Subapical Cells Research Articles

The Migration of Multipartial Nuclear and Its Regulation by DCTN1 Gene in the Mycelium Cells of Volvariella volvacea

Eight Volvariella volvacea strains (F1, F2, F3, F4, F5, F6, F7, and F8) were grown in a favorable environment, and their vegetative hyphae were stained using eosin-crystal violet. Microscopic observations showed few or nonexistent nuclei in the cells at the tip of the hyphae, whereas more nuclei were observed in the sub-apical cells. Migrating nuclei were found in most strains, except F2 and F5. These nuclei had no definite shapes as they passed through intercellular channels. Migrating nuclei had the tendency to move towards the hyphal tips. Centrosomes and cytoskeleton were occasionally observed during this process. Likewise, DCTN1 gene transcription in the hyphae was analyzed by semi-quantitative RT-PCR. The transcriptional levels of strains F2 and F5 were significantly lower than those of the other strains, which implied that the nuclear migration was regulated by DCTN1 gene. This paper provides fundamental information on the mechanisms of nuclear migration and strain degeneration in V. volvacea.

Spatial heterogeneity of glycogen and its metabolizing enzymes in Aspergillus nidulans hyphal tip cells

Glycogen is a homopolymer of glucose and a ubiquitous cellular-storage carbon. This study investigated which Aspergillus nidulans genes are involved in glycogen metabolism. Gene disruptants of predicted glycogen synthase (gsyA) and glycogenin (glgA) genes accumulated less cellular glycogen than the wild type strain, indicating that GsyA and GlgA synthesize glycogen similarly to other eukaryotes. Meanwhile, gene disruption of gphA encoding glycogen phosphorylase increased the amount of glycogen to a higher degree than wild type during the stationary phase that accompanies carbon-source limitation. GFP-tagged GsyA and GphA were distributed in the cytosol and formed punctate and filamentous structures, respectively. Carbon starvation resulted in elongated GphA-GFP filaments and increased numbers of filaments. These structures were more frequently located in the basal regions of tip cells and adjacent cells than in the apical regions of tip cells. Cellular glycogen visualized by incorporation of a fluorescent glucose analog accumulated in cytoplasmic puncta that were more prevalent in the basal regions, a pattern similar to that seen for GsyA. The colocalization of glycogen and GsyA at punctate structures in tip and sub-apical cells likely represents the cellular machinery for synthesizing glycogen. More frequent colocalization in the basal, rather than tip cell apical regions indicated that tip cells have differentiated subcellular regions for glycogen synthesis. Our findings regarding glycogen, GsyA and GphA distribution evoke the spatial heterogeneity of glycogen metabolism in fungal hyphae.

Influence of Plant Growth Retardants on Quality of Codonopsis Radix.

Plant growth retardant (PGR) refers to organics that can inhibit the cell division of plant stem tip sub-apical meristem cells or primordial meristem cell. They are widely used in the cultivation of rhizomatous functional plants; such as Codonopsis Radix, that is a famous Chinese traditional herb. However, it is still unclear whether PGR affects the medicinal quality of C. Radix. In the present study, amino acid analyses, targeted and non-targeted analyses by ultra-performance liquid chromatography combined with time-of-flight mass spectrometry (UPLC-TOF-MS) and gas chromatography-MS were used to analyze and compare the composition of untreated C. Radix and C. Radix treated with PGR. The contents of two key bioactive compounds, lobetyolin and atractylenolide III, were not affected by PGR treatment. The amounts of polysaccharides and some internal volatiles were significantly decreased by PGR treatment; while the free amino acids content was generally increased. Fifteen metabolites whose abundance were affected by PGR treatment were identified by UPLC-TOF-MS. Five of the up-regulated compounds have been reported to show immune activity, which might contribute to the healing efficacy (“buqi”) of C. Radix. The results of this study showed that treatment of C. Radix with PGR during cultivation has economic benefits and affected some main bioactive compounds in C. Radix.

Pterocladiella feldmanniisp. nov. andP. hameliisp. nov. (Gelidiales, Rhodophyta), Two New Species Uncovered in Madagascar During the Atimo Vatae Expedition

We analyzed both mitochondrial cox1 and plastid rbcL sequences from specimens of Pterocladiella from Madagascar and compared their morphologies with previously described species in the genus. Both molecular and morphological datasets demonstrated the presence of five species in Madagascar: P. australafricanensis, P. bartlettii, P. caerulescens, and two new species, P. feldmannii and P. hamelii, described here. Pterocladiella feldmannii is distinguished by plants with a size of about 6 cm with narrow axes and opposite to irregular branches, rhizines congested in medulla, tetrasporangial branchlets constricted at base of branches, tetrasporangial sori without sterile margins, and irregular arrangement of tetrasporangia. Pterocladiella hamelii is characterized by plants with a size of about 3 cm with flattened and thin erect axes and irregular branches, radial arrangement of subapical cells at tips of main axes, tetrasporangial sori with sterile margins and horizontal to irregular arrangement of tetrasporangia. Phylogenetic analyses based on cox1 and rbcL sequences revealed the sister relationship between P. feldmannii and P. hamelii, and their distant relationships to P. australafricanensis, P. bartlettii, and P. caerulescens. The Madagascan Pterocladiella is composed of two geographical elements: Madagascan endemic (P. feldmannii and P. hamelii) and species with wide distributions (P. australafricanensis, P. bartlettii and P. caerulescens).

The evolution of basal progenitors in the developing non-mammalian brain.

The amplification of distinct neural stem/progenitor cell subtypes during embryogenesis is essential for the intricate brain structures present in various vertebrate species. For example, in both mammals and birds, proliferative neuronal progenitors transiently appear on the basal side of the ventricular zone of the telencephalon (basal progenitors), where they contribute to the enlargement of the neocortex and its homologous structures. In placental mammals, this proliferative cell population can be subdivided into several groups that include Tbr2+ intermediate progenitors and basal radial glial cells (bRGs). Here, we report that basal progenitors in the developing avian pallium show unique morphological and molecular characteristics that resemble the characteristics of bRGs, a progenitor population that is abundant in gyrencephalic mammalian neocortex. Manipulation of LGN (Leu-Gly-Asn repeat-enriched protein) and Cdk4/cyclin D1, both essential regulators of neural progenitor dynamics, revealed that basal progenitors and Tbr2+ cells are distinct cell lineages in the developing avian telencephalon. Furthermore, we identified a small population of subapical mitotic cells in the developing brains of a wide variety of amniotes and amphibians. Our results suggest that unique progenitor subtypes are amplified in mammalian and avian lineages by modifying common mechanisms of neural stem/progenitor regulation during amniote brain evolution.

Differential transcriptome analysis of glandular and filamentous trichomes in Artemisia annua.

BackgroundThe medicinal plant Artemisia annua is covered with filamentous trichomes and glandular, artemisinin producing trichomes. A high artemisinin supply is needed at a reduced cost for treating malaria. Artemisinin production in bioreactors can be facilitated if a better insight is obtained in the biosynthesis of artemisinin and other metabolites. Therefore, metabolic activities of glandular and filamentous trichomes were investigated at the transcriptome level.ResultsBy laser pressure catapulting, glandular and filamentous trichomes as well as apical and sub-apical cells from glandular trichomes were collected and their transcriptome was sequenced using Illumina RNA-Seq. A de novo transcriptome was assembled (Trinity) and studied with a differential expression analysis (edgeR).A comparison of the transcriptome from glandular and filamentous trichomes shows that MEP, MVA, most terpene and lipid biosynthesis pathways are significantly upregulated in glandular trichomes. Conversely, some transcripts coding for specific sesquiterpenoid and triterpenoid enzymes such as 8-epi-cedrol synthase and an uncharacterized oxidosqualene cyclase were significantly upregulated in filamentous trichomes. All known artemisinin biosynthesis genes are upregulated in glandular trichomes and were detected in both the apical and sub-apical cells of the glandular trichomes. No significant differential expression could be observed between the apical and sub-apical cells.ConclusionsOur results underscore the vast metabolic capacities of A. annua glandular trichomes but nonetheless point to the existence of specific terpene metabolic pathways in the filamentous trichomes. Candidate genes that might be involved in artemisinin biosynthesis are proposed based on their putative function and their differential expression level.

Trichome isolation with and without fixation using laser microdissection and pressure catapulting followed by RNA amplification: Expression of genes of terpene metabolism in apical and sub-apical trichome cells of Artemisia annua L.

The aim of this project was to evaluate the effect of fixation on plant material prior to Laser Microdissection and Pressure Catapulting (LMPC) and to identify an appropriate method for preserving good RNA quality after cell isolation. Therefore, flower buds from Artemisia annua L. were exposed to either the fixative formaldehyde or a non-fixative buffer prior to cell isolation by LMPC. Proteinase K was used after cell isolation from fixed plant tissue, in an attempt to improve the RNA yield. The ability to detect gene expression using real-time quantitative PCR with or without previous amplification of RNA from cells isolated by LMPC was also evaluated. Conclusively, we describe a new technique, without fixation, enabling complete isolation of intact glandular secretory trichomes and specific single trichome cells of A. annua. This method is based on LMPC and preserves good RNA quality for subsequent RNA expression studies of both whole trichomes, apical and sub-apical cells from trichomes of A. annua. Using this method, expression of genes of terpene metabolism was studied by real-time quantitative PCR. Expression of genes involved in artemisinin biosynthesis was observed in both apical and sub-apical cells.

Influence of Root Zone Calcium on Shoot Tip Necrosis and Apical Dominance of Potato Shoot: Simulation of This Disorder by Ethylene Glycol Tetra Acetic Acid and Prevention by Strontium

The important roles of calcium on plant growth and development including cell division and cell elongation is well documented. The purpose of the present study was to determine the impact of root zone calcium on the growth and health of potato apical meristem and on the maintenance of apical dominance. For this purpose, single-node potato cuttings (Solanum tuberosum L. cv. Dark Red Norland) were grown in sterilized modified Murashige and Skoog (MS) media containing varying concentrations of calcium (1 to 3000 μM). After 13 to 30 d of growth, plantlets were harvested and data for height of the main shoot and for the number of axillary shoots produced were recorded. Plantlets were ashed and tissue calcium concentration was determined. Shoot height was retarded with decreasing concentration of calcium in the media. Calcium deficiency induced shoot tip injury and loss of apical dominance. Tip injury was followed by the development of axillary shoots. The number of axillary shoots increased from one to 21 as calcium concentration in the media decreased from 3000 to 1 μM. At calcium concentration of 1500 μM or higher, there was a single main shoot with no axillary shoots. Addition of ethylene glycol tetra acetic acid (EGTA), a calcium chelator, to the media with 2720 μM calcium (sufficient calcium) resulted in the development of shoot injury and in the formation of axillary shoots. Calcium deficiency injury symptoms were prevented by the addition of a calcium analog, strontium, to MS media deficient in calcium. Strontium has been reported to strongly bind to plant cell walls and the inclusion of strontium prevented injury in shoots of plants grown on calcium-deficient media. These results suggest that strontium is able to mimic the role of calcium in the maintenance of cell wall integrity and supports previous studies that showed that calcium deficiency results from cell wall collapse of the subapical cells.

Chaetomorpha philippinensis (Cladophorales, Chlorophyta), a new marine microfilamentous green alga from tropical waters

Leliaert F., Payo D.A., Calumpong H.P. and De Clerck O. 2011. Chaetomorpha philippinensis (Cladophorales, Chlorophyta), a new marine microfilamentous green alga from tropical waters. Phycologia 50: 384–391. DOI: 10.2216/10-76.1A new marine microfilamentous green alga, Chaetomorpha philippinensis Leliaert sp. nov., is described as an epiphyte on Chaetomorpha vieillardii from shallow subtidal habitats in the Philippines. Phylogenetic analyses of large subunit rDNA and rDNA internal transcribed spacer (ITS) sequences show that the new tropical species is sister to the cold-water Chaetomorpha norvegica, from which it is genetically clearly distinct but morphologically almost indistinguishable. Chaetomorpha philippinensis is characterized by minute, straight or curved, unbranched, erect filaments up to 300 µm long and 7–17 µm in diameter, attached by a basal, hapteroid holdfast. Filaments in culture are similar in morphology but grow considerably longer with slightly larger cells. The cylindrical cells are multinucleate with up to eight nuclei (up to 18 in culture). Cells contain a single, parietal, lobed chloroplast with numerous small perforations and one to several pyrenoids. Zoosporangia develop by transformation of apical and subapical cells with zoids emerging through a domed pore in the apical, middle or basal part of the cell.

Localization of ENaC subunit mRNAs in adult bullfrog skin

Adult amphibian skin has served as a model for the investigation of Na+-transporting epithelia, such as mammalian renal tubules. The amiloride-blockable epithelial Na+ channel (ENaC), which is located in the apical membrane of the outer living cell layer, regulates Na+ transport across the epithelium. ENaC is thought to develop during the terminal differentiation of epidermal cells, but the details are unclear. Here, we used in situ hybridization to examine the localization of the ENaC subunit mRNAs in skin of adult bullfrogs, to clarify the development of ENaC. We found that α-ENaC mRNA was expressed within the cells of the Stratum granulosum, the Stratum spinosum, and the Stratum germinativum, while β-ENaC mRNA was expressed within the cells of the S. granulosum and the S. spinosum. However, we could not detect expression of γ-ENaC mRNA, possibly for technical reasons. α- and β-ENaC mRNAs, at least, were present in the sub-apical cells, in which ENaC protein is not necessary for amphibian skin to possess its Na+-transport function. Our results may mean that the sub-apical cells are already producing the ENaC subunit mRNAs prior to the final step in their differentiation.

Three dynamin-related protein 5B genes are related to plastid division in Physcomitrella patens

Dynamin family proteins in eukaryotic cells assemble into rings or spirals on the surface of membranes and pinch the membranes. We found 21 dynamin-related protein (DRP) genes in the Physcomitrella patens genome. Phylogenetic analysis indicated that three of them ( PpDRP5B-1, PpDRP5B-2, and PpDRP5B-3) showed robust monophyly with Arabidopsis thaliana DRP5B and Cyanidioschyzon merolae CmDnm2, both of which are related to plastid division. Quantitative RT-PCR analysis showed that the amounts of DRP5B-3 transcripts were 14-fold and 8-fold higher than those of DRP5B-1 and DRP5B-2, respectively. We generated PpDRP5B knockout transformants for each of these genes. Subapical protonemata cells in wild-type plants had an average of 47 chloroplasts. The cells in the PpDRP5B-3 knockout transformant had slightly enlarged chloroplasts, with an average chloroplast number of 28, whereas the PpDRP5B-1 and 5B-2 knockout lines had no effect on chloroplast number in P. patens. To analyze function of each PpDRP5B gene, we generated double- and triple-knockout lines. Whereas there were 32 chloroplasts in a cell of the PpDRP5B-1/5B-2 double-knockout lines, the triple-knockout line had only a few macrochloroplasts. A transient expression assay with the triple-knockout line demonstrated that the PpDRP5B-3 gene could recover the normal chloroplast phenotype.

Autophagy Deficiency Promotes β-Lactam Production in Penicillium chrysogenum

We have investigated the significance of autophagy in the production of the β-lactam antibiotic penicillin (PEN) by the filamentous fungus Penicillium chrysogenum. In this fungus PEN production is compartmentalized in the cytosol and in peroxisomes. We demonstrate that under PEN-producing conditions significant amounts of cytosolic and peroxisomal proteins are degraded via autophagy. Morphological analysis, based on electron and fluorescence microscopy, revealed that this phenomenon might contribute to progressive deterioration of late subapical cells. We show that deletion of the P. chrysogenum ortholog of Saccharomyces cerevisiae serine-threonine kinase atg1 results in impairment of autophagy. In P. chrysogenum atg1 cells, a distinct delay in cell degeneration is observed relative to wild-type cells. This phenomenon is associated with an increase in the enzyme levels of the PEN biosynthetic pathway and enhanced production levels of this antibacterial compound.

Ultrastructure of glandular hairs of <i>Sigesbeckia jorullensis</i> Kunth (Asteraceae)

Multicellular long-stalked glandular hairs of the outer involucral bracts of Sigesbeckia jorullensis Kunth were investigated with transmission electron micros-copy. The apical layer of head cells was characterized by many irregular leucoplasts, a well developed and in the secretory phase mostly agranular endoplasmic reticu-lum, few dictyosomes, and one calcium oxalate crystal per cell. The prevalent part of the secretion seems to originate from leucoplasts of apical head cells and is secreted into a subcuticular space after crossing cytoplasm and the plasma membrane. The sheath of this sub-cuticular space was formed exclusively by a thin cuticle, which was detached from the cell wall in the secretory phase. Some small wall protuberances were found between the sub-apical and apical cell layer. Whether the three to six layers of sub-apical head cells, containing many chloroplasts, contribute to the biogenesis of the lipophilic secretion or not, remains unclear. Light microscopy demonstrated that there was a...

Ectopic expression of the Brassica SHOOTMERISTEMLESS attenuates the deleterious effects of the auxin transport inhibitor TIBA on somatic embryo number and morphology

The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) is a useful compound for investigating the role of auxin flow during plant growth and development. In Arabidopsis lines, applications of TIBA during the induction phase of somatic embryogenesis inhibit embryo development and induce the differentiation of the meristematic cells of the shoot apical meristem (SAM), leading to the fusion of the cotyledons. These abnormalities were associated to changes in the expression levels of auxin transporter genes ( PINs) and endogenous distribution of IAA. Treatments of TIBA caused a rapid accumulation of IAA within the epidermal and cortical root cells of the explants (bent-cotyledon zygotic embryos), as well as in the apical and sub-apical cells of the callus generated by the surface of the cotyledons of the explants. Within the callus only a few cells acquired meristematic characteristics, and this was associated to low expression levels of genes involved in embryogenic cell fate acquisition, such as WUSCHEL ( WUS), LEAFY COTYLEDON 1 and 2. All these deleterious effects were attenuated when TIBA was administered to lines over-expressing SHOOT MERISTEMLESS ( STM) isolated from Brassica oleracea ( Bo), B. napus ( Bn), and B. rapa ( Br). Of interest, TIBA-treated explants of Arabidopsis lines over-expressing the Brassica STM were able to produce a large number of embryogenic cells and somatic embryos which exhibited a normal morphology and two distinct cotyledons. A proposed reason for this behaviour was ascribed to the ability of the transformed tissue to retain a normal distribution of auxin in the presence of TIBA. Proper localization of auxin might be required for the normal expression of several genes needed for the acquisition of embryogenic competence and formation of somatic embryos.

Peroxisomes Are Required for Efficient Penicillin Biosynthesis in Penicillium chrysogenum

In the fungus Penicillium chrysogenum, penicillin (PEN) production is compartmentalized in the cytosol and in peroxisomes. Here we show that intact peroxisomes that contain the two final enzymes of PEN biosynthesis, acyl coenzyme A (CoA):6-amino penicillanic acid acyltransferase (AT) as well as the side-chain precursor activation enzyme phenylacetyl CoA ligase (PCL), are crucial for efficient PEN synthesis. Moreover, increasing PEN titers are associated with increasing peroxisome numbers. However, not all conditions that result in enhanced peroxisome numbers simultaneously stimulate PEN production. We find that conditions that lead to peroxisome proliferation but simultaneously interfere with the normal physiology of the cell may be detrimental to antibiotic production. We furthermore show that peroxisomes develop in germinating conidiospores from reticule-like structures. During subsequent hyphal growth, peroxisome proliferation occurs at the tip of the growing hyphae, after which the organelles are distributed over newly formed subapical cells. We observed that the organelle proliferation machinery requires the dynamin-like protein Dnm1.

Mating‐type orthologous genes in the primarily homothallic Moniliophthora perniciosa, the causal agent of Witches' Broom Disease in cacao

The cacao-pathogenic Moniliophthora perniciosa C-biotype is a primarily homothallic Agaricomycete of which the genome has recently become available. Searching of the genome sequence with mating type proteins from other basidiomycetes detected one or possibly two potential genes for HD1 homeodomain transcription factors, 7 or possibly 8 genes for potential pheromone receptors and five genes for putative pheromone precursors. Apparently, the fungus possesses gene functions encoded in the tetrapolar basidiomycetes in the A and B mating loci, respectively. In the tetrapolar species, the A and B mating type genes govern formation of clamp cells at hyphal septa of the dikaryon and their fusion with sub-apical cells as well as mushroom production. The C-biotype forms fused clamp cells and also basidiocarps on mycelia germinated from basidiospores and their development might be controlled by the detected genes. It represents the first example of a primarily homothallic basidiomycete where A - and B -mating-type-like genes were found. Various strategies are discussed as how self-compatibility in presence of such genes can evolve. An A -mating-type like gene for an HD2 homeodomain transcription factor is, however, not included in the available sequence representing estimated 69% coverage of the haploid genome but there are non-mating genes for other homeodomain transcription factors of currently unknown function that are conserved in basidiomycetes and also various ascomycetes.

Localization of enzymes of artemisinin biosynthesis to the apical cells of glandular secretory trichomes of Artemisia annua L.

A method based on the laser microdissection pressure catapulting technique has been developed for isolation of whole intact cells. Using a modified tissue preparation method, one outer pair of apical cells and two pairs of sub-apical, chloroplast-containing cells, were isolated from glandular secretory trichomes of Artemisia annua. A. annua is the source of the widely used antimalarial drug artemisinin. The biosynthesis of artemisinin has been proposed to be located to the glandular trichomes. The first committed steps in the conversion of FPP to artemisinin are conducted by amorpha-4,11-diene synthase, amorpha-4,11-diene hydroxylase, a cytochrome P450 monooxygenase (CYP71AV1) and artemisinic aldehyde Δ11(13) reductase. The expression of the three biosynthetic enzymes in the different cell types has been studied. In addition, the expression of farnesyldiphosphate synthase producing the precursor of artemisinin has been investigated. Our experiments showed expression of farnesyldiphosphate synthase in apical and sub-apical cells as well as in mesophyl cells while the three enzymes involved in artemisinin biosynthesis were expressed only in the apical cells. Elongation factor 1α was used as control and it was expressed in all cell types. We conclude that artemisinin biosynthesis is taking place in the two outer apical cells while the two pairs of chloroplast-containing cells have other functions in the overall metabolism of glandular trichomes.

Influence of Root Zone Calcium on Subapical Necrosis in Potato Shoot Cultures: Localization of Injury at the Tissue and Cellular Levels

Shoot tip necrosis has been attributed to calcium deficiency in in vitro cultures, resulting in death of the stem tip, the loss of apical dominance, and axillary branch development. Using an in vitro shoot culture system with Solanum tuberosum L. cv. Dark Red Norland, we studied the development of injury symptoms at the microscopic and tissue levels at a range of media calcium concentrations varying from 6.8 to 3000 μm. Light and electron microscopic studies revealed that the primary injury due to calcium deficiency was the death and collapse of expanding pith cells below the shoot apex. The structure and organization of the shoot apical meristem was the same when plants were cultured on sufficient or suboptimal media calcium concentrations. However, the apical meristem senesced following subapical shoot tissue collapse. Death of the shoot apical meristem was a secondary effect of calcium deficiency, resulting in loss of apical dominance. Studies with 45Ca indicated that calcium was distributed in a gradient along the shoot, with highest concentration at the base and the lowest at the apex. Shoot tip necrosis developed after 20 days of culture on the suboptimal calcium concentration medium. The development of these symptoms and axillary shoot growth was associated with the lack of calcium accumulation in the shoots. Our results provide evidence that a primary injury of calcium deficiency is localized in the expanding pith cells below the shoot apical meristem and this injury results in the collapse of subapical cells. Death of the shoot apical meristem is a secondary injury resulting from calcium deficiency.

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

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. 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. 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. 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.

Cell wall architecture of Physcomitrella patens is revealed by atomic force microscopy

The moss Physcomitrella patens (Hedw.) Bruch & Schimp. in B.S.G. serves as a nonvascular plant model system suitable for studying many plant developmental phenomena. The tip-growing filamentous protonemal stage of its life cycle exhibits polarized growth and various tropic responses. Conventional staining and light microscopy (LM) were used to provide the first direct evidence that protonemal cells of P. patens lack a cuticle. Atomic force microscopy (ATM) images reveal detailed surface structures identified by scanning electron microscopy (SEM). The cell wall ultrastructure is characterized by rounded protrusions that are uniformly distributed along each caulonemal filament, and longer fibrillar structures, which are disorganized at the apex, but become oriented in longitudinal arrays parallel to the growth axis in more proximal regions of caulonemal apical cells. The subapical cells are characterized by a polylamellated texture. There was no difference in gross surface ultrastructure between light-grown and dark-grown filaments, but the dimensions of the rounded protrusions at the apices of caulonemata cultured in the light and in darkness were significantly different. The convex and concave cell wall surfaces of a curved, gravitropically responding dark-grown caulonema appear structurally different. This investigation is the first to use AFM to probe the cell wall ultrastructure of a bryophyte. The data further elaborate a simple model of cell wall development in the caulonemata of P. patens that was proposed for other tip-growing filamentous plants.