The origin of the sporophyte shoot in land plants: a bryological perspective.

BACKGROUND: Land plants (embryophytes) are monophyletic and encompass four major clades: liverworts, mosses, hornworts and polysporangiophytes. The liverworts are resolved as the earliest divergent lineage and the mosses as sister to a crown clade formed by the hornworts and polysporangiophytes (lycophytes, monilophytes and seed plants). Alternative topologies resolving the hornworts as sister to mosses plus polysporangiophytes are less well supported. Sporophyte development in liverworts depends only on embryonic formative cell divisions. A transient basal meristem contributes part of the sporophyte in mosses. The sporophyte body in hornworts and polysporangiophytes develops predominantly by post-embryonic meristematic activity. SCOPE: This paper explores the origin of the sporophyte shoot in terms of changes in embryo organization. Pressure towards amplification of the sporangium-associated photosynthetic apparatus was a major driver of sporophyte evolution. Starting from a putative ancestral condition in which a transient basal meristem produced a sporangium-supporting seta, we postulate that in the hornwort-polysporangiophyte lineage the basal meristem acquired indeterminate meristematic activity and ectopically expressed the sporangium morphogenetic programme. The resulting sporophyte body plan remained substantially unaltered in hornworts, whereas in polysporangiophytes the persistent meristem shifted from a mid-embryo to a superficial position and was converted into an ancestral shoot apical meristem with the evolution of sequential vegetative and reproductive growth. CONCLUSIONS: The sporophyte shoot is interpreted as a sterilized sporangial axis interpolated between the embryo and the fertile sporangium. With reference to the putatively ancestral condition found in mosses, the sporophyte body plans in hornworts and polysporangiophytes are viewed as the product of opposite heterochronic events, i.e. an anticipation and a delay, respectively, in the development of the sporangium. In either case the result was a pedomorphic sporophyte permanently retaining juvenile characters.

Major transitions in the evolution of early land plants: a bryological perspective.

Background Molecular phylogeny has resolved the liverworts as the earliest-divergent clade of land plants and mosses as the sister group to hornworts plus tracheophytes, with alternative topologies resolving the hornworts as sister to mosses plus tracheophytes less well supported. The tracheophytes plus fossil plants putatively lacking lignified vascular tissue form the polysporangiophyte clade. Scope This paper reviews phylogenetic, developmental, anatomical, genetic and paleontological data with the aim of reconstructing the succession of events that shaped major land plant lineages. Conclusions Fundamental land plant characters primarily evolved in the bryophyte grade, and hence the key to a better understanding of the early evolution of land plants is in bryophytes. The last common ancestor of land plants was probably a leafless axial gametophyte bearing simple unisporangiate sporophytes. Water-conducting tissue, if present, was restricted to the gametophyte and presumably consisted of perforate cells similar to those in the early-divergent bryophytes Haplomitrium and Takakia. Stomata were a sporophyte innovation with the possible ancestral functions of producing a transpiration-driven flow of water and solutes from the parental gametophyte and facilitating spore separation before release. Stomata in mosses, hornworts and polysporangiophytes are viewed as homologous, and hence these three lineages are collectively referred to as the 'stomatophytes'. An indeterminate sporophyte body (the sporophyte shoot) developing from an apical meristem was the key innovation in polysporangiophytes. Poikilohydry is the ancestral condition in land plants; homoiohydry evolved in the sporophyte of polysporangiophytes. Fungal symbiotic associations ancestral to modern arbuscular mycorrhizas evolved in the gametophytic generation before the separation of major present-living lineages. Hydroids are imperforate water-conducting cells specific to advanced mosses. Xylem vascular cells in polysporangiophytes arose either from perforate cells or de novo. Food-conducting cells were a very early innovation in land plant evolution. The inferences presented here await testing by molecular genetics.

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis.

BACKGROUND AND AIMS: Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells. METHODS: Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins. KEY RESULTS: The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls. CONCLUSIONS: The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.

The dawn of symbiosis between plants and fungi.

The colonization of land by plants relied on fundamental biological innovations, among which was symbiosis with fungi to enhance nutrient uptake. Here we present evidence that several species representing the earliest groups of land plants are symbiotic with fungi of the Mucoromycotina. This finding brings up the possibility that terrestrialization was facilitated by these fungi rather than, as conventionally proposed, by members of the Glomeromycota. Since the 1970s it has been assumed, largely from the observation that vascular plant fossils of the early Devonian (400 Ma) show arbuscule-like structures, that fungi of the Glomeromycota were the earliest to form mycorrhizas, and evolutionary trees have, until now, placed Glomeromycota as the oldest known lineage of endomycorrhizal fungi. Our observation that Endogone-like fungi are widely associated with the earliest branching land plants, and give way to glomeromycotan fungi in later lineages, raises the new hypothesis that members of the Mucoromycotina rather than the Glomeromycota enabled the establishment and growth of early land colonists.

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.

Frogs, sentinels of DNA damage induced by pollution in Naples and the neighbouring Provinces.

Many DNA mutation-based diseases recognised in Campania have recently been related to toxic substances in illegal dumping areas. We performed a comet assay on edible frog erythrocytes to evaluate DNA damage. Differences in genotoxic parameters were observed among populations. We show that severe DNA damage occurred in the north Campania where the emergence of environmental waste exploded recently. Although a similar magnitude of genotoxic damage was observed in some southern populations, it is attributable to a massive pesticide pollution related to intensive farming. The frog species analysed seems to be a good bioindicator for detecting genotoxic effects of chemical environmental hazards.

Novel localization of callose in the spores of Physcomitrella patens and phylogenomics of the callose synthase gene family.

BACKGROUND AND AIMS: Callose involvement in spore development is a plesiomorphic feature of land plants. Correlated light, fluorescence and immuno-electron microscopy was conducted on the developing spores of Physcomitrella patens to probe for callose. Using a bioinformatic approach, the callose synthase (PpCalS) genes were annotated and PpCalS and AtCalS gene families compared, testing the hypothesis that an exine development orthologue is present in P. patens based on deduced polypeptide similarity with AtCalS5, a known exine development gene. METHODS: Spores were stained with aniline blue fluorescent dye. Capsules were prepared for immuno-light and immuno-electron microscopy by gold labelling callose epitopes with monoclonal antibody. BLAST searches were conducted using the AtCalS5 sequence as a query against the P. patens genome. Phylogenomic analysis of the CalS gene family was conducted using PAUP (v.4.1b10). KEY RESULTS: Callose is briefly present in the aperture of developing P. patens spores. The PpCalS gene family consists of 12 copies that fall into three distinct clades with AtCalS genes. PpCalS5 is an orthologue to AtCalS5 with highly conserved domains and 64 % similarity of their deduced polypeptides. CONCLUSIONS: This is the first study to identify the presence of callose in moss spores. AtCalS5 was previously shown to be involved in pollen exine development, thus making PpCalS5 a suspect gene involved in moss spore exine development.

Cellular differentiation in moss protonemata: a morphological and experimental study.

BACKGROUND AND AIMS: Previous studies of protonemal morphogenesis in mosses have focused on the cytoskeletal basis of tip growth and the production of asexual propagules. This study provides the first comprehensive description of the differentiation of caulonemata and rhizoids, which share the same cytology, and the roles of the cytoskeleton in organelle shaping and spatial arrangement. METHODS: Light and electron microscope observations were carried out on in vitro cultured and wild protonemata from over 200 moss species. Oryzalin and cytochalasin D were used to investigate the role of the cytoskeleton in the cytological organization of fully differentiated protonemal cells; time-lapse photography was employed to monitor organelle positions. KEY RESULTS: The onset of differentiation in initially highly vacuolate subapical cells is marked by the appearance of tubular endoplasmic reticulum (ER) profiles with crystalline inclusions, closely followed by an increase in rough endoplasmic reticulum (RER). The tonoplast disintegrates and the original vacuole is replaced by a population of vesicles and small vacuoles originating de novo from RER. The cytoplasm then becomes distributed throughout the cell lumen, an event closely followed by the appearance of endoplasmic microtubules (MTs) in association with sheets of ER, stacks of vesicles that subsequently disperse, elongate mitochondria and chloroplasts and long tubular extensions at both poles of the nucleus. The production of large vesicles by previously inactive dictysomes coincides with the deposition of additional cell wall layers. At maturity, the numbers of endoplasmic microtubules decline, dictyosomes become inactive and the ER is predominantly smooth. Fully developed cells remain largely unaffected by cytochalasin; oryzalin elicits profound cytological changes. Both inhibitors elicit the formation of giant plastids. The plastids and other organelles in fully developed cells are largely stationary. CONCLUSIONS: Differentiation of caulonemata and rhizoids involves a remarkable series of cytological changes, some of which closely recall major events in sieve element ontogeny in tracheophytes. The cytology of fully differentiated cells is remarkably similar to that of moss food-conducting cells and, in both, is dependent on an intact microtubule cytoskeleton. The disappearance of the major vacuolar apparatus is probably related to the function of caulonema and rhizoids in solute transport. Failure of fully differentiated caulonema and rhizoid cells to regenerate is attributed to a combination of endo-reduplication and irreversible tonoplast fragmentation. The formation of giant plastids, most likely by fusion, following both oryzalin and cytochalasin treatments, suggests key roles for both microtubules and microfilaments in the spatial arrangement and replication of plastids.

Development of water-conducting cells in the antipodal liverwort Symphyogyna brasiliensis (Metzgeriales).

The thallus of the metzgerialean liverwort Symphyogyna brasiliensis Nets contains a strand of dead thick-walled tells with helicoidally-arranged pits that arc presumably involved in water transport. During the first phase of differentiation these cells undergo a 13-16-fold elongation while remaining thin-walled and almost unchanged in diameter. During subsequent maturation the walls become strongly thickened by deposition of highly electron-opaque material on extraplasmodesmal areas and of transparent material forming collars around plasmodesmata. Whilst the growing wall shows an ordered microribrillar texture and is strongly reactive to PATAg staining for carbohydrates, the material associated with plasmodesmata is amorphous and PATAg-negative. A dense cortical array of microtobules (MTs) overlies the growing wall except in proximity to plasmodesmata, which are closely associated with tubular endoplasmic reticulum (ER). During cellular maturation plasmodesmata undergo extensive secondary elongation by incorporation of cortical ER supposedly continuous with desmotubules. Quantitative analysis of plasmodesmal frequencies in relation to cellular elongation and wall thickness indicates that there is no de novo formation of plasmodesmata. Cortical MTs, wall microfibrils and secondarily-modified plasmodesmata are consistently co-aligned, all forming helices of about 45°. During maturation the Golgi apparatus proliferates and a vast number of vesicles containing PATAg-positive material are produced from a membrane domain interpreted as trans Golgi network, whilst PATAg-negative vesicles are formed along the fenestrated margins of C& and media) dictyosomal cisternae. Exocytosis of PATAg-positive vesicles is confined to extraplasmodesmal areas. In ageing cells abundant fibrillar material, also positive to PATAg-test, accumulates within pleomorphic membrane-bounded tubules. Final cytoplasmic dissolution involves the lysis of all cellular membranes and the liberation of the membrane-bounded fibrillar material, that is subsequently deposited onto the walls. The eventual dissolution of the plugs of amorphous electron-transport material results in the formation of open pits. Similarities in the cytological mechanisms underlying pore development in water-conducting cells of Symphyogyna and in the sieve elements of angiosperms are discussed.

Development of the leafy shoot in Sphagnum (Bryophyta) involves the activity of both apical and subapical meristems.

This light- and electron-microscope study of four species of Sphagnum reveals that stem elongation involves meristematic activities unique to the group and hitherto unrecognized. The internal tissue of the mature stem arises by the concerted activity of an apical (primary) and a subapical (secondary) meristem. The primary meristem comprises the immediate derivatives of the single apical cell. Following a small number of divisions, the primary derivatives differentiate into highly vacuolate parenchymatous cells with a storied arrangement. Subsequently, the large vacuoles are replaced by numerous small vacuoles and the cells then divide repeatedly, by transverse septa, producing files of about nine short cells. Finally, ninefold elongation of these secondary cells is responsible for extension growth of the main stem below the mature capitulum. An early step in primary differentiation is the confinement of pre-existing plasmodesmata to distinct pitted areas. Further enlargement of the cells during primary and secondary differentiation involves the thickening of non-pitted wall areas, followed by expansion and thinning out, while the pitted areas remain virtually unchanged. A cortical array of microtubules is regularly found in association with non-pitted wall areas, while the unexpanded pitted areas are associated with smooth endoplasmic reticulum showing continuity with desmotubules. Though sharing much the same cytology as the conducting cells in bryoid mosses, in terms of their development the central stem cells in Sphagnum are not homologous with those of other mosses. The unique mode of stem development may be an important factor in the ecological success of Sphagnum.

The leafy stems of Sphagnum (Bryophyta) contain highly differentiated polarized cells with axial arrays of endoplasmic microtubules.

Contrary to the long-held belief that, internal to the cortical sterome, the central region of Sphagnum stems comprises unspecialized parenchyma, the present light- and electron-microscope study has revealed that these cells in fact have a highly specialized cytoplasmic organization. Their key features are: (a) the absence of large central vacuoles; (b) a spindle-shaped nucleus positioned internally; (c) a prominent axial system of endoplasmic microtubules associated with the nucleus, mitochondria, pleomorphic vacuoles, and membrane-bounded tubules and vesicles; (d) a distinct cytoplasmic polarization, with the cellular region near the capitulum being richer in organelles than the basal region; and (e) a high frequency of plasmodesmata in the cross walls with an enlarged median region containing no discernible desmotubule. Such a distinctive combination of cytological features has been hitherto only described for putative food-conducting cells in bryoid mosses. The results introduce a major new character common to Sphagnum and bryoid mosses and strongly suggest that this cytological organization underlines cellular specialization in symplasmic transport.

Subterranean gametophytic axes in the primitive liverwort Haplomitrium harbour a unique type of endophytic association with aseptate fungi.

• Haplomitrium, a primitive liverwort taxon with only remote affinities to other liverwort groups, develops root-like subterranean axes harbouring fungal endophytes. Here we report on the fungal association in H. gibbsiae and H. ovalifolium, using light and electron microscopy. • The epidermal cells of subterranean axes secrete abundant mucilage that harbours aseptate fungal hyphae. The fungus penetrates the epidermal cells and forms intracellular arbuscules invested by the host cytoplasm. Infection is restricted to epidermal cells in H. gibbsiae, whereas in H. ovalifolium the fungus also infects the cortical cells immediately adjacent, where it forms prominent swellings ('lumps'). In H. gibbsiae similar fungal swellings are formed in the epidermal cells along with arbuscules. In both species the lumps undergo cytoplasmic degeneration and collapse, showing a shorter lifespan than the arbuscules. • The fungal infection in Haplomitrium presents affinities with symbiotic associations with glomeromycotean fungi in higher plants (arbuscular mycorrhizas) and thalloid liverworts. However, the pattern of fungal morphogenesis in Haplomitrium has no precedent in bryophytes nor in higher plants. • Considering the Glomeromycota as the most ancient lineage of mycorrhizal fungi, and Haplomitrium as basal in land plant phylogenies, the association described here may be the most primitive land plant-fungal symbiosis documented to date.

Diversity in the distribution of polysaccharide and glycoprotein epitopes in the cell walls of bryophytes: new evidence for the multiple evolution of water-conducting cells.

• Although histologically much simpler than higher plants, bryophytes display a considerable degree of tissue differentiation, notably in those groups that possess an internal system of specialized water-conducting cells (WCCs). Here, using a battery of monoclonal antibodies, we examined the distribution of cell wall polysaccharide and glycoprotein carbohydrate epitopes in the gametophyte of four hepatics and eight mosses, with special reference to water-conducting cells. • CCRC-M7, an antibody against an arabinogalactan epitope, gave a highly consistent and generally specific labelling of WCCs; more variable results were obtained with other antibodies. The labelling patterns indicate that bryophytes exhibit cell and tissue complexity with respect to cell wall components on a par with higher plants. • A remarkable diversity in the immunocytochemical characteristics of WCCs was observed not only when comparing major bryophyte groups but also within the relatively small and well-circumscribed moss order Polytrichales, indicating that the cell wall biochemistry of WCCs may have been finely tuned in response to specific evolutionary pressures. The immunocytochemical data strengthen the notion that the WCCs in Takakia are not homologous with the hydroids of other mosses nor with the WCCs in Haplomitrium and metzgerialean liverworts. • The presence of several carbohydrate epitopes in hydroid walls runs strongly counter to the notion that their maturation involves hydrolysis of noncellulosic polysaccharides.

A novel ascomycetous endophytic association in the rhizoids of the leafy liverwort family, Schistochilaceae (Jungermanniidae, Hepaticopsida).

Liverworts form diverse associations with endophytic fungi similar to mycorrhizas in vascular plants. Whereas the widespread occurrence of glomeromycotes in the basal liverwort lineages is well documented, knowledge of the distribution of ascomycetes and basidiomycetes in derived thalloid and leafy clades is more fragmented. Our discovery that the ramified and septate rhizoids of the Schistochilaceae, the sister group to all other ascomycete-containing liverworts, are packed with fungal hyphae prompted this study on the effects of the fungi on rhizoid morphology, host specificity, the cytology of the association, and a molecular analysis of the endophytes. Two species of Pachyschistochila and their fungi were grown axenically. Axenic rhizoids were unbranched and nonseptate. Reinfected with their own fungus and that from the other species, both Pachyschistochila species produced branched and septate rhizoids identical to those in nature. Woronin bodies and simple septa identified the fungus as an ascomycete referable, according to phylogenetic analyses of ITS sequences, to the Rhizoscyphus (Hymenoscyphus) ericae aggregate, also found in other liverwort-ascomycete associations and in mycorrhizas in the Ericales. Healthy hyphae and host cytoplasm suggest that the Schistochila-fungus association reflects a balanced mutualistic relationship. The recent dating of the divergence of the Jungermanniales from the fungus-free Porellales in the Permian and the origins of the Schistochilaceae in the Triassic indicate that these associations in liverworts predate the appearance of the Ericales.

Desiccation tolerance in the moss Polytrichum formosum: physiological and fine-structural changes during desiccation and recovery.

BACKGROUND AND AIMS: This study explores basic physiological features and time relations of recovery of photosynthetic activity and CO(2) uptake following rehydration of a desiccation-tolerant moss in relation to the full temporal sequence of cytological changes associated with recovery to the normal hydrated state. It seeks reconciliation of the apparently conflicting published physiological and cytological evidence on recovery from desiccation in bryophytes. METHODS: Observations were made of water-stress responses and recovery using infrared gas analysis and modulated chlorophyll fluorescence, and of structural and ultrastructural changes by light and transmission electron microscopy. KEY RESULTS: Net CO(2) uptake fell to zero at approx. 40 % RWC, paralleling the fluorescence parameter PhiPSII at 200 micromol m(-2) s(-1) PPFD. On re-wetting the moss after 9-18 d desiccation, the initially negative net CO(2) uptake became positive 10-30 min after re-wetting, restoring a net carbon balance after approx. 0.3-1 h. The parameter F(v)/F(m) reached approx. 80 % of its pre-desiccation value within approx. 10 min of re-wetting. In the presence of the protein-synthesis inhibitors chloramphenicol and cycloheximide, recovery of F(v)/F(m) (and CO(2) exchange) proceeded normally in the dark, but declined rapidly in the light. Though initial recovery was rapid, both net CO(2) uptake and F(v)/F(m) required approx. 24 h to recover completely to pre-desiccation values. The fixation protocols produced neither swelling of tissues nor plasmolysis. Thylakoids, grana and mitochondrial cristae remained intact throughout the drying-re-wetting cycle, but there were striking changes in the form of the organelles, especially the chloroplasts, which had prominent lobes and lamellar extensions in the normally hydrated state, but rounded off when desiccated, returning slowly to their normal state within approx. 24 h of re-wetting. Sub-cellular events during desiccation and re-wetting were generally similar to those seen in published data from the pteridophyte Selaginella lepidophylla. CONCLUSIONS: Initial recovery of respiration and photosynthesis (as of protein synthesis) is very rapid, and independent of protein synthesis, suggesting physical reactivation of systems conserved intact through desiccation and rehydration, but full recovery takes approx. 24 h. This is consistent with the cytological evidence, which shows the thylakoids and cristae remaining intact through the whole course of dehydration and rehydration. Substantial and co-ordinated changes in other cell components, which must affect spatial relationships of organelles and metabolic systems, return to normal on a time span similar to full recovery of photosynthesis. Comparison of the present data with recently published results suggests a significant role for the cytoskeleton in desiccation responses.

Desiccation tolerance in the moss Polytrichum formosum: physiological and fine-structural changes during desiccation and recovery.

BACKGROUND AND AIMS: This study explores basic physiological features and time relations of recovery of photosynthetic activity and CO2 uptake following rehydration of a desiccation-tolerant moss in relation to the full temporal sequence of cytological changes associated with recovery to the normal hydrated state. It seeks reconciliation of the apparently conflicting published physiological and cytological evidence on recovery from desiccation in bryophytes. METHODS: Observations were made of water-stress responses and recovery using infrared gas analysis and modulated chlorophyll fluorescence, and of structural and ultrastructural changes by light and transmission electron microscopy. KEY RESULTS: Net CO2 uptake fell to zero at approx. 40 % RWC, paralleling the fluorescence parameter PhiPSII at 200 micromol m(-2) s(-1) PPFD. On re-wetting the moss after 9-18 d desiccation, the initially negative net CO2 uptake became positive 10-30 min after re-wetting, restoring a net carbon balance after approx. 0.3-1 h. The parameter Fv/Fm reached approx. 80 % of its pre-desiccation value within approx. 10 min of re-wetting. In the presence of the protein-synthesis inhibitors chloramphenicol and cycloheximide, recovery of Fv/Fm (and CO2 exchange) proceeded normally in the dark, but declined rapidly in the light. Though initial recovery was rapid, both net CO2 uptake and Fv/Fm required approx. 24 h to recover completely to pre-desiccation values. The fixation protocols produced neither swelling of tissues nor plasmolysis. Thylakoids, grana and mitochondrial cristae remained intact throughout the drying-re-wetting cycle, but there were striking changes in the form of the organelles, especially the chloroplasts, which had prominent lobes and lamellar extensions in the normally hydrated state, but rounded off when desiccated, returning slowly to their normal state within approx. 24 h of re-wetting. Sub-cellular events during desiccation and re-wetting were generally similar to those seen in published data from the pteridophyte Selaginella lepidophylla. CONCLUSIONS: Initial recovery of respiration and photosynthesis (as of protein synthesis) is very rapid, and independent of protein synthesis, suggesting physical reactivation of systems conserved intact through desiccation and rehydration, but full recovery takes approx. 24 h. This is consistent with the cytological evidence, which shows the thylakoids and cristae remaining intact through the whole course of dehydration and rehydration. Substantial and co-ordinated changes in other cell components, which must affect spatial relationships of organelles and metabolic systems, return to normal on a time span similar to full recovery of photosynthesis. Comparison of the present data with recently published results suggests a significant role for the cytoskeleton in desiccation responses.

Glomeromycotean associations in liverworts: a molecular, cellular, and taxonomic analysis.

Liverworts form endophytic associations with fungi that mirror mycorrhizal associations in tracheophytes. Here we report a worldwide survey of liverwort associations with glomeromycotean fungi (GAs), together with a comparative molecular and cellular analysis in representative species. Liverwort GAs are circumscribed by a basal assemblage embracing the Haplomitriopsida, the Marchantiopsida (except a few mostly derived clades), and part of the Metzgeriidae. Fungal endophytes from Haplomitrium, Conocephalum, Fossombronia, and Pellia were related to Glomus Group A, while the endophyte from Monoclea was related to Acaulospora. An isolate of G. mosseae colonized axenic thalli of Conocephalum, producing an association similar to that in the wild. Fungal colonization in marchantialean liverworts suppressed cell wall autofluorescence and elicited the deposition of a new wall layer that specifically bound the monoclonal antibody CCRC-M1 against fucosylated side groups associated with xyloglucan and rhamnogalacturonan I. The interfacial material covering the intracellular fungus contained the same epitopes present in host cell walls. The taxonomic distribution and cytology of liverwort GAs suggest an ancient origin and multiple more recent losses, but the occurence in widely separated liverwort taxa of fungi related to glomeromycotean lineages that form arbuscular mycorrhizas in tracheophytes, notably the Glomus Group A, is better explained by host shifting from tracheophytes to liverworts.

A highly differentiated glomeromycotean association with the mucilage-secreting, primitive antipodean liverwort Treubia (Treubiaceae): clues to the origins of mycorrhizas.

Thallus anatomy in three species of the primitive liverwort genus Treubia (Metzgeriidae, Treubiales) was studied by light and electron microscopy. The thallus exudes copious mucilage, a feature shared elsewhere in liverworts only with the mycotrophic subterranean axes of the allied genus Haplomitrium. The central strand in the thallus midrib has a unique histological organization and harbors an intra- and intercellular infection by a glomeromycotean fungus that is far more highly differentiated than most of the glomeromycotean associations described to date. The fungus enters the thallus via clefts in the ventral epidermis along the midrib and colonizes the parenchyma above, forming intracellular coils and prominent, relatively short-lived, hyphal swellings. Above the zone with intracellular colonization is a tissue area containing mucilage-filled intercellular spaces; here the fungus is entirely intercellular and forms abundant pseudoparenchymatous structures and, in more mature parts of the thalli, large hyphae with thick multistratose walls. Mucilage in Treubia differs in histochemistry and origin from that produced by apical papillae, via hypertrophied Golgi, in all other bryophytes. Remarkable parallels between fungal associations in Treubia, Haplomitrium, and Lycopodium, all members of very ancient lineages, suggest that these associations epitomize very early stages in the evolution of glomeromycotean symbioses.

Effects of de- and rehydration on food-conducting cells in the moss Polytrichum formosum: a cytological study.

BACKGROUND AND AIMS: Moss food-conducting cells (leptoids and specialized parenchyma cells) have a highly distinctive cytology characterized by a polarized cytoplasmic organization and longitudinal alignment of plastids, mitochondria, endoplasmic reticulum and vesicles along endoplasmic microtubules. Previous studies on the desiccation biology of mosses have focused almost exclusively on photosynthetic tissues; the effects of desiccation on food-conducting cells are unknown. Reported here is a cytological study of the effects of de- and rehydration on food-conducting cells in the desiccation-tolerant moss Polytrichum formosum aimed at exploring whether the remarkable subcellular organization of these cells is related to the ability of mosses to survive desiccation. METHODS: Shoots of Polytrichum formosum were dehydrated under natural conditions and prepared for transmission and scanning electron microscopy using both standard and anhydrous chemical fixation protocols. Replicate samples were then fixed at intervals over a 24-h period following rehydration in either water or in a 10 microM solution of the microtubule-disrupting drug oryzalin. KEY RESULTS: Desiccation causes dramatic changes; the endoplasmic microtubules disappear; the nucleus, mitochondria and plastids become rounded and the longitudinal alignment of the organelles is lost, though cytoplasmic polarity is in part retained. Prominent stacks of endoplasmic reticulum, typical of the hydrated condition, are replaced with membranous tubules arranged at right angles to the main cellular axis. The internal cytoplasm becomes filled with small vacuoles and the plasmalemma forms labyrinthine tubular extensions outlining newly deposited ingrowths of cell wall material. Whereas plasmodesmata in meristematic cells at the shoot apex and in stem parenchyma cells appear to be unaffected by dehydration, those in leptoids become plugged with electron-opaque material. Starch deposits in parenchyma cells adjoining leptoids are depleted in desiccated plants. Rehydration sees complete reestablishment over a 12- to 24-h period of the cytology seen in the control plants. Oryzalin effectively prevents leptoid recovery. CONCLUSIONS: The results point to a key role of the microtubular cytoskeleton in the rapid re-establishment of the elaborate cytoplasmic architecture of leptoids during rehydration. The reassembly of the endoplasmic microtubule system appears to dictate the time frame for the recovery process. The failure of leptoids to recover normal cytology in the presence of oryzalin further underlines the key role of the microtubules in the control of leptoid cytological organization.

Distribution of cell-wall xylans in bryophytes and tracheophytes: new insights into basal interrelationships of land plants.

Xylans are known to be major cellulose-linking polysaccharides in secondary cell walls in higher plants. We used two monoclonal antibodies (LM10 and LM11) for a comparative immunocytochemical analysis of tissue and cell distribution of xylans in a number of taxa representative of all major tracheophyte and bryophyte lineages. The results show that xylans containing the epitopes recognized by LM10 and LM11 are ubiquitous components of secondary cell walls in vascular and mechanical tissues in all present-living tracheophytes. In contrast, among the three bryophyte lineages, LM11 binding was detected in specific cell-wall layers in pseudoelaters and spores in the sporophyte of hornworts, while no binding was observed with either antibody in the gametophyte or sporophyte of liverworts and mosses. The ubiquitous occurrence of xylans containing LM10 and LM11 epitopes in tracheophytes suggests that the appearance of these polysaccharides has been a pivotal event for the evolution of highly efficient vascular and mechanical tissues. LM11 binding in the sporophyte of hornworts, indicating the presence of relatively highly substituted xylans (possibly arabinoxylans), separates these from the other bryophytes and is consistent with recent molecular data indicating a sister relationship of the hornworts with tracheophytes.