Formation of specialized propagules resistant to desiccation and cryopreservation in the threatened moss Ditrichum plumbicola (Ditrichales, Bryopsida).

BACKGROUND AND AIMS: Successful cryopreservation of bryophytes is linked to intrinsic desiccation tolerance and survival can be enhanced by pre-treatment with abscisic acid (ABA) and sucrose. The pioneer moss Ditrichum plumbicola is naturally subjected to desiccation in the field but showed unexpectedly low survival of cryopreservation, as well as a poor response to pre-treatment. The effects of the cryopreservation protocol on protonemata of D. plumbicola were investigated in order to explore possible relationships between the production in vitro of cryopreservation-tolerant asexual propagules and the reproductive biology of D. plumbicola in nature. METHODS: Protonemata were prepared for cryopreservation using a four-step protocol involving encapsulation in sodium alginate, pre-treatment for 2 weeks with ABA and sucrose, desiccation for 6 h and rapid freezing in liquid nitrogen. After each stage, protonemata were prepared for light and electron microscopy and growth on standard medium was monitored. Further samples were prepared for light and electron microscopy at intervals over a 24-h period following removal from liquid nitrogen and re-hydration. KEY RESULTS: Pre-treatment with ABA and sucrose caused dramatic changes to the protonemata. Growth was arrested and propagules induced with pronounced morphological and cytological changes. Most cells died, but those that survived were characterized by thick, deeply pigmented walls, numerous small vacuoles and lipid droplets in their cytoplasm. Desiccation and cryopreservation elicited no dramatic cytological changes. Cells returned to their pre-dehydration and cryopreservation state within 2 h of re-hydration and/or removal from liquid nitrogen. Regeneration was normal once the ABA/sucrose stimulus was removed. CONCLUSIONS: The ABA/sucrose pre-treatment induced the formation of highly desiccation- and cryopreservation-tolerant propagules from the protonemata of D. plumbicola. This parallels behaviour in the wild, where highly desiccation-tolerant rhizoids function as perennating organs allowing the moss to endure extreme environmental conditions. An involvement of endogenous ABA in the desiccation tolerance of D. plumbicola is suggested.

The placenta in Monoclea forsteri Hook. and Treubia lacunosa (Col.) Prosk: insights into placental evolution in liverworts.

Placental morphology is remarkably diverse between major bryophyte groups, especially with regard to the presence and distribution of transfer cells in the sporophyte and gametophyte. In contrast, with the exception of metzgerialean liverworts, placental morphology is highly conserved within major bryophyte groups. Here we examine the ultrastructure of the placenta in Monoclea forsteri and Treubia lacunosa, basal members of the marchantialean and metzgerialean liverwort lineages, respectively. In both species several layers of transfer cells are found on both sides of the placenta, with sporophytic transfer cells exhibiting prominent wall labyrinths. Consistent with previous reports of a similar placenta in other putatively basal and isolated liverwort genera such as Fossombronia, Haplomitrium, Blasia and Sphaerocarpos, this finding suggests that this type of placenta represents the plesiomorphic (primitive) condition in liverworts. Distinctive ultrastructural features of placental cells in Monoclea include branched plasmodesmata in the sporophyte and prominent arrays of smooth endoplasmic reticulum, seemingly active in secretion in the gametophyte. These arrays contain a core of narrow tubules interconnected by electron-opaque rods, structures with no precedent in plants. Analysis of the distribution of different types of placenta in major bryophyte groups provides valuable insights into their inter-relationships and possible phylogeny.

The use of soft x rays to study the ultrastructure of living biological material.

Imaging biological specimens with soft x rays offers several potential benefits over electron microscopy, and these are briefly reviewed. The disadvantages, most notably radiation-induced structural changes, have been investigated and images of irradiated algal cells (Chlorella) are presented. In soft x-ray contact microscopy the image is recorded rapidly to avoid both natural and radiation-induced movement and this technique has been used to study the ultrastructural effects of electron microscopy fixatives. In the epidermal hairs of tomato plants there are numerous strands of cytoplasm which, by light microscopy, appear to traverse the vacuole but are rarely seen by electron microscopy. However, by soft x-ray contact microscopy these strands and the organelles within them can be successfully imaged. Moreover, examination by soft x-ray contact microscopy of the cytoplasm in a fixed material shows that these strands are not present in chemically fixed material. This paper also reports the use of soft x-ray contact microscopy to examine the abscission cells found within the protonema of a moss (Bryum tenuisetum) and compares the images to those obtained by light and electron microscopy.

Rhodamine-phalloidin staining of F-actin in rhodophyta.

Rhodamine-phalloidin was used to label F-actin in unfixed cells of 13 species of filamentous and blade-forming red algae from the three families Ceramiaceae, Acrochaetiaceae and Bangiaceae. Labelling was achieved only after treatment with either beta-glucuronidase or a combination of cellulase and an extract of snail gut enzyme. Different species required different enzyme treatments and incubation times for successful labelling. All species examined showed extensive arrays of F-actin which generally are confined to the peripheral cytoplasm and are oriented longitudinally. Transverse arrays are present beside the crosswalls of Griffithsia pacifica, and Audouinella species show actin concentrations at the tips of apical cells and in developing branch initials.

A light and electron microscope study of rhizoid-ascomycete associations and flagelliform axes in British hepatics with observations on the effects of the fungi on host morphology.

Ascomycetous fungi, identified by the presence of simple septa and Woronin bodies, form a range of highly specialized associations with the unicellular rhizoids of 46 (16.2%) of the 284 species of British liverworts. The majority of these are in the two jungermannialian suborders Lepidoziineae and Cephaloziineae. Thirty three species (11.6%) also possess flagelliform axes. These extend to depths of 20-30 cm in peaty substrata and in most species bear abundant fungus-infected rhizoids. When grown in sand or water culture, isolated from their natural substrata (sandstone rocks, peat, Sphagnum, rotten wood), the hepatics are free from rhizoid infections. On returning the plants to their native substrata, the rhizoid-fungus associations are re-established. Each rhizoid or fascicle of rhizoids is infected independently and a continuous strand of fungus-containing cells is absent. Internally the flagelliform axes contain elongate parenchyma cells with abundant plasmodesmata in their transverse end walls. The apices of the axes are invested by mucilage and subapical amyloplasts appear to act as graviperceptive particles. The morphology of the flagelliform axes and the ultrastructure of the rhizoid-fungus association varies greatly between taxa. The Lepidoziaceae and Calypogeiaceae produce fascicles of rhizoids from the bases of rudimentary leaves whereas the flagelliform axes in the Cephaloziaceae are leafless. In Cladopodiella the fungus forms a pseudoparenchymatous sheath around the rips of swollen rhizoids. Development of swollen rhizoids precedes fungal infection in Lepidozia, Kurzia and Telaranea but not in other genera. Hyphae penetrating the basal walls of the rhizoids in Cephalosia, Nowellia, Calypogeia and Odantoschisma are ensheathed by host Wall material. These diverse patterns of fungal infection suggest independent, and probably relatively recent, evolution in the various genera. The flagelliform axes in hepatics appear to be major organs of assimilation and perennation structurally and functionally analogous in the former capacity to vascular plant mycorrhizas and particularly the hairs roots in Ericaceae.

Monoclonal antibodies against plant proteins recognise animal intermediate filaments.

Four monoclonal antibodies were raised against polypeptides present in a high-salt detergent-insoluble fraction from cells of Chlamydomonas reinhardtii. Indirect immunofluorescence microscopy of fibroblasts and epithelial cells grown in culture using these plant antibodies revealed staining arrays identical to those obtained with well characterised antibodies to animal intermediate filaments. Immunofluorescence microscopy of Chlamydomonas with these monoclonal antibodies and a monoclonal antibody that recognises all animal intermediate filaments (anti-IFA) gave a diffuse, patchy cytoplasmic staining pattern. Both the plant antibodies and anti-IFA stained interphase onion root tip cells in a diffuse perinuclear pattern. In metaphase through to telophase, the labelling patterns colocalised with those of microtubules. Labelling of the phragmoplast was also detected but not staining of the preprophase band. On Western blots of various animal cell lines and tissues, all the antibodies labelled known intermediate filament proteins. On Western blots of whole Chlamydomonas proteins, all the antibodies labelled a broad band in the 57,000 Mr range, and three antibodies labelled bands around 66,000 and 140,000 Mr but with variable intensities. On Western blots of whole onion root tip proteins, all the antibodies labelled 50,000 Mr (two to three bands) polypeptides and a diffuse band around 60,000 Mr and three of the antibodies also labelled several polypeptides in the 90,000-200,000 Mr range. The consistent labelling of these different bands by several different monoclonal antibodies recognising animal intermediate filaments makes these polypeptides putative plant intermediate filament proteins.

Anti-tubulin antibodies locate the blepharoplast during spermatogenesis in the fern Platyzoma microphyllum R.Br.: a correlated immunofluorescence and electron-microscopic study.

The discovery that the monoclonal anti-tubulin antibody YOL 1/34 recognizes a microtubule organizing centre, the blepharoplast (which arises de novo during the latter stages of spermatogenesis in the fern, Platyzoma microphyllum), has enabled us to follow it and associated microtubules throughout most of its ontogeny. By correlating electron-microscopic and immunofluorescence observations, YOL 1/34 is seen to stain the blepharoplast uniformly at a time when no microtubules are present within the organelle. Later, staining becomes intense at the surface, concomitant with the re-location of cylindrical channels to the periphery of the blepharoplast. During anaphase of the ultimate division of the spermatid mother cell the blepharoplast moves to the spindle poles and sharpens the otherwise barrel-shaped mitotic apparatus. Prior to this stage the blepharoplast is, however, off-centre and at variable positions around the poles. Later still, in the differentiating spermatids, the blepharoplast is the focus for radiating cytoplasmic microtubules that abut directly onto the electron-dense organelle, penetrating the ribosome-free halo. The three main conclusions are: that tubulin in a pre-microtubular form is associated with the cylindrical channels that arise de novo within the previously amorphous blepharoplast and act as a template in basal body formation; that the late appearance of the blepharoplast as a focus for the spindle poles during the final mitosis provides strong argument against its functioning during spindle pole initiation (despite its ability to sharpen the poles at anaphase); that the blepharoplast does seem to act as a microtubule organizing centre in the mitotically quiescent spermatid.

An X-ray Microanalytical Study of the Distribution of Cadmium in Roots of Zea mays L.

X-ray microanalysis of roots of Zea mays L. grown in the presence of cadmium, revealed that the absorbed metal is removed by normal electron microscope preparative procedures. Following pretreatment with either phosphate or sulphide to immobilize cadmium, the metal was detected in the walls of the sieve elements and in the middle lamella separating the endodermis from the pericycle. These findings suggest that cadmium is principally bound to ion exchange sites on pectic residues in the cell wall.

Immunofluorescence microscopy of the flagella and multilayered structure in two mosses, Sphagnum palustre L. and Polytrichum juniperinum Hedw.

Antibody against tubulin from porcine brain was used to examine the distribution of tubulin in developing spermatids of Polytrichum and mature spermatozoids of Sphagnum. Cells were prepared for indirect immunofluorescence microscopy after fixation in buffered paraformaldehyde and brief incubation in cellulase. Pretreatment with cold methanol resulted in considerably enhanced immunofluorescence but exposure to Triton X-100, with or without sonication, had no effect. The antibody showed similar immunological cross-reactivity with the flagella (both basal bodies and axonemes) and the spline microtubules of the multilayered structure. This is the first direct evidence that this rigid array of stable cytoskeletal microtubules consists of tubulin. Particularly intense fluorescence from the lamellar strata of the MLS in developing spermatids provides strong support for the notion that the lamellae comprise a highly structured microtubule organizing centre (MTOC), responsible for the ordered assembly of the overlying spline tubules. The demonstration of immunological cross-reactivity with antitubulin from porcine brain tubulin, within a plant structure other than fully formed microtubules, suggests that immunocytochemistry may have considerable potential for the detection of other MTOCs. By contrast, no detectable fluorescence emanated from the granular matrix cementing the flagellar basal bodies to the spline or the spindle-shaped sheath of fibres present in the spermatozoids of Sphagnum. Disruption of the mature gametes by sonication and treatment with Triton X-100 reveals the presence of particularly strong links between the spline and subjacent nuclear envelope.

Ultrastructure of sensory receptors on the labium of the rice brown planthopper.

The ultrastructure of the sensory receptors located on the labium of the rice brown planthopper is described; possible functions of individual receptors are suggested on morphological grounds. Uniporous chemosensilla which may or may not possess a mechanoreceptive dendrite, domed multiporous chemosensilla, and mechanoreceptive pegs are present on the flattened labial tip. Mechanoreceptive pegs are distributed over the labium. The fine structure of a multilobed sensillum located on either side of the labium is also described.

Sensory receptors associated with the stylets and cibarium of the rice brown planthopper, Nilapavarta lugens.

The stylets of Nilapavarta lugens consist of two maxillae that interlock to form separate food and salivary ducts partially surrounded by two mandibles. The ultrastructure of the sensory innervation of the stylets is described. Each maxilla possesses five neurones which extend to the tip of the stylet. The mandibles also contain five neurones, four of which are paired. The paired neurones comprise a shorter dendrite extending part of the way along the stylet and a longer one extending to the tip. The possible functions of these neurones are discussed. Gustatory receptors are located in the small passageway leading from the food duct to the cibarium. The receptors are in two distinct groups on the epipharyngeal side and one group on the hypopharyngeal side of the food canal. Two to five neurones innervate each receptor which connects to the food canal via a small pore.

An acetylcholinesterase-deficient mutant of the nematode Caenorhabditis elegans.

Within a set of five separable molecular forms of acetylcholinesterase found in the nematode Caenorhabditis elegans, previously reported differences in kinetic properties identify two classes, A and B, likely to be under separate genetic control. Using differences between these classes in sensitivity to inactivation by sodium deoxycholate, a screening procedure was devised to search for mutants affected only in class A forms. Among 171 previously isolated behavioral and morphological mutant strains examined by this procedure, one (PR946) proved to be of the expected type, exhibiting a selective deficiency of class A acetylcholinesterase forms. Although originally isolated because of its uncoordinated behavior, this strain was subsequently shown to harbor mutations in two genes; one in the previously identified gene unc-3, accounting for its behavior, and one in a newly identified gene, ace-1, accounting for its selective acetylcholinesterase deficiency. Derivatives homozygous only for the ace-1 mutation also lacked class A acetylcholinesterase forms, but were behaviorally and developmentally indistinguishable from wild type. The gene ace-1 has been mapped near the right end of the X chromosome. Gene dosage experiments suggest that it may be a structural gene for a component of class A acetylcholinesterase forms.