The roles of light and the nucleus in the regulation of reproductive onset in Acetabularia acetabulum

At reproductive onset the marine green alga Acetabularia acetabulum (L.) P.C. Silva redirects growth from vertical elongation of the axis of the plant to lateral expansion of a disk-shaped reproductive structure, the “cap.” We used amputation to synchronize cap initiation and to facilitate investigation of the light requirements during amputation-induced cap initiation. Following amputation of a nascent cap, most plants initiate one whorl of vegetative hairs and then a cap. Both hair and cap initiation required photosynthesis, as indicated by studies with 3-(3′,4′-dichlorophenyl)-1, 1-dimethylurea, but did not require the nucleus. Amputation-induced hair initiation occurred in red light, but 10 min of blue light given in a background of red light significantly increased hair initiation, supporting previous studies that hair initiation is a blue-light-triggered photomorphogenic event. Amputation-induced cap initiation also occurred in red light, but daily 10-min flashes of blue light given in a background of red light did not significantly enhance cap initiation. We also examined the light requirements of intact plants at each phase of development. In the absence of blue light, juveniles and adults with ≤13.7 ± 4.3 whorls of hairs arrested in development and failed to initiate caps. In contrast, very late adults with ≥13.7 ± 4.3 whorls of hairs initiated caps in the absence of blue light, suggesting that there is a point in late adult development beyond which cap initiation does not require blue light. Several plausible interpretations of the role of light and the nucleus in the regulation of reproductive onset are discussed to try to reconcile these data with those in the literature. Received: 18 March 1999 / Accepted: 13 May 1999     

Importance of the cap in maize root growth

A large population of primary roots of Zea mays (cv. LG 11) was selected for uniform length at zero time. Their individual growth rates were measured over an 8-h period in the vertical position (in humid air, darkness). Three groups of these roots with significantly different growth rates were then chosen and their cap length was measured. It was found that slowly growing roots had long caps whereas rapidly growing roots had short caps. The production by the cap cells of basipetally transported growth inhibitors was tested (biologically by the curvature of half-decapped roots) and found to be significantly higher for longer root caps than that for shorter ones.     

Dynamics of the actin cytoskeleton,hyphal tip growth and the movement of the two nuclei in the dikaryon ofCoprinus cinereus

We first examined the changes in distribution of F-actin during conjugate division in the apical cells of the dikaryon ofCoprinus cinereus using indirect immunofluorescence microscopy, then followed hyphal tip growth and the movement of the two nuclei in the apical cells using differential interference contrast microscopy (DIC). In apical cells with interphase nuclei, F-actin occurred solely as peripheral plaques, which were distributed along the whole length of the cell and were more concentrated at the tips, where they formed caps. In the early prophase of conjugate division, F-actin was transiently concentrated, as diffused form and plaques, at hyphal regions where the two nuclei sit, and this was accompanied by transient disappearance of the actin cap at the hyphal tip in the majority of cells. The actin cap was also present at the tips of growing clamp cells from late prophase through metaphase and disintegrated during anaphase. In telophase, actin rings formed at the future septa. DIC revealed that, in early prophase, when the F-actin array occurs around the two nuclei and the actin cap is absent at hyphal tips, hyphae kept growing and the second nucleus accelerated its forward movement to catch up with the leading nucleus, which was still moving forward.     

Microtubule formation on the nuclear surface during meiosis inAcetabularia acetabulum

Summary Microtubules (MT) are a feature of all eukaryotic cells. However, they have not been observed in the cytoplasm of the vegetative phase ofAcetabularia acetabulum. Previous investigators have reported that, in the propagative phase, MTs function as anchors in the transport of secondary nuclei to the cap. They also form elaborate arrays around nuclei during cyst formation. The life history ofA. acetabulum is marked by changes in chromatin, the nucleolus, and the perinuclear cytoplasm. In this study light microscopical features of the nucleolus and changes in chromatin, labelled with anti-histon antibodies, were used to define the developmental stages. Anti-tubulin antibodies have been used to trace the origin and development of MTs, MTs are formed on the surface of the primary nucleus. They are organized first into short thick sticks and then later elongate into thinner strands which enclose the nucleus in a dense network. Following these events on the surface of the nucleus, the spindle develops inside the nuclear membrane which remains intact throughout the mitotic division.     

AXENIC CULTURES OF ACETABULARIA (CHLOROPHYTA): A DECONTAMINATION PROTOCOL WITH POTENTIAL APPLICATION TO OTHER ALGAE1

We developed an easy, reliable decontamination protocol for caps of Acetabularia acetabulum (L.) Silva; with minimal labor hundreds of caps can be decontaminated. In addition, cysts isolated from these caps do not exhibit dormancy and can be used immediately to establish large populations of axenic cell cultures. This method consists of three successive incubations: 1) proteinase K/sodium dodecyl sulfate/1,3-bis[tris(hydroxymethyl)-methylamino]propane for 1 h, 2) mild silver protein for 5 min, and 3) an antibiotic solution (neomycin, chloramphenicol, nystatin, ampicillin, streptomycin)for 3 days. This protocol eliminates bacterial, algal, fungal, and yeast contaminants. It is useful for decontaminating caps from lab cultures and caps collected from the wild and may also be effective in decontaminating the reproductive structures of other algae. Cyst dormancy was reduced from 15 weeks to less than 1 week, which represents a 40% reduction of the life cycle of A. acetabulum. We routinely obtained 90-100% gamete release from cysts 3-14 days after they were made axenic. The component of our culturing methods that allows gamete release without a “dormant” period is unknown.     

Shape of the Outer End of the Cap Rays and Cyst Formation in Acetabularia Mediterranea

Abstract

The convex shape of the outer end of the cap rays in Acetabularia mediterranea is a typical species specific character, probably determined by a corresponding «morphogenetic substance» of nuclear origin. When cysts differentiate, the outer end of the rays looses its convexity, becoming flat or concave. This phenomenon, which is observed only in laboratory cultures, may be related to a probable fall in the internal pressure of the cell.     

Evidence that actin reinforces the extensible hyphal apex of the oomyceteSaprolegnia ferax

Summary Filamentous actin in the apices of growing hyphae of the oomyceteSaprolegnia ferax is distributed such that it could compensate for weakness in the expanding apical cell wall and thus play a role in morphogenesis of the tip. The tapered extensible portion of the hyphal tip where the cell wall is plastic contains a cap of actin which differs in organization from the actin in subapical, inextensible regions of the hypha. Rapidly growing hyphae which are expected to have a longer plastic cell wall region contain longer actin caps. Furthermore, the weakest point in the hyphal apex, demonstrated by osmotic shock-induced bursting, was within the taper where the wall is plastic but never in the extreme apex where actin was most densely packed and presumably the strongest. Treatment of hyphae with cytochalasin E/dimethyl sulphoxide induced rapid changes in actin caps. Cap disruption was accompanied by transient growth rate increases, subsequent rounding and swelling of apices and a shift of osmotically induced burst points closer to the apex. These correlated changes are consistent with a role for the actin cap in tip morphogenesis. The association between regions of plasticity in the apical cell wall, the extent of the actin cap, the location of the weakest point in the apex and the effects of damage to the actin cap suggest that the cap functions to support the apex in regions where the cell wall is weak.Abbrevations CE cytochalasin E - DMSO dimethyl sulphoxide - RP rhodamine phalloidin Dedicated to the memory of Professor Oswald Kiermayer     

The marketing ofLactarius deliciosus in Northern Spain

We report the harvesting of an average of 4,000 kg of saffron milk caps (Lactarius deliciosus Fr.) per day during four to six weeks between mid-October and mid-November in a village of 200 inhabitants in northern Spain. Nearly every inhabitant picks saffron milk caps, for which they receive an average of 2 ε/kg. A family of four could make a profit of 5,600–8,400 ε in a season (average annual income per family in the area is 18,727 ε). Pickers sell the harvested mushrooms either to a local middleman or directly to the buyer, who then takes the produce to the final point of sale, usually in Catalonia, where the demand for saffron milk caps is increasing yearly. This trade has occurred for 30 years, and began when saffron milk caps started to appear in the area after pine trees were introduced to replace the native oaks. This study provides evidence that the collection and marketing of wild edible fungi is a profitable task on a local and national scale.     

Salt stress in Mesembryanthemum crystallinum L. cell suspensions activates adaptive mechanisms similar to those observed in the whole plant

A salt-tolerant stable cell-suspension culture from the halophyte Mesembryanthemum crystallinum L. has been established from calli generated from leaves of 6-week-old well-watered plants. Optimal cell growth was observed in the presence of 200 mM NaCl, and within 7 d cells were able to concentrate Na⁺ to levels exceeding those in the growth medium. Accumulation of Na⁺ was paralled by increases in the compatible solute pinitol and myo-inositol methyl transferase (IMT), a key enzyme in pinitol biosynthesis. Increasing concentrations of NaCl stimulated the activities of tonoplast and plasma-membrane H⁺-ATPases. Immunodetection of the ATPases showed that the increased activity was not due to changes in protein amount that could be attributed to treatment conditions. A specific role for these mechanisms in salt-adaptation is supported by the inability of mannitol-induced water stress to elicit the same responses, and the absence of enzyme activity and protein expression associated with Crassulacean acid metabolism in the cells. Results demonstrate that these  M. crystallinum cell suspensions show a halophytic growth response, comparable to that of the whole plant, and thus provide a valuable tool for studying signaling and biochemical pathways involved in salt recognition and response. Received: 18 June 1998 / Accepted: 22 August 1998     

5′ and 3′ end modifications of spliceosomal RNAs in <Emphasis Type="Italic">Plasmodium falciparum</Emphasis>

5′ caps provide recognition sequences for the nuclear import of snRNAs. The 5′ and 3′ ends of snRNAs were studied in Plasmodium falciparum with a modified adapter ligation method, which showed that 5′ ends of U1, U2, U4, U5 and U6 snRNAs are capped. In P. falciparum, the 3′ ends of U1, U2, U4 and U5 snRNAs have free hydroxyl groups whereas U6 snRNA has a blocked 3′ end. An immunoprecipitation assay for trimethyl guanosine caps shows that the cap structures of parasite U1-U5 snRNAs are hypermethylated while U6 snRNA may be γ-mono-methylated. Bioinformatics analysis of proteins involved in hypermethylation and trafficking of snRNAs indicates that the methyltransferase TGS1 is present in the P. falciparum genome. PfTGS1 is larger than its orthologs and may have transmembrane domains in the C-terminus. Surprisingly, the snRNA trafficking protein Snurportin is absent from the P. falciparum genome suggesting that reminiscent of yeast, parasite snRNAs may be retained in the nucleus.     

Cytoskeletal 10 nm filaments in cells of the algal phyla Chlorophyta,Charophyta, and Chrysophyta and their developmentally regulated and species-specific association with prosomes

Summary.  10 nm diameter filaments were observed in whole-mount preparations of algae of diverse phyla: Acetabularia acetabulum and A. major (Chlorophyta), Chara australis and Nitella flexilis (Charophyta), and Poterioochromonas malhamensis (Chrysophyta). A polyclonal antibody raised against a basic, 50 kDa DNA-binding protein of A. acetabulum stains the filaments of A. acetabulum and A. major as well as of C. australis and N. flexilis. While in the perinuclear region of A. acetabulum and A. major and throughout the cytoplasm of P. malhamensis the 10 nm filaments have a smooth appearance, in the stalk of A. acetabulum and A. major they are densely covered by globular structures; in C. australis and N. flexilis they are less frequently associated with such material. The morphology of a part of the globular particles is quite reminiscent of prosomes. A monoclonal antibody elicited against prosomes isolated from A. acetabulum indeed decorates the globular particles on the A. acetabulum and A. major filaments. The possible role of these filament-particle associations is discussed. Received August 10, 2001; accepted October 30, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Max-Planck-Institut für Zellbiologie, 68526 Ladenburg, Federal Republic of Germany. E-mail: sberger@zellbio.mpg.de RID="**" ID="**" Present address: Long Island University, Southampton, New York, U.S.A. RID="+" ID="+" Present address: Leica Microsystems Wetzlar GmbH, Wetzlar, Federal Republic of Germany     

Production of micronucleoli and onset of cotton fiber growth

Experiments focused on the early development of fiber cells of cotton (Gossypium hirsutum L. cv. MD51 ne) ovules produced two novel findings: one biological, the other methodological. The first concerns a micronucleolus in the nucleus of fibers. This developmental marker appears at or a little before 4 days postanthesis (dpa) in about 10% of the fibers and increases thereafter to nearly 80% provided the fibers are growing on fertilized ovules. Micronucleoli are neither seen in nuclei of fibers at 0–2 dpa nor in nuclei of non-fiber cells. Consequently, it is postulated that they are the product of specific developmental genes associated with fiber growth. The second, methodological finding, involves a cytological means of directly counting the number of fibers produced on young ovules at 1–4 dpa. The method provides quantitative data unavailable in the past. We used this method to show that emasculation caused a temporary 24-h delay in the initiation of fibers, that 30% of the fibers are affected, and that at 3 dpa both fertilized and unfertilized ovules have about 14 500 fibers. These data indicate that the fibers on fertilized and unfertilized ovules represent the same cell populations, a finding heretofore unknown. Received: 24 October 1997 / Accepted: 5 January 1998     

Dynamic features of adherens junctions during Drosophila embryonic epithelial morphogenesis revealed by a Dα-catenin-GFP fusion protein

 Cell-cell adherens junctions (AJs), comprised of the cadherin-catenin adhesion system, contribute to cell shape changes and cell movements in epithelial morphogenesis. However, little is known about the dynamic features of AJs in cells of the developing embryo. In this study, we constructed Dα-catenin fused with a green fluorescent protein (Dα-catenin-GFP), and found that it targeted apically located AJ-based contacts but not other lateral contacts in epithelial cells of living Drosophila embryos. Using time-lapse fluorescence microscopy, we examined the dynamic performance of AJs containing Dα-catenin-GFP in epithelial morphogenetic movements. In the ventral ectoderm of stage 11 embryos, concentration and deconcentration of Dα-catenin-GFP occurred concomitantly with changes in length of AJ contacts. In the lateral ectoderm of embryos at the same stage, dynamic behaviour of AJs was concerted with division and delamination of sensory organ precursor (SOP) cells. Moreover, changes in patterns of AJ networks during tracheal extension could be followed. Finally, we utilized Dα-catenin-GFP to precisely observe the defects in tracheal fusion in shotgun mutants. Thus, the Dα-catenin-GFP fusion protein is a helpful tool to simultaneously observe morphogenetic movements and AJ dynamics at high spatio-temporal resolution. Received: 5 October 1998 / Accepted: 30 November 1998     

Blood cell induction in Xenopus animal cap explants: Effects of fibroblast growth factor, bone morphogenetic proteins, and activin

 Cultures of Xenopus blastula animal caps were used to explore the haematopoietic effects of three candidate inducers of mesoderm: basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMPs) and activin A. In response to either bFGF or activin A, explants expanded into egg-shaped structures, and beneath an outer layer of epidermis, a ventral mesodermal lining surrounded a fluid-filled cavity containing ”blood-like cells”. Immunocytochemistry identified some of these cells as early leukocytes, but erythrocytes were rare. BMP-2 or BMP-4 induced primitive erythrocytes as well as leukocytes, and a high concentration was required for these cells to differentiate in only a small proportion of explants. BMP-2 but not BMP-4 induced ventral mesoderm concomitantly. High concentrations of activin A dorsalized explants, which contained infrequent leukocytes, and an optimal combination of activin A and bFGF caused differentiation of muscle with few blood cells. By contrast, BMP-2 or BMP-4 plus activin A synergistically increased the numbers of both leukocytes and erythrocytes. Explants treated with BMPs plus activin contained a well organized cell mass in which yolk-rich cells mixed with blood cells and pigmented cells did not. BMP-2 plus bFGF also induced numerous leukocytes and fewer erythrocytes, but BMP-4 antagonized the leukopoietic effect of bFGF. The data suggest that the signalling pathways these three factors use to induce leukopoiesis overlap and that erythropoiesis may be activated when inducers are present in combination. Received: 3 August 1998 / Accepted: 7 October 1998     

Involvement of Arabidopsis thaliana phospholipase Dζ2 in root hydrotropism through the suppression of root gravitropism

Root hydrotropism is the phenomenon of directional root growth toward moisture under water-deficient conditions. Although physiological and genetic studies have revealed the involvement of the root cap in the sensing of moisture gradients, and those of auxin and abscisic acid (ABA) in the signal transduction for asymmetric root elongation, the overall mechanism of root hydrotropism is still unclear. We found that the promoter activity of the Arabidopsis phospholipase Dζ2 gene (PLDζ2) was localized to epidermal cells in the distal root elongation zone and lateral root cap cells adjacent to them, and that exogenous ABA enhanced the activity and extended its area to the entire root cap. Although pldζ2 mutant root caps did not exhibit a morphological phenotype in either the absence or presence of exogenous ABA, the inhibitory effect of ABA on gravitropism, which was significant in wild-type roots, was not observed in pldζ2 mutant roots. In root hydrotropism experiments, pldζ2 mutations significantly retarded or disturbed root hydrotropic responses. A drought condition similar to that used in a hydrotropism experiment enhanced the PLDζ2 promoter activity in the root cap, as did exogenous ABA. These results suggest that PLDζ2 responds to drought through ABA signaling in the root cap and accelerates root hydrotropism through the suppression of root gravitropism.     

Immunocytochemical localization of indole-3-acetic acid in primary roots of Zea mays

Colloidal gold-labelled antibody was used to localize indole-3-acetic acid in caps of primary roots of Z. mays cv. Kys. Gold particles accumulated on the nucleus, vacuoles, mitochondria, and some dictyosomes and dictyosome-derived vesicles. This is the first localization of indole-3-acetic acid in dictyosomes and dictyosome-derived vesicles and indicates that dictyosomes and vesicles constitute a pathway for indole-3-acetic acid movement in and secretion from root cap cells. Our findings provide cytochemical evidence to support the hypothesis that indole-3-acetic acid plays an important role in root gravitropism.     

Cellular Differentiation and Tissue Partitioning in Caps of Primary and Lateral Roots of Helianthus annuus

Cellular and tissue volumes in caps of primary and lateral rootsof Helianthus annuus have been measured in order to determinequantitatively how tissues and their functions are partitionedin root caps. Patterns of change in cellular dimensions andvolumes are similar in caps of primary and lateral roots. Significantincreases in cellular dimensions and volume occur during thedifferentiation of columella cells and the innermost peripheralcells. There are no significant changes in cellular dimensionsas either (i) the production and secretion of mucilage begins,or (ii) cells are sloughed from the cap. Tissues are partitionedsimilarly in caps of primary and lateral roots. indeed, rootcaps allocate 7–8 per cent of their volume for regeneration(i.e. calyptrogen tissue), 16–19 per cent of their volumefor graviperception (i.e. columella tissue), and approx. 38per cent of their volume for the production and secretion ofmucilage. These results are discussed relative to patterns ofcellular differentiation and tissue function in root caps. Helianthus annuus, root caps, primary root, lateral root, calyptrogen, columella, peripheral cells, tissue partitioning     

Redistribution of actin, profilin and phosphatidylinositol-4,5-bisphosphate in growing and maturing root hairs

The continuously changing polar cytoplasmic organization during initiation and tip growth of root hairs is reflected by a dynamic redistribution of cytoskeletal elements. The small G-actin binding protein, profilin, which is known to be a widely expressed, potent regulator of actin dynamics, was specifically localized at the tip of root hairs and co-distributed with a diffusely fluorescing apical cap of actin, but not with subapical actin microfilament (MF) bundles. Profilin and actin caps were present exclusively in the bulge of outgrowing root hairs and at the apex of elongating root hairs; both disappeared when tip growth terminated, indicating a tip-growth mechanism that involves profilin-actin interactions for the delivery and localized exocytosis of secretory vesicles. Phosphatidylinositol-4,5-bisphosphate (PIP₂), a ligand of profilin, was localized almost exclusively in the bulge and, subsequently, formed a weak tip-to-base gradient in the elongating root hairs. When tip growth was eliminated by the MF-disrupting inhibitor cytochalasin D, the apical profilin and the actin fluorescence were lost. Mastoparan, which is known to affect the PIP₂ cycle, probably by stimulating phospholipases, caused the formation of a meshwork of distinct actin MFs replacing the diffuse apical actin cap and, concomittantly, tip growth stopped. This suggests that mastoparan interferes with the PIP₂-regulated profilin-actin interactions and hence disturbs conditions indispensable for the maintenance of tip growth in root hairs. Received: 11 March 1999 / Accepted: 27 May 1999     

Effects of aluminium on canola roots

There is little information on the effects of aluminium (Al) on canola (Brassica napus var. napus L.), which is a commercially important crop species in many parts of the world. In this report, we describe the effects of Al on roots of canola seedlings grown hydroponically in a nutrient solution at pH 4.5. The morphological and ultrastructural changes that accompanied these growth effects were examined. Additions to the nutrient solution of Al at concentrations below 40 μM stimulated root growth of canola seedlings, increasing both the size and number of central cap cells. The stimulation of root growth did not appear to be due to the alleviation of a proton toxicity at the root surface. At concentrations of Al above 60 μM, root growth was strongly inhibited, with cellular damage being observed primarily in peripheral root cap cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Kinetics of double fertilization in Torenia fournieri based on direct observations of the naked embryo sac

Torenia fournieri Lind. has a naked embryo sac that protrudes from the micropyle. The precise time course of the entire process of double fertilization and the kinetics of fertilization events were determined in this species by the following methods: (i) without squashing, pollen tubes on the torn stylar canal were observed by fluorescence microscopy after staining with both 4′,6-diamidino-2-phenylindole (DAPI) and aniline blue; and (ii) large numbers of living embryo sacs were observed directly by differential interference microscopy before and after fertilization. The pollen began to germinate 5 min after pollination and extruded pollen tubes which elongated at a constant rate of 2.3 mm · h⁻¹. At 4.0 h after pollination, the mitotic index of the generative cell within the pollen tube reached 88% and the two sperm cells were formed. Pollen tubes began to arrive at ovules 8.9 h after pollination and directly entered one of two synergids in the naked embryo sac. The time required for transport of sperm cells in the degenerated synergid was estimated statistically to be 1.9 ± 1.8 min for transport of the first cell and 7.4 ± 1.6 min for the second. In the nucleus of the fertilized egg cell, the male nucleolus began to emerge 10 h after pollination and the female nucleolus often decreased in size. The two nucleoli fused together prior to elongation of the zygote, which began 28 h after pollination. In the central cell, the secondary nucleus migrated to a region adjacent to the egg apparatus after pollination but prior to the arrival of the pollen tube. The primary endosperm nucleus rapidly returned to the inner region after fertilization. Prior to embryogenesis, the first division of the primary endosperm began about 15 h after pollination, at a defined site, to form the chalazal haustorium. Received: 24 October 1996 / Accepted: 13 March 1997