Asymmetric synthesis of all stereoisomers of 6-methylpipecolic acids

Summary.  Asymmetric synthesis of all four stereoisomers of 6-methylpipecolic acids with high enantiomeric purity via iterative AD reaction, starting from 1,6-heptadiene, has been described. Received March 25, 2002 Accepted June 15, 2002 Published online January 30, 2003 Authors' address: Hiroki Takahata, Faculty of Pharmaceutical Sciences, Tohoku Pharmaceutical University, Sendai 981-8558, Japan, E-mail: takahata@tohoku-pharm.ac.jp RID="*" ID="*"  The stereo-configurations of 8 and 9 in Fig. 3 and 12 in Fig. 4 show only major isomers. RID="**" ID="**"  The absolute configurations of 11 and 14     

Structure, morphology, and composition of silicon biocomposites in the palm tree Syagrus coronata (Mart.) Becc.

Summary.  Syagrus coronata is an economically important palm tree grown as an ornament, for the oil extracted from its seeds, and the wax from its leaves which has several applications in industry. Silicon biocomposites were analyzed in leaves of S. coronata. Silica bodies were found as extracellular silica masses between the hypodermal-layer cell walls and in granules present in the vacuoles of palisade cells. Scanning electron microscopy of the hypodermal layer of cells showed a collection of spherical bodies embedded in enveloping cavities that outlined the general structure of the bodies. Globular subunits with sharp edges formed the spherical bodies that ranged from 6 to 10 μm in diameter (average, 7.8 μm). X-ray microanalysis detected only silicon and oxygen homogeneously distributed throughout the bodies. Vacuoles of palisade cells contained a large number of granules ranging from 20 nm to 1.2 μm in size (average, 300 nm). Transmission electron microscopy associated with electron spectroscopic imaging and electron energy loss spectroscopy were used to determine the elemental composition of the granules. Vacuolar granules were amorphous and composed of silicon and oxygen, suggesting they consist of amorphous silica biominerals. No nitrogen, indicative of organic matter, was detected in the granules. Received November 26, 2001; accepted July 1, 2002; published online October 31, 2002 RID="*" ID="*" Correspondence and reprints: Departamento de Microbiologia Geral, Instituto de Microbiologia Professor Paulo de Góes, Centro de Ciências da Saude, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazil.     

Localization of p210-related proteins in green flagellates and analysis of flagellar assembly in the green alga Dunaliella bioculata with monoclonal anti-p210

Summary.  Recently, p210 was identified as a component of the flagellar basal apparatus in the green flagellate Spermatozopsis similis. In a search for potential homologues to p210, isolated cytoskeletons of several green flagellates were probed with a monoclonal antibody, BAS4.13, against p210. In Western blots, cross-reacting bands in the molecular-mass range of 210 kDa were detected only in the quadriflagellate Spermatozopsis exsultans. As described earlier for S. similis, the flagellar transition region was decorated in Chlamydomonas reinhardtii and several other green flagellates, whereas in the marine alga Dunaliella bioculata the antigen was present in the proximal part of the axoneme. Double immunofluorescence of D. bioculata with an antitubulin antibody further revealed dotlike signals at sites where the probasal bodies are located. Since most of the antigen in D. bioculata was located in the axoneme, deflagellation offered a possibility to study the kinetics of its incorporation during flagellar regeneration. The antigen was only detected after a flagellum reached a length of 3–4 μm and its integration into the growing flagellar proceeded from proximal to distal. A similar delay in the incorporation of the antigen was also observed during flagellar assembly on new basal bodies during cell division. Thus, the antigen of BAS4.13 was incorporated late and from proximal to distal into the growing flagellum. We conclude that the pace and site by which individual proteins are integrated into the flagellum differ greatly. Received February 18, 2002; accepted May 17, 2002; published on line October 31, 2002 RID="*" ID="*" Correspondence and reprints: Botanisches Institut, Universit?t zu K?ln, Gyrhofstrasse 15, 50931 K?ln, Federal Republic of Germany     

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures

Summary.  In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H₂O₂ is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H₂O₂. Superoxide dismutase (5 U/ml) induced an increase in H₂O₂ production by 22.2%. This indicates that at least part of the H₂O₂ is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H₂O₂ under copper stress for 90%. The influence of the pH on the H₂O₂ production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress. Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan 171, 2020 Antwerp, Belgium.     

Inhibition of neutral Mg2+-dependent sphingomyelinase by ubiquinol-mediated plasma membrane electron transport

Summary.  Sphingomyelin is an abundant constituent of the plasma membranes of mammalian cells. Ceramide, its primary catabolic intermediate, has emerged as an important lipid signaling molecule. Previous work carried out by our group has documented that plasma membrane Mg²⁺-dependent neutral sphingomyelinase can be effectively inhibited by exogenous ubiquinol. In this work, we have tested whether or not plasma-membrane-associated electron transport can also achieve this inhibition through endogenous ubiquinol. Our results have shown that Mg²⁺-dependent neutral sphingomyelinase in isolated plasma membranes was inhibited by NAD(P)H under conditions where ubiquinone is reduced to ubiquinol. This inhibition was potentiated in the presence of an extra amount of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2). Depletion of plasma membranes from lipophilic antioxidants by solvent extraction abolished the inhibition by reduced pyridine nucleotides without affecting the sensitivity of the neutral sphingomyelinase to exogenous ubiquinol. Reconstitution of plasma membranes with ubiquinone restored the ability of NAD(P)H to inhibit the enzyme. Our results support that the reduction of endogenous ubiquinone to ubiquinol by NAD(P)H-driven electron transport may regulate the activity of the plasma membrane neutral sphingomyelinase. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="**" ID="**" Present address: Department of Biomedical Engineering, School of Medicine, University of Baltimore, Maryland, U.S.A. RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio C-6, Campus Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

The Commelina yellow mottle virus promoter drives companion-cell-specific gene expression in multiple organs of transgenic tobacco

Summary.  Previous work has demonstrated that some endogenous plant gene promoters are active in selective companion cells of the phloem, depending on organ types and developmental stages. Here we report that the Commelina yellow mottle virus (CoYMV) promoter is active in the companion cells of leaves, stems and roots of transgenic Nicotiana tabacum cv. Xanthi NN, using β-glucuronidase (GUS) as a reporter. Thus, the CoYMV promoter has a broad organ specificity. This promoter can be useful in molecular studies on the functions of companion cells in many aspects of phloem biology, such as regulation of long-distance transport, macromolecular traffic, plant development and interaction with pathogens. It may also be useful in engineering crops that produce specific gene products in the companion cells to block long-distance movement of pathogens. Received February 5, 2002; accepted March 27, 2002; published online July 4, 2002 RID="*" ID="*" Correspondence and reprints: Department of Plant Biology and Plant Biotechnology Center, 207 Rightmire Hall, Ohio State University, 1060 Carmack Road, Columbus, OH 43210, U.S.A.     

Differential localisation of GFP fusions to cytoskeleton-binding proteins in animal, plant, and yeast cells

Summary.  The structure and functioning of the cytoskeleton is controlled and regulated by cytoskeleton-associated proteins. Fused to the green-fluorescent protein (GFP), these proteins can be used as tools to monitor changes in the organisation of the cytoskeleton in living cells and tissues in different organisms. Since the localisation of a specific cytoskeleton protein may indicate a particular function for the associated cytoskeletal element, studies of cytoskeleton-binding proteins fused to GFP may provide insight into the organisation and functioning of the cytoskeleton. In this article, we focused on two animal proteins, human T-plastin and bovine tau, and studied the distribution of their respective GFP fusions in animal COS cells, plant epidermal cells (Allium cepa), and yeast cells (Saccharomyces cerevisiae). Plastin-GFP localised preferentially to membrane ruffles, lamellipodia and focal adhesion points in COS cells, to the actin filament cytoskeleton within cytoplasmic strands in onion epidermal cells, and to cortical actin patches in yeast cells. Thus, in these 3 very different types of cells plastin-GFP associated with mobile structures in which there are high rates of actin turnover. Chemical fixation was found to drastically alter the distribution of plastin-GFP. Tau-GFP bound to microtubules in COS cells and onion epidermal cells but failed to bind to yeast microtubules. Thus, animal and plant microtubules appear to have a common tau binding site which is absent in yeast. We conclude that the study of the distribution patterns of microtubule- and actin-filament-binding proteins fused to GFP in heterologous systems should be a valuable tool in furthering our knowledge about cytoskeleton function in eukaryotic cells. Received January 12, 2002; accepted March 7, 2002; published online June 24, 2002 RID="*" ID="*" Correspondence and reprints (present address): Institute of Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Federal Republic of Germany. Abbreviation: smRS-GFP soluble modified red-shifted GFP.     

Strict paternal transmission of mitochondrial DNA of Chlamydomonas species is explained by selection against maternal nucleoids

Summary.  The non-Mendelian inheritance of organelle DNA is common in most plants and animals. Here we examined inheritance mechanisms involved in the transfer of mitochondrial DNA. We successively backcrossed (to F₅) two interfertile strains of the unicellular isogamous haploid algae Chlamydomonas reinhardtii and Chlamydomonas smithii to match nuclear backgrounds and examine transmission patterns of mitochondrial DNA by PCR analysis of cob gene sequences. Mitochondrial DNA was strictly transmitted paternally. To investigate the behavior of parental mitochondrial DNA, we used F₅ progeny to form zygotes and isolated single zygotes. The results showed selective disappearance of maternal mitochondrial nucleoids occurred between 3 and 6 h after zygote formation. Received July 11, 2002; accepted September 28, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan.     

Proton conduction through full-length gp91phox requires histidine 115

Summary.  The NADPH oxidase of neutrophils is a transmembrane electron transfer complex, containing a flavin adenine dinucleotide and two hemes, all of which are suggested to be contained within gp91 ^phox , one of four subunits of the enzyme. The transfer of electrons through the NADPH oxidase is associated with an efflux of protons. gp91 ^phox has previously been demonstrated to function as the proton conduction pathway. The mutation of histidines 111, 115, and 119 to leucines and of histidine 115 to leucine within the N-terminal 230-amino-acid fragment of gp91 ^phox has previously been demonstrated to result in the loss of proton conduction through this N-terminal fragment. In this paper we have investigated the role of these histidines in proton conduction by the full-length gp91 ^phox . Stable CHO cell lines were established which expressed full-length gp91 ^phox in which histidines 111, 115, and 119 had been mutated to leucines (CHO91H111/115/119) and in which histidine 115 had been mutated to leucine (CHO91H115L). The expression of gp91 ^phox and its cellular localisation in these cell lines were comparable between wild-type and the mutant gp91 ^phox . The mutation of histidines 111, 115, and 119 to leucines or just histidine 115 to leucine resulted in an almost total loss of both the arachidonate-activated influx and efflux of protons, in comparison with that observed for wild-type gp91 ^phox . Therefore, histidine 115 is required for proton conduction by both full-length gp91 ^phox and the N-terminal 230-amino-acid fragment of gp91 ^phox . Histidine 115 has recently been proposed to act as a coordinating ligand for the outer heme iron of the NADPH oxidase. On the basis of observations for cytochrome c oxidase, we propose a model for this dual role of histidine 115. Received May 2, 2002; accepted July 26, 2002; published online May 21, 2003 RID="**" ID="**" Present address: Bristol Institute for Transfusion Sciences, Bristol, United Kingdom. RID="*" ID="*" Correspondence and reprints: Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, United Kingdom.     

Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves

Summary.  The plant plasma membrane (PM) contains more than one b-type cytochrome. One of these proteins has a rather high redox potential (can be fully reduced by ascorbate) and is capable of transporting electrons through the PM. Four genes encoding proteins with considerable homology to the sequences of cytochrome b ₅₆₁ proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b ₅₆₁ located in the Arabidopsis PM, first PM vesicles were purified by aqueous polymer two-phase partitioning from the leaves of 9-week-old A. thaliana. PM proteins were solubilized by nonionic detergent, and the fully ascorbate-reducible b-type cytochrome was partially purified by anion-exchange chromatography steps. Potentiometric redox titration of the fraction, containing the fully ascorbate-reducible b-type cytochrome after the first anion-exchange chromatography step, revealed the presence of two hemes with redox potentials of 135 mV and 180 mV, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing the fully ascorbate-reducible b-type cytochrome after the second anion-exchange chromatography step revealed the presence of a single polypeptide band at about 120 kDa. However, heat treatment (15 min, 90 °C) before electrophoresis was able to split the 120 kDa band into two bands with molecular masses of about 23 and 28 kDa. These values are lower than the apparent molecular mass for the fully ascorbate-reducible b-type cytochrome purified from Phaseolus vulgaris hypocotyls (about 52 kDa) but are in good agreement with those characteristic for the cytochrome b ₅₆₁ proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis genome (24–31 kDa). Received May 4, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Institute of Biophysics, BRC, Hungarian Academy of Sciences, POB 521, 6701 Szeged, Hungary.     

A marking technique for live fish eggs and larvae

 A new marking technique for live fish eggs and larvae was proposed to elucidate the larval biology and adult breeding ecology of wild fish. In the laboratory, females of a freshwater goby Rhinogobius sp. OR were abdominally injected with one of three coloring agents—brilliant blue FCF, rose Bengal, or β-carotene—before their oviposition. The rose Bengal proved lethal to adult fish. The other two dyes had little effect on adult mortality. With these two treatments, there were negative effects on neither fecundity nor egg mortality, resulting in normally developed larvae. The brilliant blue FCF stained eggs and larvae greenish blue whereas the staining effect of β-carotene was unclear. The timing of injection was important in effective staining of eggs and reducing the risk of miscarriage. In conclusion, the brilliant blue FCF was the more useful marker. We discuss what this method can show us about the ecology of wild fish and how this method can be applied to field study. Received: March 6, 2002 / Revised: July 11, 2002 / Accepted: August 14, 2002 RID="*" ID="*" Present address: Center for Marine Environmental Studies, Ehime University, 3 Bunkyo-cho, Matsuyama 790-8577, Japan (e-mail: nokuda@sci.ehime-u.ac.jp) Acknowledgments I am grateful to K. Karino, M. Kohda, and A. Moriyama for giving us valuable advice and to M. Inoue and H. Miyatake for their field assistance. This study was financially supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. Correspondence to:Noboru Okuda     

Multiple activities of a novel substance,dictyopyrone C isolated from Dictyostelium discoideum,in cellular growth and differentiation

Summary.  We report that a novel substance named dictyopyrone C (DPC) has remarkable effects on growth and differentiation of Dictyostelium discoideum Ax-2 cells, in a dose-dependent manner. In the presence of 3–15 μM DPC, differentiation of starving Ax-2 (clone MS) cells was greatly enhanced in submerged culture, when vegetative MS cells were harvested at the mid-late-exponential growth phase (>3 × 10⁶ cells per ml) and starved. In contrast, DPC above 30 μM markedly impaired the progression of differentiation including cell aggregation, most of starved cells being round after 3–4 h of DPC application and then lysed during further incubation. In the presence of 30 μM DPC however, MS cells that had been harvested at the early exponential growth phase (<5 × 10⁵ cells per ml) and starved became neither round nor lysed and exhibited rather enhanced differentiation. Essentially the same results were obtained in cultures of starved cells on nonnutrient agar. With respect to the DPC effect on MS cells growing in axenic medium, cell lysis and growth inhibition by DPC at concentrations higher than 15 μM were realized in the mid-late-exponential-growth-phase cells (>3 × 10⁶ cells per ml) but not in the early-exponential-growth-phase cells (<5 × 10⁵ cells per ml). Moreover, analysis using synchronized MS cells has demonstrated that the DPC effect changes in a cell-cycle-dependent manner. In contrast to such unique DPC actions, the pyrone ring of DPC had no effects on growth and differentiation within the range of 3–120 μM tested. These findings strongly suggested the importance of the combined structure of the pyrone ring and the linear carbon chain in revelation of the DPC activities. Received August 5, 2002; accepted November 11, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan. E-mail: ymaeda@mail.cc.tohoku.ac.jp     

Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers

Summary.  Methods for cryogenic fixation, freeze substitution, and embedding were developed to preserve the cellular structure and protein localization of secondary-wall-stage cotton (Gossypium hirsutum L.) fibers accurately for the first time. Perturbation by specimen handling was minimized by freezing fibers still attached to a seed fragment within 2 min after removal of seeds from a boll still attached to the plant. These methods revealed native ultrastructure, including numerous active Golgi bodies, multivesicular bodies, and proplastids. Immunolocalization in the context of accurate structure was accomplished after freeze substitution in acetone only. Quantitation of immunolabeling identified sucrose synthase both near the cortical microtubules and plasma membrane and in a proximal exoplasmic zone about 0.2 μm thick. Immunolabeling also showed that callose (β-1,3-glucan) was codistributed with sucrose synthase within this exoplasmic zone. Similar results were obtained from cultured cotton fibers. The distribution of sucrose synthase is consistent with its having a dual role in cellulose and callose synthesis in secondary-wall-stage cotton fibers. Received August 19, 2002; accepted November 12, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131, U.S.A. E-mail: candace.haigler@ttu.edu     

Salicylic acid changes the properties of extracellular peroxidase activity secreted from wounded wheat (Triticum aestivum L.) roots

Summary.  Wheat (Triticum aestivum L.) roots released proteins showing peroxidase activity in the apoplastic solution in response to wound stress. Preincubation of excised roots with 1 mM salicylic acid at pH 7.0 enhanced the guaiacol peroxidase activity of the extracellular solution (so-called extracellular peroxidase). The soluble enzymes were partially purified by precipitation with ammonium sulfate followed by size exclusion and ion exchange chromatography. Despite an increase in the total activity of secreted peroxidase induced by pretreatment of excised roots with salicylic acid, the specific activity of the partially purified protein was significantly lower compared to that of the control. Purification of the corresponding proteins by ion exchange chromatography indicates that several isoforms of peroxidase occurred in both control and salicylic acid-treated samples. The activities of the extracellular peroxidases secreted by the salicylic acid-treated roots responded differently to calcium and lectins compared with those from untreated roots. Taken together, our data suggest that salicylic acid changes the isoforms of peroxidase secreted by wounded wheat roots. Received June 10, 2002; accepted September 24, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Institute of Biochemistry and Biophysics, Russian Academy of Sciences, P.O. Box 30, Kazan 420111, Russia.     

Cytological evidence for preservation of mitochondrial and plastid DNA in the mature generative cells of Chlorophytum spp. (Liliaceae)

Summary.  Following 4′,6-diamidino-2-phenylindole staining of mature pollen grains of Chlorophytum comosum, fluorescence microscopy confirmed that cytoplasmic nucleoids (DNA aggregates) were present in the generative cells, which indicated the possibility of biparental cytoplasmic inheritance. Electron and immuno-electron microscopy showed that both plastids and mitochondria were present in the generative cells, and both organelles contained DNA. These results indicate that mitochondria and plastids of C. comosum have the potential for biparental inheritance. Similar results were obtained with mature pollen grains of C. chinense. Therefore, we conclude the coincident biparental inheritance for mitochondria and plastids in the members of the genus Chlorophytum. Received June 28, 2002; accepted September 26, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: College of Life Science, Peking University, Bejing 100871, People's Republic of China.     

Solution phase synthesis of the 14-residue peptaibol antibiotic trichovirin I

Summary.  The 14-residue peptaibol antibiotic trichovirin I 4A of the structure Ac-Aib-L-Asn-L-Leu-Aib-L-Pro-L-Ala-L-Val-Aib-L-Pro-Aib-L-Leu-Aib-L-Pro-L-Leuol (Aib = α-aminoisobutyric acid, Leuol = leucinol) was synthesized by stepwise conventional solution phase synthesis using the Z/OtBu(OMe) strategy and HOBt/EDC as coupling reagents. Intermediates were fully characterized and the identity of the synthetic peptide with the component 4A of the natural, microheterogeneous peptide mixture was proven by electrospray mass spectrometry, HPLC, and bioassay. Received March 25, 2002 Accepted June 14, 2002 Published online December 18, 2002 RID="*" ID="*"  Dedicated to Prof. Dr. Günther Jung. Tübingen University, on the occasion of his 65^th anniversary. Authors' address: Prof. Dr. Hans Brückner, Interdisciplinary Research Center, Institute of Nutritional Science, Department of Food Sciences, Justus-Liebig-University of Giessen, Heinrich-Buff-Ring 26, D-35392 Giessen, Germany, Fax: +49-641-99-39149, E-mail: hans.brueckner@ernaehrung.uni-giessen.de Abbreviations: Amino acids are abbreviated according to three-letter-nomenclature; Aib, α-aminoisobutyric acid (2-methylalanine); Iva (isovaline, 2-ethylalanine); Leuol, L-leucinol [(S)-2-amino-4-methyl-1-pentanol]; AAA, amino acid analysis; EI-MS, electron impact mass spectrometry; ESI-MS, electrospray ionization mass spectrometry; HPLC, high performance liquid chromatography; Z, benzyloxycarbonyl; Fmoc, 9-fluorenylmethyoxycarbonyl; OtBu, tertiary butoxy (tert-butylester); OMe, methoxy (methyl ester); OBzl, benzyloxy (benzyl ester); TDM, N,N,N′,N′-tetramethyl-4,4′-diamino-diphenylmethane (Arnold's base); for other abbreviations see Experimental.     

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.     

A novel mechanism of silicon uptake

Summary.  Crystal-like structures in vacuoles, precipitates in the cytoplasm and on the tonoplast membrane have been found to store remarkable amounts of Si in a number of higher plants. In most of the cases the final storage product is a SiO₂ gel. Accumulation inside the cells presumes a membrane and cytoplasm passage, driven by unknown transporters. Beside this uptake into the cytoplasm, Si-accumulating species possess a mechanism that does not involve a membrane and cytoplasm passage. Unusual small invaginations comprising the two membranes, plasmalemma and tonoplast, which enclose a small border of cytoplasm, were observed. The same cells contained vacuolar vesicles surrounded by two membranes, obviously derived from the invaginations. By energy-dispersive X-ray analysis and electron spectroscopic imaging, Si was shown in the invaginations and vacuolar vesicles. This novel endocytotic process allows the uptake of condensed, higher-molecular-weight Si compounds. In Zn hyperaccumulators, frequently SiO₂ precipitates were found in different cell compartments. Such plants showed the same invaginations and vacuolar vesicles, but Zn, colocalized with Si, was detected in these structures. Electron energy loss spectra confirmed the assumption that Zn-silicate is present in the vesicles. In the vacuoles the unstable Zn-silicate is degraded, forming SiO₂ precipitates, while the released Zn is bound to an unknown partner. Received January 22, 2002; accepted July 2, 2002; published online October 31, 2002 RID="*" ID="*" Correspondence and reprints: Institute of Plant Biochemistry, Weinberg 3, 06120 Halle, Federal Republic of Germany. Abbreviations: EELS electron energy loss spectroscopy; EDX energy-dispersive analysis of X-rays; ESI electron spectroscopic imaging.