Microtubules in pollen tube subprotoplasts: organization during protoplast formation and protoplast outgrowth

Summary Microtubules inNicotiana tabacum pollen tube subprotoplasts reassembled in wave-like to concentric cortical arrays. Crosslinks between microtubules were either 15 or 80 nm in length. Cortical actin filaments showed different distributions; no colocalization like that in pollen tubes was observed. Degradation of actin filaments by cytochalasin D had no influence on microtubule organization. Degradation of microtubules and/or actin filaments did not affect outgrowth of the subprotoplasts. Organization of the microtubules occurred independent of the presence of the generative cell and/or the vegetative nucleus. No relation of actin filament and microtubule organization with organelle distribution could be detected.Abbreviations AFs actin filaments - DAPI 4,6-diamidino-2-phenylindole - EGTA ethylene glycol bis (2-amino ethylether) N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MTs microtubules - SPPs subprotoplasts - TRITC tetramethyl rhodamine B isothiocyanate     

Models for the length distributions of actin filaments: II. Polymerization and fragmentation by gelsolin acting together

In a previous paper, we studied elementary models for polymerization, depolymerization, and fragmentation of actin filaments (Edelstein-Keshet and Ermentrout, 1998, Bull. Math. Biol. 60, 449–475). When these processes act together, more complicated dynamics occur. We concentrate on a particular case study, using the actin-fragmenting protein gelsolin. A set of biological parameter values (drawn from the experimental literature) is used in computer simulations of the kinetics of gelsolin-mediated actin filament fragmentation.     

Jasplakinolide reversibly disrupts actin filaments in suspension-cultured tobacco BY-2 cells

Summary. Jasplakinolide is potentially a useful pharmacological tool for the study of actin organization and dynamics in living cells, since it induces actin polymerization in vitro and, unlike phalloidin, is membrane permeative. In the present work, the effect of jasplakinolide on the actin cytoskeleton of living suspension-cultured Nicotiana tabacum ‘Bright Yellow 2’ cells was investigated. Actin filaments in the living cells were disrupted by jasplakinolide. The effect of jasplakionlide on the actin cytoskeleton was concentration and time dependent. When cells were treated with a moderate concentration (150 nM) of jasplakinolide, cortical actin filaments were disrupted preferentially, whereas actin aggregated at the perinuclear region. With concentrations higher than 400 nM and exposure times longer than 30 min, actin filaments in the cell disappeared completely. The effect of jasplakinolide on the actin cytoskeleton was reversible even at high concentration. Actin bundles appeared first in the perinuclear region within 5 min, and the cortical actin array was reestablished in 15 min, suggesting that actin filaments might be organized at this region. Received July 31, 2001 Accepted December 14, 2001     

Pyrus pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen and tubes in vitro

Summary. Pears (Pyrus pyrifolia L.) have an S-RNase-based gametophytic self-incompatibility system, and S-RNases have also been implicated in self-pollen or genetically identical pollen rejection. Tip growth of the pollen tube is dependent on a functioning actin cytoskeleton. In this study, configurations of the actin cytoskeleton in P. pyrifolia pollen and effects of stylar S-RNases on its dynamics were investigated by fluorescence and confocal microscopy. Results show that actin filaments in normal pollen grains exist in fusiform or circular structures. When the pollen germinates, actin filaments assembled around one of the germination pores, and then actin bundles oriented axially throughout the shank of the growing tube. There was a lack of actin filaments 5–15 μm from the tube tip. When self-stylar S-RNase was added to the basal medium, pollen germination and tube growth were inhibited. The configuration of the actin cytoskeleton changed throughout the culturing time: during the first 20 min, the actin configurations in the self-pollen and tube were similar to the control; after 20 min of treatment, the actin filaments in the pollen tube gradually moved into a network running from the shank to the tip; finally, there was punctate actin present throughout the whole tube. Although the actin filaments of the self-pollen grain also disintegrated into punctate foci, the change was slower than in the tube. Furthermore, the alterations to the actin cytoskeleton occurred prior to the arrest of pollen tube growth. These results suggest that P. pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen grains and tubes. Correspondence: Shao-ling Zhang, College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China.     

Dynamic Actin Controls Polarity Induction de novo in Protoplasts

Cell polarity and axes are central for plant morphogenesis. To study how polarity and axes are induced de novo, we investigated protoplasts of tobacco Nicotiana tabacum cv. BY-2 expressing fluorescently-tagged cytoskeletal markers. We standardized the system to such a degree that we were able to generate quantitative data on the temporal patterns of regeneration stages. The synthesis of a new cell wall marks the transition to the first stage of regeneration, and proceeds after a long preparatory phase within a few minutes. During this preparatory phase, the nucleus migrates actively, and cytoplasmic strands remodel vigorously. We probed this system for the effect of anti-cytoskeletal compounds, inducible bundling of actin, RGD-peptides, and temperature. Suppression of actin dynamics at an early stage leads to aberrant tripolar cells, whereas suppression of microtubule dynamics produces aberrant sausage-like cells with asymmetric cell walls. We integrated these data into a model, where the microtubular cytoskeleton conveys positional information between the nucleus and the membrane controlling the release or activation of components required for cell wall synthesis. Cell wall formation is followed by the induction of a new cell pole requiring dynamic actin filaments, and the new cell axis is manifested as elongation growth perpendicular to the orientation of the aligned cortical microtubules.     

Organismic supercategories and qualitative dynamics of systems

The representation of biological systems by means of organismic supercategories, developed in previous papers (Bull. Math. Biophysics,30, 625–636;31, 59–71;32, 539–561), is further discussed. The different approaches to relational biology, developed by Rashevsky, Rosen and by Băianu and Marinescu, are compared with Qualitative Dynamics of Systems which was initiated by Henri Poincaré (1881). On the basis of this comparison some concrete result concerning dynamics of genetic system, development, fertilization, regeneration, analogies, and oncogenesis are derived.     

Cortical actin filaments in guard cells respond differently to abscisic acid in wild-type and abi1-1 mutant Arabidopsis

Cortical actin filaments in guard cells of Commelina communis L. show signal-specific organization during stomatal movements [S.-O. Eun and Y. Lee (1997) Plant Physiol 115: 1491–1498; S.-O. Eun and Y. Lee (2000) Planta 210: 1014–1017]. To study the roles of actin in signal transduction, it is advantageous to use Arabidopsis thaliana (L.) Heynh., an excellent model plant with numerous well-characterized mutants. Using an immunolocalization technique, we found that actin deployments in guard cells of A. thaliana were basically identical to those in C. communis: actin proteins were assembled into radial filaments under illumination, and were disassembled by ABA. In addition, we examined actin organization in an ABA-insensitive mutant (abi1-1) to test the involvement of protein phosphatase 2C (PP2C) in the control of actin structure. A clear difference was observed after ABA treatment, namely, neither stomatal closing nor depolymerization of actin filaments was observed in guard cells of the mutant. Our results indicate that PP2C participates in ABA-induced actin changes in guard cells. Received: 23 June 2000 / Accepted: 20 October 2000     

Polarity establishment requires dynamic actin in fucoid zygotes

Summary.  Previous work has demonstrated that actin plays important roles in axis establishment and polar growth in fucoid zygotes. Distinct actin arrays are associated with fertilization, polarization, growth, and division, and agents that depolymerize actin filaments (cytochalasins, latrunculin B) perturb these stages of the first cell cycle. Rearrangements of actin arrays could be accomplished by transport of intact filaments and/or by actin dynamics involving depolymerization of the old array and polymerization of a new array. To investigate the requirement for dynamic actin during early development, we utilized the actin-stabilizing agent jasplakinolide. Immunofluorescence of actin arrays showed that treatment with 1–10 μM jasplakinolide stabilized existing arrays and induced polymerization of new filaments. In young zygotes, a cortical actin patch at the rhizoid pole was stabilized, and in some cells supernumerary patches were formed. In older zygotes that had initiated tip growth, massive filament assembly occurred in the rhizoid apex, and to a lesser degree in the perinuclear region. Treatment disrupted polarity establishment, polar secretion, tip growth, spindle alignment, and cytokinesis but did not affect the maintenance of an established axis, mitosis, or cell cycle progression. This study suggests that dynamic actin is required for polarization, growth, and division. Rearrangements in actin structures during the first cell cycle are likely mediated by actin depolymerization within old arrays and polymerization of new arrays. Received July 15, 2002; accepted November 27, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112-0840, U.S.A.     

Long term dynamics of Rosa multiflora in a successional system

Long term studies of invasion dynamics are critical in developing a more complete understanding of the factors that influence species spread. To address this issue, the dynamics of the non-native invasive plant, Rosa multiflora, were examined using a 40-year record of successional change. The roles of biotic and abiotic factors in regulating R. multiflora invasion were also assessed. The invasion showed an initial 9-year time lag, followed by a 20-year period of population expansion and an ultimate decline as succession progressed. During all phases of R. multiflora’s invasion, there was continuous turnover within plots. Rainfall during the previous season was found to increase R. multiflora colonization during population expansion while tree species inhibited the invader’s growth. During expansion and decline of R. multiflora, common associated species were often positively or negatively correlated with changes in R. multiflora cover. Though early population dynamics were regulated by propagule pressure, the major influence on R. multiflora late in succession was canopy closure. Although the invasion of this species was largely self-limiting in this system, the species is likely to persist within late successional systems and may require management intervention.     

The 135-kDa actin-bundling protein from lily pollen tubes arranges F-actin into bundles with uniform polarity

 A plant 135-kDa actin-bundling protein (P-135-ABP) isolated from pollen tubes of Lilium longiflorum (Thunb.) binds stoichiometrically to F-actin filaments and bundles them in vitro (E. Yokota et al., 1998, Plant Physiol. 116: 1421–1429). To further understand the mechanism of actin-filament bundle formation by P-135-ABP, the polarity of each F-actin filament in bundles was examined using myosin subfragment 1 (S-1). Dissociation of F-actin filaments from bundles organized by P-135-ABP was induced by S-1. However, F-actin filaments that remained in a bundle and decorated by S-1 showed uniform polarity. These results indicate that P-135-ABP arranges F-actin filaments into bundles with uniform polarity and consequently plays a key role in the orientation of cytoplasmic streaming in pollen tubes. Received: 23 February 1999 / Accepted: 22 April 1999     

Comparison of engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> for the production of an optically pure keto alcohol

In this study, the production of enantiomerically pure (1R,4S,6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one ((−)-2) through stereoselective bioreduction was used as a model reaction for the comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli as biocatalysts. For both microorganisms, over-expression of the gene encoding the NADPH-dependent aldo-keto reductase YPR1 resulted in high purity of the keto alcohol (−)-2 (>99% ee, 97–98% de). E. coli had three times higher initial reduction rate but S. cerevisiae continued the reduction reaction for a longer time period, thus reaching a higher conversion of the substrate (95%). S. cerevisiae was also more robust than E. coli, as demonstrated by higher viability during bioreduction. It was also investigated whether the NADPH regeneration rate was sufficient to supply the over-expressed reductase with NADPH. Five strains of each microorganism with varied carbon flux through the NADPH regenerating pentose phosphate pathway were genetically constructed and compared. S. cerevisiae required an increased NADPH regeneration rate to supply YPR1 with co-enzyme while the native NADPH regeneration rate was sufficient for E. coli. Nádia Skorupa Parachin and Magnus Carlquist have contributed equally to the paper.     

Plantlet regeneration of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> var. adik-adik by tissue culture

Three color morphotypes of Kappaphycus alvarezii var. adik-adik (brown, green and red) collected from a farming area in Tictauan Is., Zamboanga City, Philippines were used as explants in the study in order to micropropagate ‘new’ plants. Individual sections of sterile Kappaphycus alvarezii var. adik-adik, initially cultured in a 48-well culture plate containing ESS/2 + E3 + PGR, released callus cells after 4–5 days of incubation at 23–25°C, 13:11H LD cycle and 10–15 μmol photons m⁻² s⁻¹ light intensity. True calli were formed after 29–35 days following dense formation of filaments or undifferentiated round cells at the medullary and inner cortical layers of the section. Plantlets (2–3 mm long) of Kappaphycus alvarezii var. adik-adik were able to regenerate after 98, 150 and 177 days in-vitro among the reds, greens, and browns, respectively. This study established successful methods for the production and regeneration of tissue explants of Kappaphycus alvarezii var. adik-adik which can possibly be used to mass produce ‘new’ cultivars for land- and sea-based nurseries as sources for commercial farming. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

A two-step procedure for adventitious shoot regeneration on excised leaves of lowbush blueberry

An efficient system to regenerate shoots on excised leaves of greenhouse-grown wild lowbush blueberry (Vaccinium angustifolium Ait.) was developed in vitro. The effect of thidiazuron (TDZ) on adventitious bud and shoot formation from apical, medial, and basal segments of the leaves was tested. Leaf cultures produced multiple buds and shoots with or without an intermediary callus phase on 2.3–4.5 μM TDZ within 6 wk of culture initiation. The greatest shoot regeneration came from young expanding basal leaf segments positioned with the adaxial side touching the culture medium and maintained for 2 wk in darkness. Callus development and shoot regeneration depended not only on the polarity of the explants but also on the genotype of the clone that supplied the explant material. TDZ-initiated cultures were transferred to medium containing 2.3–4.6 μM zeatin and produced usable shoots after one additional subculture. Elongated shoots were dipped in 39.4 mM indole-3-butyric acid powder and planted on a peat:perlite soilless medium at a ratio of 3:2 (v/v), which yielded an 80–90% rooting efficiency. The plantlets were acclimatized and eventually established in the greenhouse with 75–85% survival.     

Plant regeneration capacity of mesophyll protoplasts from Brassica napus and related species

Protoplasts from a total of thirty-six genotypes of Brassica species – B. napus, B. campestris (syn. B. rapa), B. juncea, and three distant relatives, Orychophragmus violaceus, Isatis indigotica and Xinjiang wild rape – were analysed for shoot regeneration using a feeder culture system. With the exception of B. campestris and Xinjiang wild rape, some genotypes of all the species could regenerate plants with high efficiency (above 20% of isolated calli initiating shoots). Several genotypes with high regeneration ability were elite breeding lines. Culture conditions as well as genotype had a significant impact on shoot regeneration frequency. In particular, silver nitrate added to the regeneration medium at doses of 6 and 30 μM improved shoot regeneration frequency to 25.4% and 52.2% of isolated calli, respectively, compared to 7.3% percent shoot regeneration without silver nitrate in seven responsive genotypes. Addition of silver nitrate to the regeneration medium also induced shoot regeneration in non-responsive genotypes. Intact plants could be obtained within three months from protoplast isolation in the regenerative genotypes using the current culture system. Advantages of mesophyll protoplasts as compared to protoplasts isolated from hypocotyls for genetic manipulation in Brassica species are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Actin filaments connected with the microtubules of lipotubuloids, cytoplasmic domains rich in lipid bodies and microtubules

Summary. Lipotubuloids, i.e., cytoplasmic domains containing an agglomeration of lipid bodies surrounded by half-unit membrane, entwined and held together by a system of microtubules, have been found in the ovary epidermis of Ornithogalum umbellatum. Ultrastructural studies demonstrated thin filaments in lipotubuloids that are probably actin filaments arranged parallel to microtubules. It is suggested that interaction of actin filaments with the microtubules determines the driving force for the rotary motion characteristic of lipotubuloids, as this movement is sensitive to cytochalasin B. Correspondence: Department of Cytophysiology, University of Łódź, Pilarskiego 14, 90-231 Łódź, Poland.     

Actin filaments predominate in morphogenic cell stages, whereas plaques predominate in non-morphogenic cell stages in Peronosporomycetes

We investigated the structural distribution of both types of actin arrays, filaments and plaques, in a soil-borne phytopathogenic peronosporomycete (oomycete), Aphanomyces cochlioides, under standardized host-free bioassays. The phenomenon was monitored during progression through all the asexual developmental processes of the organism. It was noted that the filamentous-form of actin was predominant during the morphogenic (morphologically active) stages of development. Conversely, during non-morphogenic (morphologically quiescent) stages, plaques dominated. From these analyses, we proposed a criterion that predominance of an actin form relates to, and precedes the morphological behaviour of a cellular stage in Peronosporomycetes. A decrease in the quantity of plaques in the encysted zoospore (non-morphogenic stage) during its developmental progression into morphogenic stages, both in germination and regeneration processes, asserted the notion that plaques function as the organization centres and are related to the reorganization of cell structure and the transition of the cell into a new stage. Furthermore, polymerization of filamentous-form during emergence stages in zoospore regeneration process revealed that filaments render motility to a developing zoospore. This unprecedented function of filaments in the developing zoospores was demonstrated using nicotinamide (0.8 × 10⁻⁶ m), which did not cause actin disruption, but could induce zoospore encystment, and its further replacement with water triggered the zoospore emergence process. Additionally, by using latrunculin B, an actin polymerization inhibitor, we also demonstrated the functional necessity of actin during various developmental processes in Aphanomyces.     

Cytoskeletal alterations in interphase cells of the green alga Spirogyra decimina in response to heavy metals exposure: I. The effect of cadmium

Summary. The aim of the study was to elucidate the effect of cadmium ions on the arrangement of the actin and tubulin cytoskeleton, as well as the relationships between cytoskeletal changes and growth processes in the green filamentous alga Spirogyra decimina. Batch cultures of algae were carried out under defined conditions in the presence of various cadmium concentrations. In control cells, the cytoskeleton appeared to be a transversely oriented pattern of both microtubules and actin filaments of various thickness in the cell cortex; colocalization of cortical microtubules and actin filaments was apparent. Microtubules were very sensitive to the presence of cadmium ions. Depending on the cadmium concentration and the time of exposure, microtubules disintegrated into short rod-shaped fragments or they completely disappeared. A steep increase in cell width and a decrease in growth rate accompanied (and probably ensued) a very rapid disintegration of microtubules. Actin filaments were more stable because they were disturbed several hours later than microtubules at any cadmium concentration used. When cadmium ions were washed out, the actin cytoskeleton was rebuilt even in cells in which actin filaments were completely disintegrated at higher cadmium concentrations (40 or 100 μM). The much more sensitive microtubules were regenerated after treatment with lower cadmium concentrations (10 or 15 μM) only. Correspondence and reprints: Centre of Phycology, Institute of Botany, Academy of Sciences of the Czech Republic, Dukelská 135, 379 82 Třeboň, Czech Republic.     

A mathematical model for the dynamics of large membrane deformations of isolated fibroblasts

In this paper we develop and extend a previous model of cell deformations, initially proposed to describe the dynamical behaviour of round-shaped cells such as keratinocytes or leukocytes, in order to take into account cell pseudopodial dynamics with large amplitude membrane deformations such as those observed in fibroblasts. Beyond the simulation (from a quantitative, parametrized model) of the experimentally observed oscillatory cell deformations, a final goal of this work is to underline that a set of common assumptions regarding intracellular actin dynamics and associated cell membrane local motion allows us to describe a wide variety of cell morphologies and protrusive activity. The model proposed describes cell membrane deformations as a consequence of the endogenous cortical actin dynamics where the driving force for large-amplitude cell protrusion is provided by the coupling between F-actin polymerization and contractility of the cortical actomyosin network. Cell membrane movements then result of two competing forces acting on the membrane, namely an intracellular hydrostatic protrusive force counterbalanced by a retraction force exerted by the actin filaments of the cell cortex. Protrusion and retraction forces are moreover modulated by an additional membrane curvature stress. As a first approximation, we start by considering a heterogeneous but stationary distribution of actin along the cell periphery in order to evaluate the possible morphologies that an individual cell might adopt. Then non-stationary actin distributions are considered. The simulated dynamic behaviour of this cytomechanical model not only reproduces the small amplitude rotating waves of deformations of round-shaped cells such as keratinocytes [as proposed in the original model of Alt and Tranquillo (1995, J. Biol. Syst. 3, 905–916)] but is furthermore in very good agreement with the protrusive activity of cells such as fibroblasts, where large amplitude contracting/retracting pseudopods are more or less periodically extended in opposite directions. In addition, the biophysical and biochemical processes taken into account by the cytomechanical model are characterized by well-defined parameters which (for the majority) can be discussed with regard to experimental data obtained in various experimental situations.     

Survivorship of a monocarpic bamboo grass,Sasa kurilensis,during the early regeneration process after mass flowering

The survivorship of a monocarpic bamboo grass,Sasa kurilensis, during the early regeneration process was documented by a 10 year observation of the seedling population after mass flowering in the Hakkoda Mountains, northern Japan. Three phases were recognized: the establishment, density-stable and thinning phases. The mortality of the densely germinated seedlings (932.9m⁻² in aBetula ermanii forest and 1222.3 m⁻² in aSasa grassland) was high, up to 0.5 year⁻¹, in the establishment phase (0–1 year after germination) and low in the density-stable phase (1–3 years after germination). After reaching full density state, the seedling population showed a nearly constant mortality of 0.18 year⁻¹ due to self-thinning (the thinning phase). The high C/F ratio presumably caused suppressed seedlings to die. Recovery of theS. kurilensis population was estimated to requireca 20 years in the study plots, judging from the height growth and the decrease in culm density of the seedling population. The illuminance on the ground was higher in the flowered population than in the unflowered one for 5 years after mass death. The duration of high ground illuminance is an important factor affecting the dynamics of forests withSasa undergrowth, because tree seedlings need to establish under high ground illuminance for the successful regeneration of the forests.