Fine structural observations of embryo development inPelargonium XHortorum bailey: with normal and mutant plastids

Summary The ultrastructural changes in the cotyledon, radicle and suspensor haustorium ofPelargonium, containing either normal or mutant plastids, are investigated from the heart stage of embryogenesis to the mature seed. The fine structure of parenchymatous cells from the cotyledon and radicle is essentially similar whereas that of the suspensor haustorium is very different.The cotyledon and radicle develop into one massive storage tissue possessing numerous lipid and several protein bodies per cell, and well developed starch grains. The suspensor haustorium has no storage function, rather it acts as a transitory tissue which dies off as the seed matures. The extensive chloroplast development suggests that, in addition to its traditional role, the suspensor haustorium also acts as a photosynthetic booster for the developing embryo.The development of surviving mutant embryos is similar to normal ones except that in cotyledon and radicle cells plastids develop only to vesicles, which associate into loose prolamellar bodies and sometimes small fenestrated thylakoids, and in the suspensor haustorium cells, only to small compact grana.     

Fine structural observations of the cotyledons in germinating seeds ofPelargonium XHortorum Bailey: With normal and mutant plastids

Summary The fine structural changes in cotyledon cells of germinatingPelargonium seeds are studied on the first to fifth day of seedling emergence. Initially there is a rapid change in the cell fine structure, marked most conspicuously by the progressive liberation of the lipid and protein food reserves and the formation of an extensive thylakoid system within the plastids, but gradually the cells start to senesce. The subcellular changes in mutant cells are similar except that the plastids lack the usual thylakoid system and develop only deranged prolamellar bodies. They may store starch and they possess plastoglobuli, but seem not to contain plastid ribosomes. In rare mixed cells normal and mutant plastids remain quite distinct.     

Mitochondrial DNA decreases during pollen development in rapeseed (Brassica napus L.), but mitochondrial linear-plasmid-encoded RNA polymerase persists in mature pollen

Summary. Mitochondrial DNA in the male reproductive cells of rapeseed (Brassica napus L.) was monitored by fluorescence microscopy of Technovit 7100 resin sections double-stained with 4,6-diamidino-2-phenylindole and 3,3-dihexyloxacarbocyanine iodide. Mitochondrial DNA progressively decreased during pollen development and disappeared in mature pollen. This result corresponds well with the maternal inheritance of mitochondria in rapeseed determined by previous genetic analyses. To better characterize the mode of inheritance of the mitochondrial linear plasmid in rapeseed, which is transmitted through pollen, we analyzed by indirect immunofluorescence microscopy the expression and localization of ORF6 protein, a putative RNA polymerase encoded by the plasmid. ORF6 protein was expressed in mature pollen and specifically localized in the cytoplasm of sperm cells in the mature pollen. This suggests that the genes encoded by the plasmid DNA are transcribed in the mature pollen by its own RNA polymerase (ORF6 protein) and that the gene expression in the generative cells may be needed for transmission of plasmid DNA through the pollen.Present address: Laboratory of Plant Molecular Biology, Nara Institute of Science and Technology, Ikoma, Nara, Japan.Correspondence and reprints: Department of Plant Biotechnology, National Institute of Agrobiological Sciences, 2-1-2 Kan-non-dai, Tsukuba 305-8602, Japan.     

The light-induced reduction of the gravitropic growth-orientation of seedlings of Arabidopsis thaliana (L.) Heynh. is a photomorphogenic response mediated synergistically by the far-red-absorbing forms of phytochromes A and B

Hypocotyls of dark-grown seedlings of Ara bidosis thaliana exhibit a strong negative gravitropism, which is reduced by red and also by long-wavelength, far-red light treatments. Light treatments using phytochrome A (phyA)- and phytochrome B (phyB)-deficient mutants showed that this response is controlled by phyB in a red/far-red reversible way, and by phyA in a non-reversible, very-low-fluence response. Crosses of the previously analyzed phyB-1 allele (in the ecotype Landsberg erecta background) to the ecotype Nossen wild-type (WT) background resulted in a WT-like negative gravitropism in darkness, indicating that the previously described gravitropic randomization observed with phyB-1 in the dark is likely due to a second mutation independent of that in the PHYB gene.Abbreviations FR long-wavelength far-red light - phyA phytochrome A (holoprotein) - phyB phytochrome B (holoprotein) - Pr red-absorbing form of phytochrome - WT wild type We thank Dr. A. Nagatani (RIKEN Institute, Wako-City, Japan) and Dr. M. Furuya (Hitachi, Hatoyama, Japan) for the phyA-201/phyB-5 double mutant. The work was supported by Deutsche Forschungsgemeinschaft and Human Frontier Science Program grants to E.S.     

Heat shock pre-treatment enhances the response of Arabidopsis thaliana leaves and Cucurbita pepo cotyledons to benzyladenine

The effects of heat shock (HS) pre-treatment on the response tobenzyladenine were studied in two plant model systems (1) retardation ofsenescence of Arabidopsis thaliana L. Heyhn rosette leavesand (2) induction of greening of detached Cucurbita pepoL.cotyledons. N⁶-benzyladenine (BA) retarded senescence of rosetteleaves of Arabidopsis thaliana (L) Heyhn and briefpre-treatment with HS (3 at 37)essentially enhanced this cytokinin effect. BA stimulated cotyledon greening inCucurbita pepo L due to the activation of chlorophyllsynthesis. Brief cotyledon pre-heating at moderate temperatures (3 at 33–35) also enhanced thiscytokinin effect.     

Fungal coil formation ofRhizoctonia repens in seedlings ofGaleola septentrionalis (orchidaceae)

Protocorms or protocorms with roots of an achlorophyllous orchidGaleola septentrionalis were inoculated with isolates ofRhizoctonia repens, R. solani, andRhizoctonia spp. The seedlings were infected with eight of twelve isolates ofR. repens. Fungal coils were formed in the cells, which was suggestive of a symbiotic association. The other isolates caused soft rot or no infection to the protocorms or the protocorms with a root. Contribution No. 97, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.     

Gestörte Thylakoidbildung in Chloroplasten einer xantha-Mutante von Arabidopsis thaliana (L.) Heynh.

Zusammenfassung In den Palisadenzellen von Keim- und Rosettenblättern einer rezessiven xantha-Mutante von Arabidopsis thaliana fehlt den Plastiden die für normale, grüne Chloroplasten typische Schichtenstruktur. Licht- und elektronenmikroskopische Untersuchungen ergaben, daß sich dieser genabhängige Differenzierungsdefekt während der Plastidenentwicklung beim Übergang von den Tubuli des Prolamellarkörpers zu Thylakoiden manifestiert. Es entstehen aus dem persistierenden Prolamellarkörper nur einzelne abnorme Thylakoide, die zuweilen napfoder taschenartig verformt oder zu Magnograna vereinigt sein können. Besonders charakteristisch für die Mutante aber ist, daß solchen Thylakoiden häufig parallel laufende Züge einzelner Tubuli oder Fibrillen beidseitig dicht aufliegen. Auf Grund Dieser Anordnung wird die Möglichkeit eines Thylakoidwachstums durch laminalen Einbau von Tubuli erörtert.In den Blättern der Mutante wurden nur Carotinoide, jedoch keine Chlorophylle festgestellt. Zugleich finden sich in den entwicklungsgehemmten Plastiden, neben Phytoferritin-Partikeln, zahlreiche osmiophile Globuli sowie Matrixeinschlüsse unbekannter Natur. Daher wird die Frage diskutiert, ob die beobachteten Störungen der Thylakoid-Differenzierung in der Mutante auf den Chlorophyllmangel oder auf eine blockierte Strukturproteid-Synthese zurückgehen.Alle übrigen Zellorganelle sind in der Mutante ebenso gestaltet wie in der grünen Normalform. Besonders wird auf das Vorkommen von dense bodies hingewiesen und ihre Beziehung zu Sphärosomen erörtert.

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Disturbed thylakoid-formation in chloroplasts of a xantha-mutant of Arabidopsis thaliana (L.) Heynh.

Summary A monohybrid recessive xantha-mutant of Arabidopsis thaliana was grown aseptically on mineral agar +2% sucrose up to maturity. The cotyledons and rosette leaves were non-green during all developmental stages, their yellow colour bleaching with age.Palisade cells of various leaves of mutant plants were investigated light- and electron microscopically. All of the organelles of these cells, except the plastids, had a normal appearance. Special attention was paid to the existence of dense bodies, and their relationship to sphaerosomes and lysosomes was outlined.In the mutant plastids the typical thylakoid differentiation present in the chloroplasts of the green Arabidopsis plant is entirely lacking. Within the palisade cells the plastids are normal in number and size. Under the light microscope they appear as hyaline vesicles with usually two primary grana. Their outline is characterized by numerous invaginations and protrusions; however, it is still uncertain to what extent this amoeboidy is only an artifact.The specific gene dependent block in the plastid development is apparently effective during the conversion of tubules of the prolamellar body to thylakoids. From the persistent prolamellar bodies in the mutant only single abnormal thylakoids originate. These often have a deformed cup- or ventricle-like shape and occasionally are piled up as magnograna. In a characteristic manner tubules and fibrils are closely associated in parallel rows with the outsides of the thylakoids. The fibrils having a mean diameter of 75 Å are connected with the tubules which are derived from the prolamellar body. From this striking aggregation the possibility of a laminal fusion of tubules as a process of normal thylakoid growth is deduced.By pigment analysis of mutant leaves no chlorophyll but only carotenoids were detected. However, many osmiophilic globules as well as large bodies of unknown composition (proteinaceous?) appear in the plastid matrix. Therefore the question is discussed whether the disturbances of the thylakoid differentiation is due primarily to a blocked chlorophyll formation or to an inhibition of the synthesis of some structural membrane proteids.Phytoferritin particles which up to now have not been observed either in normal or in mutant plastids of Arabidopsis were frequently found in the plastid matrix of this xantha-mutant.

     

Organellar DNA synthesis in permeabilized soybean cells

Summary Cultured cells of Glycine max (L.) Merr. v. Corsoy were permeabilized by treatment with L--lysophosphatidylcholine (LPC). The permeabilized cells were capable of uptake and incorporation of deoxynucleoside triphosphates into DNA. Incorporation of exogenous nucleotides into DNA was linear for at least 90 minutes and the initial rate of incorporation approached 50% of the theoretical in vivo rate of DNA synthesis. However, DNA synthesis in the permeabilized cells was unaffected by the potent DNA polymerase inhibitor, aphidicolin. Analysis of newly synthesized DNA by molecular hybridization revealed that only organellar DNA was synthesized by the permeabilized cells. The LPC treated cells were also permeable to a protein as large as DNase I. The permeabilized cells were capable of RNA and protein synthesis as indicated by incorporation of radiolabeled UTP and leucine, respectively, into acid-precipitable material.     

Species ofUrocystis (Ustilaginomycetes) onAnemone in Japan

Three species ofUrocystis onAnemone (Ranunculaceae) are reported based on comparative morphology with specimens collected in Japan.Urocystis anemones, U. japonica, andU. pseudoanemones sp. nov. are separated by the number of ustilospores and sterile cells surrounding the ustilospores in the spore balls. Morphological characteristics, host plants and geographical distribution of these three species are also reported. Contribution No. 148, Laboratory of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan.     

Xenorhabdus japonicus sp. nov. associated with the nematode Steinernema kushidai

A new species, Xenorhabdus japonicus, is proposed as the bacterial symbiont of Steinernema kushidai isolated from field soil in Shizuoka Prefecture, Japan. Xenorhabdus japonicus could be distinguished phenotypically and genetically from other Xenorhabdus spp. The type strain of the species, SK-1, a Gram-negative, facultative anaerobe and peritrichously flagellated rod, has colonies with primary and secondary forms. The strain can be differentiated from the type strain of Xenorhabdus nematophilus by several characters, including the formation of arginine dehydrolase, phenylalanine deaminase and lysine decarboxylase, the assimilation of inosine and L-proline and acid production from inositol. The major cellular fatty acids are 16:0, cyclo 17:0 and 18:1. The ubiquinone system is Q-8. The G+C content of DNA is 45.9 mol%. The DNA of strain SK-1 has 20 to 58% homology with that of the type strains of other Xenorhabdus spp.Y. Nishimura, A. Hagiwara and T. Suzuki are with the Department of Applied Biological Science, Science University of Tokyo, Noda 278, Japan, and SDS Biotech K.K., Tsukuba Technology Centre, Tsukuba 300-26, Japan     

Changes in Ultrastructure of Phaseolus Vulgaris L. Cotyledons Associated with their Modulated Life Span

French bean (Phaseolus vulgaris L.) cotyledons lost most of their reserve substances during several early days of germination and turned green. In cotyledon mesophyll cells of one-week-old seedlings, plastids were represented predominantly by amyloplasts (starch grains) and chloroamyloplasts, and the cells appeared to be metabolically highly active. Cell heterogeneity associated with distance of the cells from cotyledon vascular bundles was evident. Only mesophyll cells near to the bundles were rich in plastids. In two-weeks-old intact bean plants, the cotyledons were yellow and shrunken, and their cells were nearly "empty". The plastids in them were represented by senescent plastids (gerontoplasts) only. In the gerontoplasts as well as freely in cytosol, fluorescent lipoid inclusions were accumulated. This cotyledon development was more or less independent of irradiance. In "decapitated" bean plants, senescence of mesophyll cells and plastids was slowed down considerably, and the life span of the cotyledons was prolonged.     

Clathrin is involved in organization of mitotic spindle and phragmoplast as well as in endocytosis in tobacco cell cultures

Summary. We previously identified a 175 kDa polypeptide in Lilium longiflorum germinating pollen using a monoclonal antibody raised against myosin II heavy chain from Physarum polycephalum. In the present study, the equivalent polypeptide was also found in cultured tobacco BY-2 cells. Analysis of the amino acid sequences revealed that the 175 kDa polypeptide is clathrin heavy chain and not myosin heavy chain. After staining of BY-2 cells, punctate clathrin signals were distributed throughout the cytoplasm at interphase. During mitosis and cytokinesis, clathrin began to accumulate in the spindle and the phragmoplast and then was intensely concentrated in the cell plate. Expression of the C-terminal region of clathrin heavy chain, in which light chain binding and trimerization domains reside, induced the suppression of endocytosis and the formation of an aberrant spindle, phragmoplast, and cell plate, the likely cause of the observed multinucleate cells. These data strongly suggest that clathrin is intimately involved in the formation of the spindle and phragmoplast, as well as in endocytosis. Correspondence and reprints: Department of Life Science, Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo 678-1297, Japan. Present address: RIKEN Plant Science Center, Yokohama, Kanagawa, Japan.     

Transmission of plastids by pollen in Antirrhinum majus

Summary Up to now Antirrhinum was classified as a typical example for a uniparentalmaternal inheritance of the plastids. However, the findings reported here prove that also the male gametophyte of Antirrhinum may occasionally transmit plastids into the egg. This conclusion is based on genetic experiments involving a form of the plastom mutant prasinizans which is described as gelbgrüne prasinizans. In contrast to all other plastid mutations known in Antirrhinum majus this mutant originated in Sippe 50 is completely viable. In plants containing plastids of this mutant type only, the mutant character is manifested during early growth stages. Cotyledons and first foliage leaves which are initially white or white yellow, slowly turn green and become indistinguishable from normal Sippe 50. Reciprocal crosses of green Sippe 50 with gelbgrüne prasinizans gave few variegated descendants; the others were exclusively plants identical with the maternal parent as far as leaf colour is concerned (Table). The variegated individuals cannot be gene mutants since selfing and crossing experiments showed non-mendelian inheritance. Furthermore it could be ruled out that in the cross Sippe 50 x gelbgrüne prasinizans the three variegated descendants represent spontaneous new plastom mutants because the pale tissue in these plants turned green in the same way as the paternal parent. Because of the typical greening of this mutant and since plastid mutations could be ruled out we have to conclude that plastids were transmitted by the pollen parent into the egg. There these plastids multiplied together with the maternal plastids giving rise to the chimeras after sorting-out of the two plastid types. This interpretation is supported by the observation of mixed cells in tissues where the leaf variegation is finely mosaiced. The results were possible only because the plastids of the pollen parent can be unequivocally recognised.     

Vesicular-arbuscular mycorrhizal fungi in coastal dune plant communities II. Spore formation ofGlomus spp. predominates under geographically separated patches ofElymus mollis

Five study sites in Japan with pure patches ofElymus mollis near to the shoreline were chosen. Abundance of spore formation ofGlomus spp. underE. mollis patches were studied at four sites to understand the factors determining the development of VA mycorrhizal fungal communities at the edge of coastal sand dune vegetation. At each study site, eight soil samples from two soil depths were collected four times (three times for Niigata) during a year. Spores ofGlomus spp. predominated at every site, but species composition ofGlomus spp. differed from site to site. In a further experiment to measure the soil depth of the highest spore densities underE. mollis, the highest density was found at depths where rhizomes ofE. mollis were present.Contribution No. 119, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Conjoined mitochondria and plastids in the barley mutant ‘albostrians’

The intercalary meristem and surrounding tissues of the gene induced plastome mutant albostrians of Hordeum vulgare L. were examined in the electron microscope for ultrastructural evidence of membrane continuities between plastids and mitochondria. In well developed tissues the ribosome-deficient plastids were usually in close proximity or appressed to mitochondria of normal appearance. In some sections through the meristemmatic region however the relationship between the two organelles was observed to be of a fused nature. These conjoinings are thought to be similar to those reported in normal living cells using cinephotomicrography but never before observed by transmission electron microscopy.     

Host plant suitability of a recently naturalized thistle Cirsium vulgare (Asteraceae) for a phytophagous ladybird beetle,Epilachna pustulosa (Coleoptera: Coccinellidae)

The phytophagous ladybird beetle, Epilachna pustulosa Kôno occurs mainly on the thistle Cirsium kamtschaticum Ledeb. In recent years, we have confirmed feeding and oviposition by E.pustulosa, and some degree of larval development of this beetle, on a naturalized congeneric thistle, Cirsium vulgare (Savi) Tenore. In this study, we assessed the host suitability of C.vulgare for E.pustulosa, using larval rearing and simple food choice tests, by comparing the performance on, and preference for, their legitimate host C.kamtschaticum. All experiments were initiated in July, when the utilization of C.vulgare by E.pustulosa became obvious. For larval performance, E.pustulosa showed a lower eclosion rate, shorter developmental duration and smaller body size when reared on C.vulgare than on C.kamtschaticum. Cirsium vulgare appeared to be a less adequate larval food than C.kamtschaticum. Adult feeding preference was altered seasonally; females preferred C.kamtschaticum to C.vulgare in a food choice test conducted in early July, whereas they showed the opposite preference in tests conducted in mid and late July. The utilization of C.vulgare by E.pustulosa appeared to be induced by qualitative deterioration of C.kamtschaticum leaves in July, which resulted in increased dispersion of adult beetles.     

Notes on three Japanese Agaricales

Three species of Agaricales from Japan are treated here. 1) From the examination of the types and additional specimens,Lyophyllum nigrescens is conspecific withHydropus nigrita from Cuba. 2)Hydropus kansaiensis sp. nov. from Otsu appears close toTrogia mellea from Malaysia. 3)Alnicola lactariolens sp. nov. from Otsu is a distinctive species in this genus because of the purple color of its spore print.This work has been supported by a grant to one of us (H. C.) by the Japanese Society for the Promotion of Science in 1988/89.     

Recognizing scientific excellence in the biology of cell adhesion

The prestigious 2005 Japan Prize for Cell Biology has been awarded to Dr. Masatoshi Takeichi, Director of RIKEN Developmental Biology, Kobe, Japan, and Dr. Erkki Ruoslahti, Distinguished Professor, The Burnham Institute, La Jolla, USA for their "Fundamental contribution in elucidating the molecular mechanisms of cell adhesion". The award is scheduled to be presented to the scientists in ceremonies in Tokyo on April 20, 2005 as part of a week-long celebration of "Japan Prize Week".     

Endosymbiotic Microflora of the Vestimentiferan Tubeworm (<Emphasis Type="Italic">Lamellibrachia</Emphasis> sp.) from a Bathyal Cold Seep

Gutless vestimentiferan tubeworms are known to harbor endosymbiotic bacteria in a specialized tissue, the trophosome, which consists of lobules. The endosymbionts of vestimentiferans inhabiting sulfide-rich hydrothermal vents are monospecific for their host. In contrast, previous studies suggest that vestimentiferas of methane-rich seeps may host multispecific symbionts. Phylogenetic analysis and dot-blot hybridization of 16S ribosomal RNA genes (16S rDNA) detected 4 operational taxonomic units (OTUs) in the trophosome of the vestimentifera Lamellibrachia species from a bathyal methane-seep. The OTUs were closely related to 16S rDNA of the species belonging to -Proteobacteria (Sulfitobacter), -Proteobacteria (Janthinobacterium), and -Proteobacteria (Acinetobacter and Pseudomonas). Localizations of the 4 OTUs within the trophosome were confirmed by in situ hybridization (ISH). ISH signals of the -proteobacterial OTU were observed in the innermost zone of the trophosome lobules. In contrast, ISH signals of the - and -proteobacterial OTUs were observed at the periphery of the lobules; however, whether they occur inside or outside the lobules remains unclear. These results support the possibility that the studied methane-seep tubeworm has a microflora composed of multispecific endosymbionts. Present address (Yukimasa Higashide): Noto Marine Center, 3-47 Uchiura, Suzu, Ishikawa 927-0552, Japan Present address (Hiroyuki Kimura): Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 6 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki 305-8566 Japan