Outer Cap Research Articles

A PROPOSAL FOR A NEW RED ALGAL ORDER, THE THOREALES

The freshwater red algal genera Nemalionopsis and Thorea are currently classified in the family Thoreaceae of the Batrachospermales. However, these two genera differ from other members of the order in having multiaxial thalli. The phylogenetic relationships of the genera were investigated using a combination of DNA sequence analysis (rbcL and 18S rRNA genes) and transmission electron microscopy. In both gene trees, Nemalionopsis and Thorea form well supported clades (87–100% bootstrap values) separate from other members of the Batrachospermales as well as taxa from the Acrochaetiales, Palmariales, and Nemaliales. In addition, analysis of the secondary structure of the 18S rRNA gene in Nemalionopsis and Thorea reveals an additional helix, which is not present in any of the other taxa within the Rhodophyta. Pit plugs of the Thoreaceae also differ from those of other members of the Batrachospermales in not having an outer cap layer that is not universally domed. Based on these findings, it is proposed that Nemalionopsis and Thorea represent a distinct taxonomic group that should be recognized as a new order, the Thoreales.

Directional cell-to-cell communication in theArabidopsis root apical meristem III. Plasmodesmata turnover and apoptosis in meristem and root cap cells during four weeks after germination

Plasmodesmata frequency and distribution in root cap cells ofArabidopsis thaliana root tips were characterized during four weeks after germination to understand the symplasmic control of apoptosis. Apoptotic cells in some of the root apical-meristem cells and in root cap cells were identified by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling reaction and characterized by electron microscopy. Starting at the second week after germination, cells in the outermost layers of the root cap showed typical apoptotic features, including nuclear DNA fragmentation, chromatin condensation, cytoplasmic vacuolation, and organelle destruction. Intercellular connections, indicated by the frequency and number of plasmodesmata per cell length, were significantly reduced in the walls of outer root cap cells. This shows that cells become symplasmically isolated during the apoptosis process. In apoptotic root cap cells, the majority of nonfunctional plasmodesmata were observed to be associated with degenerated endoplasmic reticulum; this state was prior to the detection of any nuclear DNA fragmentation. Other nonfunctional plasmodesmata were sealed by heterogeneous cell wall materials. However, in immature epidermal and cortical cells in 4-week-old arrested roots the endoplasmic reticulum associated with plasmodesmata became disconnected as a result of protoplast condensation and shrinkage. No degenerated endoplasmic reticulum was observed in these cells. These observations suggest that the apoptotic processes in the root body and the root cap are different.

Cytological and ultrastructural response of Nicotiana tabacum L. roots to manganese stress

ABSTRACT The effects of manganese on the morphology and ultrastructure of root cells were studied in in vitro selected plantlets tolerant to high concentrations (5 mM) of Mn which, in a previous investigation, had shown a significant reduction in root growth when compared to the control (0.1 mM Mn). A decrease in the length of the root cap and meristematic region was observed. The most serious effects of Mn toxicity occurred in the outer root cap and outer meristematic cells where extensive cytoplasmic disorganisation was observed. The plasma membrane, in particular, displayed ruptures associated with the formation of vesicular and multi-lamellar bodies. Mitochondria also revealed strong structural alterations. However, the cells in the inner regions of the meristem showed better preserved subcellular components and exhibited normal mitotic activity. These last conditions suggest a possible resistance strategy which permits root development to continue. The primary root structure was not affected by the metal, except the central cylinder where 5 mM Mn induced precocious xylem differentiation (protoxylem and metaxylem), while in the corresponding zone control roots showed protoxylem elements only. Moreover, the epidermis-esoderm cell morphology seemed to be influenced by the metal.

Phylogenesis of pit-plug-associated features in the Rhodophyta: inferences from molecular systematic data

Small-subunit ribosomal RNA sequence data are presented for Rhodogorgon carriebowensis Norris et Bucher (Rhodogorgonales, Rhodophyta) and Galaxaura marginata (Ellis et Solander) Lamouroux (Nemaliales, Rhodophyta). Data for these species were included in a matrix consisting of small-subunit ribosomal RNA sequences for 70 taxonomically diverse red algal species. Distance, parsimony, and maximum-likelihood analyses of these data all strongly support (100% of bootstrap replicates for distance and parsimony) a close alliance between the Rhodogorgonales and Corallinales. Our alignment includes 19 species representing six florideophyte orders (Acrochaetiales, Batrachospermales, Corallinales, Nemaliales, Palmariales, Rhodogorgonales) in which pit plugs are characterized by two cap layers. Distance and parsimony analyses resolve a monophyletic lineage including all "two-cap-layer" species. Our parsimony-derived molecular phylogeny was used to test a published hypothesis of pit-plug evolution. Four aspects of Pueschel's hypothesis are consistent with the results of our molecular systematic study: (i) "naked" plugs represent the ancestral type, (ii) outer cap layers are homologous structures, (iii) domed outer caps are ancestral to plate-like outer caps, and (iv) cap membranes are a derived feature within the two-cap-layer lineage. Directions for future research are discussed. Key words: Acrochaetiales, Batrachospermales, Corallinales, molecular systematics, Nemaliales, Palmariales, phylogeny, pit plug, Rhodogorgonales, Rhodophyta.

Use of radioactive tracers for detecting cracks in steel structural welds in seawater : Jones, J.E.; Natalie, CA.; Burns, M. Welding Journal, Vol. 67, No. 9, pp. 47–53 (Sep. 1988)

This report summarises the feasibility of a system where, during fabrication, a radioactive tracer is included in the interior passes of a multiple pass weldment, but not in the outer cap and root passes. The feasibility has been examined using only chemical tracers. When a stress corrosion or corrosion fatigue crack penetrates through these outer passes and into the tracer-laden interior passes, the tracer will be released into the adjoining seawater environment setting off a detector. With the proposed technology, a radiation detector would be capable of detecting the presence of corrosion in exposed cracks that penetrate beyond a predetermined critical depth and, thus, warrant repair.

Distribution of acetylcholinesterase activity in the deutocerebrum of the sphinx moth Manduca sexta

We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the deutocerebrum of the brain of the sphinx moth Manduca sexta. To distinguish between extra- and intracellular pools of the enzyme, some brains were treated prior to histochemical staining with echothiophate, an irreversible AChE inhibitor which penetrates cell membranes very slowly and, therefore, inhibits only extracellular AChE. In the antennal nerve, fascicles of presumably mechanosensory fibers show echothiophate-insensitive AChE activity. They bypass the antennal lobe and project to the antennal mechanosensory and motor center of the deutocerebrum. In the antennal lobe, fibers in the coarse neuropil, cell bodies in the lateral cell group, and all glomeruli exhibit AChE activity. In most ordinary glomeruli, echothiophate-sensitive AChE activity is concentrated in the outer cap regions, corresponding to the terminal arborizations of olfactory afferents. A previously unrecognized glomerulus in the ventro-median antennal lobe shows uniform and more intense AChE-specific staining that the other glomeruli. No AChE activity appeared to be associated with male-specific pheromone-sensitive afferents in the macroglomerular complex. About 67 interneurons with somata in the lateral cell group of the antennal lobe show echothiophate-insensitive AChE activity. These neurons seem to be members of two types of antennal-lobe projection neurons with fibers passing through the outer-antennocerebral tract to the protocerebrum. AChE-stained arborizations of these neurons appear to invade all glomeruli, including three distinguishable subunits of the male-specific macroglomerular complex. In echothiophate-treated animals, the projections of one of these types of fiber form large terminals in the lateral horn of protocerebrum, which partly protrude into the adjacent glial cell layer. The results suggest that extracellularly accessible AChE is associated with ordinary olfactory receptor terminals but apparently not with pheromone-sensitive afferents. Intracellular AChE appears to be present in antennal mechanosensory fibers and in two types of olfactory projection neurons of the antennal lobe. The study provides further evidence for cholinergic neurotransmission of most antennal afferents. The AChE-containing interneurons might be cholinergic as well or use the enzyme for functions unrelated to hydrolysis of acetylcholine.

Immunolocalization of an annexin-like protein in corn

Although calcium has been proposed to be an important regulatory element in plant gravitropic growth, as yet no specific function of Ca 2+ in growth regulation has been discovered. Our recent studies on a Ca 2+-binding protein in pea seedlings called p35 indicate that it is a member of the annexin family of proteins and may play a key role in growth regulation through its function in delivering polysaccharides needed for wall construction. We previously reported the isolation of p35 from pea plumules and the production of polyclonal antibodies to it. Immunolocalization analyses of p35 in pea tissues revealed high levels of staining in secretory cell types such as developing vascular cells and outer root cap cells. To test how general was the occurrence and distribution of this annexin-like protein in plant cells we initiated an analysis of annexins in the monocot corn using immunological techniques. Our results indicate the immunochemical properties and localization of corn annexins are very similar to those reported for pea. They are consistent with the postulate that annexins may play a general role in the regulation of the secretion of wall polysaccharides needed for growth, and thus could be an important target of calcium action during gravitropic growth.

Gamma radiation from pulsar magnetospheric gaps

We investigate the production of gamma rays in two pulsar emission models: the 'polar cap' model and the 'outer cap' model. For the former, we have performed detailed simulations of energetic electrons flowing in the vacuum dipole open field line region. In the outer gap case, we generate light curves for various magnetosphere geometries. Using data from radio and optical observations, we construct models for specific viewing angles appropriate to the Crab and Vela pulsars. Phase-resolved spectra are also computed in the polar cap case and provide signatures for testing the models. The calculations have been extended to include millisecond pulsars, and we have been able to predict fluxes and spectra for populations of recycled pulsars, which are compared to COS B data for globular cluster populations.

Electron microscopical studies ofThorea ramosissima (Thoreaceae, Rhodophyta): Taxonomic implications ofThorea pit plug ultrastructure

The thallus ofThorea ramosissima was studied electron microscopically. The cells of the medulla, the cortex and the assimilatory hairs differ not only in size and number of plastids and their equipment with thylakoids but also in cell wall structure, the number of mitochondria and the activity of the Golgi apparatus, with dictyosomes transforming complete cisternae into Golgi vesicles with mucilaginous contents in the outer region of the cortex. The pit connections have plugs with a distinct plate—like (not dome-like) outer cap layer. BecauseT. riekei was reported to have dome-like outer cap layers and because this character was the main reason to place theThoreaceae into theBatrachospermales (Pueschel & Cole 1982),T. riekei was reinvestigated, too. A distinct outer cap could not be detected. The reliability of pit plug structure as a taxonomic character and the taxonomic position ofThorea is discussed.

Unusual morphological and cytochemical features of pit plugs inClathromorphum circumscriptum(Rhodophyta, Corallinales)

Ultrastructural and cytochemical study of pit plugs in Clathromorphum circumscriptum revealed two distinctive features. The large outer cap layer of the pit plugs was irregular in morphology and often deeply lobed. Some plug caps were poorly consolidated, consisting of disjunct pieces that were not in contact with the rest of the pit plug. As in other Corallinales, the outer cap layer was reactive to periodic acid Schiff, a carbohydrate stain, and mercuric bromophenol blue, a protein stain; it was also sensitive to digestion by two proteolytic enzymes. In addition, a lipid stain, Sudan black B, stained the outer caps. This was the first demonstration, in any species, of pit plug outer caps with a significant lipid component. Morphological and cytochemical features of pit plugs could be of value in elucidating relationships among some species of Clathromorphum.

Cytochemical evidence for homology of the outer cap layer of red algal pit plugs

Abstract A comparative cytochemical study of the outer cap layer of pit plugs in five orders of red algae (Rhodophyta) was performed in order to address the question of homology of this systematically important feature. Two or more representatives of the Acrochaetiales, Nemaliales, Palmariales, Batrachospermales, and Corallinales were examined. Outer caps large enough to be resolved by light microscopy were found to be reactive to the periodic acid Schiff procedure, specific for carbohydrates, and to a protein stain, mercuric bromophenol blue, but not to Sudan black B, a lipid stain. Using transmission electron microscopy, it was found that the proteolytic enzyme, proteinase K, removed much of the material constituting the outer cap. These results, together with similarities of phosphotungstic-chromic acid staining, suggest that the outer cap layer of pit plugs in all five orders is composed of glycoproteins. These findings provide evidence, but not definitive proof, that the outer caps of red algal pit p...

Cytochemistry of pit plugs in Bossiella californica (Corallinales, Rhodophyta)

The chemical composition of pit plugs in the articulated coralline red alga Bassiella californica (Decaisne) Silva (Rhodophyta) was investigated using light and electron microscopic staining methods, enzyme digestion, and FITC-labelled lectin probes. Light microscopic cytochemical techniques indicated that the outer cap layer was composed of glycoproteins. It gave a reaction with the periodic acid-Schiff procedure, specific for carbohydrates, and with mercuric bromophenol blue, a protein stain. The pit plug core was reactive only with the protein stain. Twenty-two enzymes were tested for their effects on the ultrastructure of pit plugs, but only three proteolytic enzymes, namely proteinase K, pancreatin and pronase E, caused any discernible change. All three enzymes digested the outer plug cap layer, first removing reactivity to phosphotungstic acid-chromic acid, a selective ultrastructural stain, and then removing the cap layer from the section. The plug core and inner cap layer were not affected. The ten lectins tested showed no binding to any region of the pit plugs.

AN EXPANDED SURVEY OF THE ULTRASTRUCTURE OF RED ALGAL PIT PLUGS1

ABSTRACTThe fine structure of pit plugs in 90 species of red algae was examined, bringing the total number of species in the continuing survey to 153. The organization of plug caps was confirmed to be a stable, predictable trait within thalli, between generations in heteromorphic life histories, and within the presently recognized orders, with one exception—the Acrochaetiales. Two forms of the outer cap were found in this group, a thin plate, as in the Nemaliales and Palmariales, and a dome, as in Batrachospermales and Corallinales. Variation of pit plug structure indicates that the Acrochaetiales are a heterogeneous assemblage and that pit plugs will be useful in reappraising their systematics. The systematic affinities of several species of uncertain affinities are clarified. Schmitziella endophloea Bornet et Batters is excluded from both orders, Corallinales and Acrochaetiales, with which it previously was allied. Although other ordinal attributions are not precluded by pit plug structure alone, pit plug structure is consistent with placement of Apophlaea sinclairii Harvey and Hildenbrandia rivularis (Liebman) J. Agardh in the Hildenbrandiales, Plagiospora gracilis Kuckuck, Schmitziella endophloea, and Wurdemannia miniata (Duby) J. Feldmann et Hamel in the Gigartinales, and Pseudorhododiscus nipponicus Masuda in the Palmariales.

Some unusual staining properties of tannic acid in plants

Maize root tips were fixed in glutaraldehyde fixatives containing tannic acid and then processed for electron microscopy. Under these conditions, tannic acid selectively stained the contents of the Golgi apparatus secretory vesicles of some outer root cap cells, the cell walls of all cells, and substances in, and adjacent to, intercellular connections of mature primary walls and of secondary walls. Intercellular connections of the young primary walls were not stained. Plasma membranes, and substances associated with the outer leaflets of the plasma membranes, were also stained. Tannic acid-positive material was associated with the cell plate vesicles of forming walls but very little, or none, was associated with the Golgi apparatus vesicles of dividing cells.

The development of the fungal sheath on Cistus incanus short roots

Sheath development of mycorrhizae of Cistus incanus L. ssp. incanus + Tuber melanosporum Vitt. synthesized under gnotobiotic conditions was studied by anatomical and ultrastructural techniques. Root systems examined at different times after inoculation exhibited short lateral roots which could be categorized as follows: (i) uninfected; (ii) with hyphae in the dead outer root cap cells; (iii) with developed sheaths. In the last case the inner hyphae of the sheath penetrated through remains of dead root cap cells which were trapped in the inner sheath. Remains of root cells were also found in the outer sheath. Growth of hyphae into the dead layers of the root cap leads to formation and development of the sheath.

Morphometric analysis of ultrastructural changes induced by Periconia circinata toxin in the outer root cap of sorghum

Outer root cap cells of sorghum seedlings treated with the host-specific toxin produced by Periconia circinata were analyzed morphometrically to detect changes in the quantities of cytomembranes and numbers of organelles and thus extend our observations of qualitative cytological responses to the toxin. In seedlings susceptible to the pathogen, brief (0.25 h) treatment with the toxin resulted in a marked and permanent decrease in the amounts of secretory vesicle membrane. By 2 h treatment, only secretory vesicle membrane was decreased, but longer treatments led to an increased amount of endoplasmic reticulum (4 h), which later decreased together with the amount of dictyosome membrane, while the amount of tonoplast increased (8 h). In resistant seedlings treated with the toxin, early but transient increases were detected in the quantities of plasma membrane, secretory vesicle membrane, dictyosome membrane, and endoplasmic reticulum and in the number of dictyosomes. Insensitivity to the toxin may involve the ability of resistant genotypes to recover from the toxic effects.

Ultrastructural and developmental alterations induced by Periconia circinata toxin in the root tip of sorghum

Cytological and developmental effects induced by Periconia circinata toxin were examined to better understand the mechanism of action for this toxin. Roots of sorghum seedlings susceptible and resistant to P. circinata were incubated in 500 ng toxin/mL (treated) or water (controls). Root cap cells of resistant seedlings treated with the toxin were cytologically similar to those of controls, although the toxin caused a transient inhibition of mitosis in cells of the primary root tip. In outer root cap cells of susceptible seedlings treated for 0.25 h, hypersecretory activity was lost, secretory vesicles were fewer, and secretory product accumulated between the plasma membrane and cell wall. Also, inner root cap cells showed increased vacuolation. Longer treatments caused increased vacuolation, loss of starch, increased numbers of lipid bodies, pleomorphic amyloplasts, regularly stacked endoplasmic reticulum, apparent changes in the amounts of cytomembranes, dispersion of heterochromatin, and autolysis. Mitochondrial morphology was normal, but lesions in the tonoplast occurred before autolysis. The toxin also inhibited expansion and sloughing off of root cap cells and mitotic activity in the root tip. Stacked endoplasmic reticulum, nonhypersecretory dictyosomes, fewer secretory vesicles, increased vacuolation, reorganization of heterochromatin, and increased secretory product outside the protoplast were induced by P. circinata toxin and by cyanide. These data suggested that a cyanogenic compound is biologically active in cells treated with P. circinata toxin. Our results suggest that the toxin transiently affects resistant seedlings and in susceptible seedlings alters vacuolar expansion, secretory activity, and endomembrane flow, although other processes may also be affected.

The Ultrastructure of the Salt Glands of Cynodon and Distichlis (Poaceae)

The epidermal salt glands of the grasses Cynodon and Distichlis consist of a small outer cap cell and a large, flask-shaped basal cell. The wall of the basal cell is contiguous with those of the adjacent epidermal cells and underlying mesophyll cells. The basal cell is connected symplastically with all adjoining cells via plasmodesmata. The outer, protruding portion of the glands is covered by a cuticle continuous with that of the adjoining epidermal cells. However, the lateral cell walls of the glands are not incrusted by this cuticle. The cap cell wall has a loose, mottled appearance quite different from the compact striated appearance of the basal cell wall. The cap cell is characterized by dense cytoplasm containing many organelles and a varying number of small vacuoles. The basal cell cytoplasm is distinguished by the presence of an intricate system of paired membranes that are closely associated with mitochondria and microtubules. These membranes are infoldings of the plasmalemma that originate adjacent to the wall separating the cap and basal cells. The space enclosed by the paired membranes, therefore, is an extracellular channel that is open only in the direction of secretory flow. The consistent orientation of this system of paired membranes suggests that it represents a structural specialization which is directly and functionally involved in the secretory process. The close association of mitochondria and microtubules with the paired membranes implies that these structures are also functionally related to the secretory process. Finally, the results of this study indicate that these glands are ultrastructurally similar to those of Spartina and that the glands of these three grasses are structurally distinct from those of dicotyledonous plants.

THE ULTRASTRUCTURE OF THE SALT GLANDS OF CYNODON AND DISTICHLIS (POACEAE)

The epidermal salt glands of the grasses Cynodon and Distichlis consist of a small outer cap cell and a large, flask‐shaped basal cell. The wall of the basal cell is contiguous with those of the adjacent epidermal cells and underlying mesophyll cells. The basal cell is connected symplastically with all adjoining cells via plasmodesmata. The outer, protruding portion of the glands is covered by a cuticle continuous with that of the adjoining epidermal cells. However, the lateral cell walls of the glands are not incrusted by this cuticle. The cap cell wall has a loose, mottled appearance quite different from the compact striated appearance of the basal cell wall. The cap cell is characterized by dense cytoplasm containing many organelles and a varying number of small vacuoles. The basal cell cytoplasm is distinguished by the presence of an intricate system of paired membranes that are closely associated with mitochondria and microtubules. These membranes are infoldings of the plasmalemma that originate adjacent to the wall separating the cap and basal cells. The space enclosed by the paired membranes, therefore, is an extracellular channel that is open only in the direction of secretory flow. The consistent orientation of this system of paired membranes suggests that it represents a structural specialization which is directly and functionally involved in the secretory process. The close association of mitochondria and microtubules with the paired membranes implies that these structures are also functionally related to the secretory process. Finally, the results of this study indicate that these glands are ultrastructurally similar to those of Spartina and that the glands of these three grasses are structurally distinct from those of dicotyledonous plants.

RHODOPHYCEAN PIT PLUGS: AN ULTRASTRUCTURAL SURVEY WITH TAXONOMIC IMPLICATIONS

The fine structure of pit plugs was examined in 63 species of red algae representing 34 families. The number of plug cap layers was found to be a taxonomically reliable character when tested against a recent revision of the Rhodymeniales which established the order Palmariales. In a broader survey involving all the orders of red algae possessing pit plugs, three basic plug cap configurations were detected. These differ in having one, two, or no plug cap layers. Some taxa have plug caps in which the outer cap layer is greatly elaborated, however, this layer was shown to be positionally and cytochemically identical, thus probably homologous, to the outer cap layer of the typical two‐layered plug cap. The pit plug character confirms the validity of the order Palmariales and indicates that the Nemaliales and Cryptonemiales are heterogeneous assemblages. Revision of these groups along lines indicated by the plug character is supported by significant features of morphology and reproduction. These correlations lead us to propose that Rhodophysema and Coriophyllum be transferred to the Palmariales, that the Gelidiales be resurrected, that the Bonnemaisoniales (including the Naccariaceae) is deserving of recognition, that the Batrachospermaceae be raised to a higher rank which will also receive the Lemaneaceae and the Thoreaceae, and that the Corallinaceae and Hildenbrandiaceae both be raised to ordinal status.