop differential wall thickenings, form a pore, and die. Guard cells collapse inwardly, increase in surface area, and remain perched over a substomatal cavity and network of intercellular spaces that is initially fluid filled. Following pore formation, the sporophyte dries from the outside inwardly and continues to do so after guard cells die and collapse. Spore tetrads develop in spore mother cell walls within a mucilaginous matrix, both of which progressively dry before sporophyte dehiscence. A lack of correlation between guard cell size and DNA content, lack of arabinans in cell walls, and perpetually open pores are consistent with the inactivity of hornwort stomata. Stomata are expendable in hornworts, as they have been lost twice in derived taxa. Guard cells and epidermal cells of hornworts show striking similarities with the earliest plant fossils. Our findings identify an architecture and fate of stomata in hornworts that is ancient and common to plants without sporophytic leaves.

Patterning of stomata in the moss Funaria: a simple way to space guard cells.

BACKGROUND AND AIMS: Studies on stomatal development and the molecular mechanisms controlling patterning have provided new insights into cell signalling, cell fate determination and the evolution of these processes in plants. To fill a major gap in knowledge of stomatal patterning, this study describes the pattern of cell divisions that give rise to stomata and the underlying anatomical changes that occur during sporophyte development in the moss Funaria. METHODS: Developing sporophytes at different stages were examined using light, fluorescence and electron microscopy; immunogold labelling was used to investigate the presence of pectin in the newly formed cavities. KEY RESULTS: Substomatal cavities are liquid-filled when formed and drying of spaces is synchronous with pore opening and capsule expansion. Stomata in mosses do not develop from a self-generating meristemoid as in Arabidopsis, but instead they originate from a protodermal cell that differentiates directly into a guard mother cell. Epidermal cells develop from protodermal or other epidermal cells, i.e. there are no stomatal lineage ground cells. CONCLUSIONS: Development of stomata in moss occurs by differentiation of guard mother cells arranged in files and spaced away from each other, and epidermal cells that continue to divide after stomata are formed. This research provides evidence for a less elaborated but effective mechanism for stomata spacing in plants, and we hypothesize that this operates by using some of the same core molecular signalling mechanism as angiosperms.

Arabinogalactan proteins and arabinan pectins abound in the specialized matrices surrounding female gametes of the fern Ceratopteris richardii.

MAIN CONCLUSION: Both male and female gametes of archegoniates are highly specialized cells surrounded by an extraprotoplasmic matrix rich in AGPs, which are speculated to facilitate development and gamete fusion through Ca 2+) oscillations. An additional layer, the egg envelope, forms around the egg periphery, except at the fertilization pore, and contains arabinose-rich polymers that presumably impart flexibility for the rapidly growing zygote and embryo. The abundant AGPs and arabinan pectins associated with the eggs of C. richardii not only are integral to development, fertilization, and early embryogenesis, but also may be involved in desiccation tolerance important to the survival of the reproductive gametophyte. A defining feature of gametogenesis in archegoniates is the deposition of a special matrix outside of the plasmalemma of both egg and sperm cells that displaces the primary cell wall away from the protoplasm. It is within this matrix that gamete differentiation occurs. In leptosporangiate ferns, maturation of the egg cell involves the deposition of a second specialized wall, the so-called egg envelope that surrounds the cell except at the fertilization pore, a narrow site where gamete fusion takes place. We provide the first conclusive evidence of the macromolecular constituents in the unique structures surrounding fern egg cells before and after fertilization. To test the hypotheses that the egg extracellular matrix contains arabinogalactan proteins (AGPs) as does the sperm cell matrix, and that cell wall polysaccharides, especially pectins, are components of the egg envelope, we examined the expression patterns of AGPs and cell wall constituents during oogenesis in Ceratopteris richardii. Utilizing histochemical stains for callose, cellulose and AGPs coupled with immunogold localizations employing a suite of monoclonal antibodies to cell wall components (JIM13, JIM8, LM2, LM5, LM6, LM19, LM20 and anticallose), we demonstrate that AGPs, but not pectins, are abundant in the matrix around egg cells and degrading neck canal and ventral canal cells during archegonial development. A striking finding is that both AGPs and (1,5)-α-L-arabinan pectin epitopes are principle components of the egg envelope before and after fertilization, suggesting that they are important in both egg maturation and gamete fusion.

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.

Multiflagellated sperm cells of Ceratopteris richardii are bathed in arabinogalactan proteins throughout development.

UNLABELLED: • PREMISE OF THE STUDY: Sperm cell differentiation in ferns involves the origin of an elaborate locomotory apparatus, including 70+ flagella, and the structural modification of every cellular component. Because arabinogalactan proteins (AGPs) are implicated in molecular signaling and in regulation of plant development, we speculated that these glycoproteins would be present during spermiogenesis in ferns.• METHODS: Using ß-glucosyl Yariv reagents that specifically bind to and inhibit AGPs and immunogold localizations with monoclonal antibodies JIM13, JIM8, and LM6, we examined the specific expression patterns of AGPs and inhibited their function during sperm cell development in the model fern Ceratopteris richardii.• KEY RESULTS: Developing sperm cells stained intensely with Yariv phenylglycosides, demonstrating the presence of AGPs. JIM13-AGP epitopes were widespread throughout development in the expanding extraprotoplasmic matrix (EPM) in which flagella elongate, cytoplasm is eliminated, and spherical spermatids become coiled. JIM8 and LM6 epitopes localized to the plasmalemma on growing flagella and on the rapidly changing sperm cell body. Spermatids treated with ß-glucosyl lacked an EPM and formed fewer, randomly arranged flagella.• CONCLUSIONS: We demonstrated that AGPs are abundant in the EPM and along the plasmalemma and that the three AGP epitopes have specific expression patterns during development. Coupled with inhibition studies, these results identify AGPs as critical to the formation of an extraprotoplasmic matrix and the consequent origin and development of flagella in an orderly and precise fashion around the cell. We speculate that AGPs may play additional roles as signaling molecules involved in cell shaping, cytoskeletal development, vesicle trafficking, and cytoplasmic elimination.