Development and organization of the central vacuole of Acetabularia acetabulum

* Here we analyzed the shape of the central vacuole of Acetabularia acetabulum by visualizing its development during diplophase (from juvenility through reproduction) and haplophase (from meiosis through mating). * Light microscopy and whole-organism applications of a pH-sensitive dye, neutral red, were used to visualize the anatomy of the central vacuole. We studied connectivity within the thallus by locally applying dye to morphologically distinct regions (rhizoid, stalk, apex, hairs) and observing dye movements. * In vegetative thalli most of the rhizoid, stalk and young hairs stained with dye. In reproductive structures (caps, gametangia) dye also stained the majority of the interiors. When applied to small areas, dye moved at different rates through each region of the thallus (e.g. within the stalk). Dye moved from younger hairs, but not from older hairs, into the stalk. Errors in incorporation of central vacuole into gametangia occurred at <10(-5). * These data indicate that the central vacuole of A. acetabulum is a ramified polar organelle with, potentially, a gel-like sap that actively remodels its morphology during development.     

Growth inhibition and changes in morphology and actin distribution in Acetabularia acetabulum by phalloidin and phalloidin derivatives

Summary.  Effects on morphology and microfilament structure caused by phalloidin, phallacidin, and some semisynthetic phalloidin derivatives were studied in vegetative cells of the green alga Acetabularia acetabulum (L.) Silva. All phalloidin derivatives (except for phalloidin itself) caused growth stop of the alga after 1 day and (except for the fluorescein-labeled phalloidin) death of the cells after 4–7 days. Hair whorl tip growth and morphology as screened by light microscopy, as well as microfilament structure in tips, suggested that growth stop is correlated with a disorganization of actin filaments similar to that recently described for jasplakinolide (H. Sawitzky, S. Liebe, J. Willingale-Theune, D. Menzel, European Journal of Cell Biology 78: 424–433, 1999). Using rabbit muscle actin as a model target protein, we found that the toxic effects in vivo did not correlate with actin affinity values, suggesting that permeation through membranes must play a role. Indeed, the most lipophilic phalloidin derivatives benzoylphalloidin and dithiolanophalloidin were the most active in causing growth stop at ca. 100 μM. In comparison to the concentration of jasplakinolide required to cause similar effects (<3 μM), the two most active phalloidin derivatives exhibited an activity ca. 30 times lower. Nonetheless, lipophilic phalloidin derivatives can be used in algae, and probably also other cells, to modulate actin dynamics in vivo. In addition, we found that the fluorescent fluorescein isothiocyanate-phalloidin is able to enter living algal cells and stains actin structures brightly. Since it does not suppress actin dynamics, we suggest fluorescein isothiocyanate-phalloidin as a tool for studying rearrangements of actin structures in live cells, e.g., by confocal laser scanning microscopy. Received November 5, 2001; accepted August 8, 2002; published online November 29, 2002     

An analysis of morphogenesis of the reproductive whorl of Acetabularia acetabulum

Acetabularia acetabulum (Linn.) P.C. Silva, is a useful system for studying changes in shape because it is large, morphologically complex unicell. The middle, or gametophore lobe of the cap grows radially from the stalk axis as a disc and the fully grown cap can be one of several shapes: flat, concave, convex, and saddle. The shape of the cap normally changes during the first three and a half weeks of reproductive development: individual caps within a population change shape in a stereotypical progression, with the majority proceeding from concave to flat to saddle. Marking the existing surface of caps with carbon grains revealed that the majority of growth occurs near the center, not at the perimeter, of caps. The shape of the mature cap appeared to be independent of algal height, number of gametophores per cap, and final cap diameter. Removing the rhizoid, which contains the nucleus, suggested that the contribution of the nucleus may be important for changes in shape during early cap growth. Based on these data, we present a simple model of cap shape development that suggests both differential growth and biophysical factors may contribute to the final shape of caps of A. acetabulum. Received: 7 January 1998 / Accepted: 7 March 1998     

Spectroscopic and biochemical analysis of regions of the cell wall of the unicellular 'mannan weed', Acetabularia acetabulum

Although the Dasycladalean alga Acetabularia acetabulum has long been known to contain mannan-rich walls, it is not known to what extent wall composition varies as a function of the elaborate cellular differentiation of this cell, nor has it been determined what other polysaccharides accompany the mannans. Cell walls were prepared from rhizoids, stalks, hairs, hair scars, apical septa, gametophores and gametangia, subjected to nuclear magnetic resonance and Fourier transform infrared spectroscopy, and analyzed for monosaccharide composition and linkage, although material limitations prevented some cell regions from being analyzed by some of the methods. In diplophase, walls contain a para-crystalline mannan, with other polysaccharides accounting for 10-20% of the wall mass; in haplophase, gametangia have a cellulosic wall, with mannans and other polymers representing about a quarter of the mass. In the walls of the diplophase, the mannan appears less crystalline than typical of cellulose. The walls of both diploid and haploid phases contain little if any xyloglucan or pectic polysaccharides, but appear to contain small amounts of a homorhamnan, galactomannans and glucogalactomannans, and branched xylans. These ancillary polysaccharides are approximately as abundant in the cellulose-rich gametangia as in the mannan-rich diplophase. In the diplophase, different regions of the cell differ modestly but reproducibly in the composition of the cell wall. These results suggest unique cell wall architecture for the mannan-rich cell walls of the Dasycladales.     

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     

A vacuolar ATPase and pyrophosphatase in Acetabularia acetabulum.

Vacuole-rich fractions were isolated from Acetabularia acetabulum by Ficoll step gradient centrifugation. The tonoplast-rich vesicles showed ATP-dependent and pyrophosphate-dependent H(+)-transport activities. ATP-dependent H(+)-transport and ATPase activity were both inhibited by the addition of a specific inhibitor of vacuolar ATPase, bafilomycin B1. A 66 kDa polypeptide present in the preparation cross-reacted with the anti-IgG fractions against the alpha and beta subunits of Halobacterium halobium ATPase and with the antibody against the A subunit (68 kDa subunit) of mung bean vacuolar ATPase. A 56 kDa polypeptide present in the vacuole preparation showed cross-reactivity with the antibody against the B subunit (57 kDa) of mung bean vacuolar ATPase but not with the anti-beta subunit of H. halobium ATPase. A 73 kDa polypeptide cross-reacted with the antibody against inorganic pyrophosphatase of mung bean vacuoles. These results suggest that vacuolar membrane of A. acetabulum equipped energy transducing systems similar to those found in other plant vacuoles.     

Twofold effect of blue light on a circadian rhythm in Acetabularia

Abstract

The circadian chloroplast migration in Acetabularia mediterranea was monitored by continuously measuring the transmission of the cells near the apex. Under continuous red light the amplitude of the rhythm decreased rapidly within a few days. However, circadian changes of chloroplast density were still detectable even after 28 days of red light, indicating the persistence of the rhythm. When blue light was added after red light preirradiation of several days phase shifts were observed which were expressed as advances as well as delays. The period of the rhythm proved to be strongly dependent on the intensity of the continuous blue light which was given in addition to red light. Different red light intensities did not change the period. The occurrence of both effects indicates that the sensory transduction of blue light photoreception in Acetabularia works in two different ways: quanta counting processes and processes of light intensity measurement.     

Temperature Dependency of Circadian Period in a Single Cell (Acetabularia)

The circadian rhythm in the oxygen production of 30 individual Acetabularia cells has been studied at different temperatures. The temperature induced period variation was continuously evaluated over the whole data record of each individual cell with an advanced spectral analysis technique. The observed circadian periods of O₂ production displayed a well established region of temperature compensation between 25 °C and 30 °C with a Q₁₀, value of 0.9, whereas between 15°C and 22°C a positive temperature coefficient was measured (Q₁₀ at 22 °C 0.9, Q₁₀ at 20°C 0.8, Q₁₀at 17°C 0.7).     

Decreasing Period-Length of the Endogenous Circadian Rhythm of Oxygen Evolution in Acetabularia and Its Possible Relation to Aging

Endogenous circadian rhythms observed under constant conditions normally show period length variations. However, a general trend is difficult to identify when cells or organisms are entrained with the usual 24-h-period light/dark cycles. Therefore, these variations in time have been considered as fluctuations. In order to gain more insight into this phenomenon, individual Acetabularia cells were exposed to light/dark cycles of 16 h (LD 8:8) and 33.6 h (LD 16.8:16.8), respectively, i.e., periods which lie distinctly outside the range of the normal circadian entrainment. Employing a high-resolution procedure for data analysis, decreasing period lengths could consistently be detected when cells were kept under constant conditions for several weeks. Possible causes of this decrease are discussed.     

Oscillations in ultraweak photon emission of Acetabularia acetabulum (L.)

Ultraweak photon emission of dark-incubated A. acetabulum cells were measured with the use of a sensitive electronphotomultiplier of the Hamamatsu 550 type. The photon count series were subjected to Fourier analysis for 2-1020 sec period range. The average level of photon emission in samples containing 50 cells was approximately. 40% above background. Cell cultures were prepared at least 24 hr before the photon emission measurements and kept un-disturbed ("established cultures"). This paper reports results of Fourier analysis of a number of samples of Acetabularia cells. In a single population cells periodicity of light emission was not defined directly from Fourier transformation. A large number of analyses, however, if they are combined and compared with background data, reveal a cell-culture specific frequency pattern. The results suggest the idea that established cell-cultures are characterized by higher intensities of long period (minutes) oscillations occurs, while a relative decrease was observed in the short period (few seconds) range. The long period oscillations were not detected in cell cultures that were prepared within 1 hr before the photon emission measurements. It is concluded that Fourier analysis of ultraweak photon emission, even with relatively low signals, appears to be possible. It may serve as a non-invasive tool for monitoring the physiological state of cells, or for studying the control of intercellular dynamics.     

Classification of Chinese herbs based on the cluster analysis of delayed luminescence

Traditional Chinese material medica are an important component of the Chinese pharmacopeia. According to the traditional Chinese medicinal concept, Chinese herbal medicines are classified into different categories based on their therapeutic effects, however, the bioactive principles cannot be solely explained by chemical analysis. The aim of this study is to classify different Chinese herbs based on their therapeutic effects by using delayed luminescence (DL). The DL of 56 Chinese herbs was measured using an ultra‐sensitive luminescence detection system. The different DL parameters were used to classify Chinese herbs according to a hierarchical cluster analysis. The samples were divided into two groups based on their DL kinetic parameters. Interestingly, the DL classification results were quite consistent with classification according to the Chinese medicinal concepts of ‘cold’ and ‘heat’ properties. In this paper, we show for the first time that by using DL technology, it is possible to classify Chinese herbs according to the Chinese medicinal concept and it may even be possible to predict their therapeutic properties. Copyright © 2015 John Wiley & Sons, Ltd.     

气候条件对海滨锦葵(Kosteletzkya virginica) 延迟自花传粉的影响

延迟自交被认为是一种生殖适应性,因为它保证了传粉者稀少情况下的种子生产,但当传粉者丰富时,又允许异交占优势。海滨锦葵花在传粉者不足或缺乏时能通过主动的柱头裂片弯曲运动成功实现延迟自花传粉。2002-2005年对江苏盐城滩涂海滨锦葵栽培群体花中发生延迟自花传粉花所占的百分比进行了观测,并于2005年对花内不同停止类型的柱头裂片比进行了调查,结果表明,2002-2005年间发生延迟自花传粉花所占百分比分别为13.50%±1.62%、14.39%±1.50%、14.05%±1.82%和13.47%±1.51%,不同年份间并无显著差异（F（3,189）=6.128,P=0.085）。发生延迟自花传粉花所占百分比与气候条件有密切关系,不利天气（多云/雨）（22.11%±0.94%）明显高于有利天气（晴天）（5.43%±0.43%）（F（1,189）=8009.780,P〈0.001）。花内通过花部运动实现自花传粉而停止运动的柱头裂片比在有利天气（晴天）下（18.64%±1.61%）明显低于不利天气（多云/雨天）（70.58%±2.06%）（U=0.000,W=465.000,P〈0.001）。海滨锦葵这种直接响应于影响传粉者环境之气候条件的延迟自花传粉策略为从个体或花水平上验证繁殖保障假说提供了可能。     

Induction of Actin-Based Cytoplasmic Contraction in the Siphonous Green Alga Acetabularia (Chlorophyceae) by Locally Restricted Calcium Influx

Perfused cell segments dissected from the stalk or from detached cap ray chambers of Acetabularia were used as an experimental system to study the induction of cytoplasmic contractions and concurrent cytoskeletal changes in plant cells. Immunofluorescence microscopy revealed that the actin cytoskeleton quickly rearranges upon induction of contraction by forming bundles oriented circumferentially around the affected area, whereas microtubules were not detected. Contraction is blocked by cytochalasin D or N-ethylmaleimide but is unaffected by microtubule specific inhibitors. Contraction requires external Ca²⁺ at concentrations of 1 μM or more, but fails to occur below 0.1 μM. Higher concentrations of Ca²⁺ up to 10 mM have no adverse effect. Contraction is prevented in the presence of micromolar Ca²⁺ by either 1 mM of the calcium channel blocker LaCl₃ or 10 μM of the calmodulin inhibitor fluphenazine. Calcium ionophore A 23187 (1 μM) does not perturb wound contraction per se but causes the entire cytoplasm of wounded or unwounded cells to contract slowly. These data suggest that a localized influx of calcium ions at the wound edge causes major rearrangements in the distribution of cytoskeletal actin prior to contraction in Acetabularia. An involvement of calmodulin in calcium signaling is proposed.     

Anesthetic and postanesthetic effects of isoflurane on neuronal activity in the rat hippocampus

In this study, we explored anesthetic and postanesthetic effects of isoflurane on GABA-ergic and glutamatergic systems in the rat hippocampus. Our results demonstrate that different neuronal targets affected by isoflurane recover from anesthesia at dissimilar rates. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 371–373, July–October, 2007.