Iron-containing particles accumulate in organelles and vacuoles of leaf and root cells in the nicotianamine-free tomato mutantchloronerva

Summary The mutantchloronerva ofLycopersicon esculentum Mill is the only known plant mutation that leads to a complete loss of the endogenous iron chelator nicotianamine. The mutant exhibits several morphological alterations and a permanent activation of the strategy I reactions of iron uptake as well as iron accumulation in roots and leaves. The electron microscopic energy loss technique of energy spectroscopic imaging (ESI) was used to localise the iron accumulated in the organs of wild-type and mutant plants. Iron-containing particles were detected in the chloroplast stroma and in vacuoles of mutant leaves, and in root cells in vacuoles and in mitochondria. In wild-type organs such particles were found at the same sites but they were smaller in size and occurred less frequently. The findings indicate that these compartments are preferential sites of iron storage or deposition in tomato tissues. It is discussed that the iron-containing particles detected are the result of iron release by oxidative stress. Application of nicotianamine to mutant plants, which reverts the mutant phenotype, led to a significant decrease of the iron-containing particles. This is seen as an indication that they may serve as intermediate iron stores and emphasises the crucial role of nicotianamine for the normal iron distribution in cells and organs.Dedicated Prof. Dr. K. Müntz on the occasion of his 65th birthday     

Association of particles that contain double-stranded RNAs with algal chloroplasts and mitochondria

Linear dsRNAs (double-stranded RNAs) belonging to several distinct size classes were found to be localized in chloroplasts and mitochondria of Bryopsis spp., raising the possibility that these dsRNAs are prokaryotic in nature. The algal cytosol and nuclei did not contain dsRNAs. The amount of the dsRNAs in the organelles appeared constant, and there were about 500 copies per chloroplast. The four major dsRNAs from Bryopsis chloroplasts were about 2 kbp (kilobase pairs) in length and originated from discrete isometric particles of about 25 nm diameter. These virus-like particles were purified by CsCl density gradient centrifugation after extraction from isolated chloroplasts with chloroformbutanol and subsequent precipitation with polyethylene glycol. They had a buoyant density of about 1.40 g · cm^–3 and contained four major and three minor proteins. Mitochondrial dsRNAs were about 4.5 kbp in length and formed less-stable particles of about 40 nm in diameter with a buoyant density of 1.47 g · cm^–3. Some observations support the hypothesis that vertical transmission of the protein-coated, non-infectious dsRNAs occurs within cell organelles. Double-stranded RNAs of various sizes were found in most green, red, and brown algae. The characteristics of the algal dsRNAs are compared with those of dsRNAs from higher plants and the biological significance of the dsRNAs in cell organelles is discussed.Abbreviations dsRNA double-stranded RNA - kbp kilobase pairs - SDS sodium dodecyl sulfate - SSC 0.15 M NaCl 0.015 M sodium citrate - PAGE polyacrylamide gel electrophoresis The authors would like to express their gratitude to Dr. T. Natsuaki, Utsunomiya University, and Dr. D. Hosokawa, Tokyo University of Agriculture and Technology, for their helpful suggestions throughout this research. They are also much indebted to Dr. B. Wang, Institute of Genetics, Academia Sinica, Beijing, PRC, for his suggestions on rice dsRNA, and to Dr. T. Kohbara, Senshu University, on Bryopsis cells. Sincere thanks are also due to Dr. T. Misonou, Yamanashi University, and Dr. K. Masuda, Akita Prefectural College of Agriculture, for supplying plant materials; to Dr. N. Sonoki, Azabu University, for nucleotide analysis of dsRNAs; and to Y. Koshino for technical assistance. This research was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

Mating System and Clonal Architecture: A Comparative Study in Two Marine Angiosperms

In the present study, we compared the clonal architecture between two seagrass species, the dioecious Cymodocea nodosa and the hermaphroditic, self-compatible Zostera noltii, in order to verify the hypothesis that clonal growth strategies and resulting genet architecture are associated with mating system in clonal plants. It is expected that self-incompatible species should be associated to a guerrilla growth form, because of pollen limitation due to obligate outbreeding, while the ecologically advantageous phalanx strategy can be adopted in self-compatible species. Genotypic diversity and heterozygosity were also assessed in the two species. Sampling has been conducted in mixed stands, collecting shoots of the two species at the same points of the sampling grid, in order to even out any effects of environmental heterogeneity. Species-specific microsatellite loci have been used as molecular markers to identify clones and assess their spatial distribution in both species. As expected, we found an intermingled configuration of genets in the dioecious C. nodosa while Z. noltii was characterized by a clumped, `phalanx-type' distribution of clones. C. nodosa was characterized by higher genotypic diversity with regard to Z. noltii, while heterozygosity levels were comparable in the two species. Coordinating Editor: Dr J. Tuomi     

Endoplasmic reticulum derived decorated tubules in the sieve elements ofNymphaea

Summary Bundles of decorated tubules found in the sieve elements ofNymphaea have been studied with the transmission electron microscope. Comparatively straight tubules (100 nm in diameter) arise from the endoplasmic reticulum during early stages of sieveelement development and subsequently associate into bundles of up to 100 tubules that parallel the longitudinal cell axis. From the start of their formation the tubules are structurally distinct from other ER profiles due to their dense decoration with particles. High magnifications reveal an orderly array of the particles (about 24 surround a 100 nm tubule) and suggest a modification of their membrane so that it is no longer dissolvable into a regular three-layered structure. Later during sieve-element ontogeny the decorated tubules get invaginated by smooth ER membranes, thereby squeezing out the intratubular (extracytoplasmic) space. As a result a double mantle is formed that surrounds a plasmatic cylinder. Decorated 100 nm tubules with inner membranes are present in enucleate mature sieve elements ofNymphaea alba andN. tuberosa. Considerably larger tubules (about 200 nm in diameter) were found inN. Candida andN. tetragona and occasionally also inNuphar and Barclaya, two other genera from the same family. The decoration of the tubules and their subsequent invagination by smooth membranes are discussed with respect to the controlled autolysis of sieve elements.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Molecular characterization of a seed transmitted clavicipitaceous fungus occurring on dicotyledoneous plants (Convolvulaceae)

Ergoline alkaloids (syn. ergot alkaloids) are constituents of clavicipitaceous fungi (Ascomycota) and of one particular dicotyledonous plant family, the Convolvulaceae. While the biology of fungal ergoline alkaloids is rather well understood, the evolutionary and biosynthetic origin of ergoline alkaloids within the family Convolvulaceae is unknown. To investigate the possible origin of ergoline alkaloids from a plant-associated fungus, 12 endophytic fungi and one epibiotic fungus were isolated from an ergoline alkaloid-containing Convolvulaceae plant, Ipomoea asarifolia Roem. & Schult. Phylogenetic trees constructed from 18S rDNA genes as well as internal transcribed spacer (ITS) revealed that the epibiotic fungus belongs to the family Clavicipitaceae (Ascomycota) whereas none of the endophytic fungi does. In vitro and in vivo cultivation on intact plants gave no evidence that the endophytic fungi are responsible for the accumulation of ergoline alkaloids in I. asarifolia whereas the epibiotic clavicipitaceous fungus very likely is equipped with the genetic material to synthesize these compounds. This fungus resisted in vitro and in vivo cultivation and is seed transmitted. Several observations strongly indicate that this plant-associated fungus and its hitherto unidentified relatives occurring on different Convolvulaceae plants are responsible for the isolated occurrence of ergoline alkaloids in Convolvulaceae. This is the first report of an ergot alkaloid producing clavicipitaceous fungus associated with a dicotyledonous plant.Data deposition: The sequences reported in this paper have been deposited in the GenBank (accession numbers are given in the text and in Fig. 3) Dedicated to Dr. Dr. h. c. mult. Albert Hofmann, the great pioneer of ergot research, on the occasion of his 100th birthday     

Dynamics of activity and structure of the tonoplast vacuolar-type H+-ATPase in plants with differing CAM expression and in a C3 plant under salt stress

Summary Differences in the activity and structure of the vacuolar H⁺-ATPase (V-ATPase, EC 3.6.1.3) were investigated in the C₃/CAM intermediate plantKalanchoë blossfeldiana Poellnitz cv. Tom Thumb, with lower or higher expression of CAM, andHordeum vulgare cv. Carina, grown with or without 150 mM NaCl. InK. blossfeldiana ATP-hydrolysis and H⁺-transport activity were higher with higher expression of CAM than in plants with very weak CAM. This was mainly due to a larger amount of V-ATPase. Statistical analysis of the diameter of intramembrane particles (IMPs) on freeze-fractures of tonoplast vesicles showed that IMPs were larger in tonoplast vesicle preparations ofK. blossfeldiana with strong CAM expression (9.1 nm) than in preparations ofK. blossfeldiana with low CAM expression (7.3 nm). As there is evidence that the majority of IMPs on freeze-fractures of tonoplast vesicles corresponds to the V₀ domain of V-ATPase, the higher activity of V-ATPase inK. blossfeldiana with stronger CAM could be a result of additional structural changes in its membrane-integral domain. The higher activity of V-ATPase inK. blossfeldiana with stronger CAM is discussed in relation to the requirement for a higher proton pumping capacity for nocturnal malate accumulation in the vacuole. The ATP-dependent H⁺-pumping activity inH. vulgare was higher under salt stress than in control plants, while the rates of ATP-hydrolysis and the size of IMPs were not affected by the salt treatment. The data presented here indicate that different mechanisms might increase the transport capacity of V-ATPase to meet the higher requirements of secondary active transport related to CAM expression and adaptation to salt stress.Abbrevations ATP adenosine triphosphate - CAM crassulacean acid metabolism - IMP intramembrane particles - V-ATPase vacuolar proton-translocating adenosine triphosphatase - V₀ domain membrane-integral domain of V-ATPase - V₁ domain membrane-peripheral domain of V-ATPase Dedicated to Prof. Dr. Eberhard Schnepf on the occasion of his retirement     

Die Fagus-Sippen Europas und ihre geographisch-soziologische Korrelation zur Verbreitung der Assoziationen des Fagion s.l.

Studies of the beeches and beech woods of eastern central Europe revealed, that in the postglacial period not only Fagus sylvatica (L.) emend. reimmigrated from the refugial territories in the SE-as has generally been accepted, but also the transitional taxa, which originated from hybrids with F. orientalis Lipsky. The NW area limit of these intermediate taxa must be revised. The presence in central Europe of these taxa-which form own Fagion alliances and associations in the SE (Fagus intermedia ssp. moesiaca and ssp. taurica) as well as the presence of other southeastern species in central European beach woods shows, that their postglacial development is parallel to, but different from other areas. The taxon Fagus intermedia (ssp. neglecta and ssp. transitus) became differential taxa of a central European region of the Fagion medioeuropaeum. Plant sociologists are therefore requested to make new and critical analyses of the beech woods in Europe, with special attention to the Fagus taxa, in order to establish in detail the geographical distribution and phytosociological significance of Fagus intermedia.The author offers to determine or revise Fagus material (herbarium collections).

Danksagung. Ich möchte meinen tiefsten und allzeitigen Dank besonders den Herren Prof. Dr. A. O. Horvat (Pécs), Prof. Dr. Ch. Moulopoulos, Prof. Dr. B. Jovanovié, Prof. Dr. I. Dumitriu-Tataranu, Prof. Dr. R. Bornkamm, Prof. Dr. M. A. Kotschkin, Prof. Dr. P. Fukarek, Prof. Dr. K. Browicz für gewährte Unterstützung aussprechen und ganz besonders unserem unvergeßlichen verstorbenen Prof. Dr. Drs. h.c. R. Tüxen, der mir zur Fortführung dieser Studien Mut machte.     

Expression of enzymatically active and correctly targeted strictosidine synthase in transgenic tobacco plants

Strictosidine, a precursor to over 1000 indole alkaloids including the anti-tumor drugs vinblastine, vincristine, and camptothecin, is produced by the condensation of tryptamine and secologanin. Strictosidine synthase, the enzyme responsible for this condensation, is the first committed step in the indole-alkaloid pathway. We have introduced a modified cDNA encoding Strictosidine synthase from Catharanthus roseus (L.) Don. (McKnight et al. 1990, Nucl. Acids Res. 18, 4939) driven by the CaMV 35S promoter into tobacco (Nicotiana tabacum L.). Transgenic tobacco plants expressing this construct had from 3 to 22 times greater strictosidinesynthase activity than C. roseus plants. Ultrastructural immunolocalization demonstrated that strictosidine synthase is a vacuolar protein in C. roseus and is correctly targeted to the vacuole in transgenic tobacco. Immunoblot analysis of strictosidine synthase showed that two distinct forms of the enzyme were produced in transgenic tobacco plants but that only a single form was made in C. roseus. This observation indicates that the second form of the protein is not simply a result of overexpression in tobacco, but may reflect differences in protein processing between tobacco and C. roseus.Abbreviations cDNA complementary DNA - TLC thin-layer chromatography We thank Dr. C.A. Roessner for providing the E. coli strain expressing strictosidine synthase, Dr. J. Balsevich for providing alkaloid standards, and Dr. L. Cloney for assisting with antibody preparation. This work was supported by a National Institutes of Health Biomedical Research Support Grant to T.D.M and by a grant from the US Department of Agriculture, Competitive Research Grants Office (90-37262-5375) to C.L.N.     

A new putative cellulose-synthesizing complex ofColeochaete scutata

Summary Cells of the charophycean alga,Coleochaete scutata active in cell wall formation were freeze fractured in the search for cellulose synthesizing complexes (TCs) since this alga is considered to be among the most advanced and a progenitor to land plant evolution. We have found a new TC which consists of two geometrically distinctive particle complexes complementary to one another in the plasma membrane and occasionally associated with microfibril impressions. In the E-fracture face is found a cluster of 8–50 closely packed particles, each with a diameter of 5–17 nm. Most of these particles are confined within an 80 nm circle. In the P-fracture face is found an 8-fold symmetrical arrangement of 10 nm particles circumferentially arranged around a 28 nm central particle. The TCs ofC. scutata are quite distinctive from the rosette/globule TCs of land plants. The 5.5×3.1 nm microfibril inC. scutata is also distinctive from the 3.5×3.5 nm microfibril typical of land plants. The phylogenetic implications of this unique TC in land plant evolution are discussed.     

Established cell lines ofDrosophila hydei

Summary Four cell lines have been isolated fromDrosophila hydei embryos. Three lines have a normal XY karyotype, the fourth has an XO karyotype with an additional small heterochromatic fragment. The cells contain presumable cytoplasmic virus like particles. This work was supported by a travel grant to Dr. N. H. Lubsen from the Netherlands Organization for the Advancement of Pure Research (Z. W. O.).     

Tn5 insertion mutants of Pseudomonas sp. 267 defective in siderophore production and their effect on clover (Trifolium pratense) nodulated with Rhizobium leguminosarum bv. trifolii

Pseudomonas sp. strain 267 isolated from soil promoted growth of different plants under field conditions and enhanced symbiotic nitrogen fixation in clover under gnotobiotic conditions. This strain produced pyoverdine-like compound under low-iron conditions and secreted vitamins of the B group. The role of fluorescent siderophore production in the beneficial effect of strain 267 on nodulated clover plants was investigated. Several non-fluorescent (Pvd^-) Tn5 insertion mutants of Pseudomonas sp. strain 267 were isolated and characterized. The presence of Tn5 insertions was confirmed by Southern analysis of EcoRI digested genomic DNA of each derivative strain. The siderophore-negative mutants were compared to the parental strain with respect to their growth promotion of nodulated clover infected with Rhizobium leguminosarum bv. trifolii 24.1. We found that all isolated Pvd^- mutants stimulated growth of nodulated clover plants in a similar manner to the parental strain. No consistent differences were observed between strain 267 and Pvd^- derivatives strains with respect to their plant growth promotion activity under gnotobiotic conditions.Dr Deryto died in august 1994     

Genetic transformation through the use of hyperhydric tobacco meristems

Exposed shoot meristems from normal and hyperhydric (vitrified) tobacco, Nicotiana tabacum, were bombarded with gold particles either coated with plasmid DNA containing neomycin phosphotransferase (NPTII), rolC and -glucuronidase (GUS) genes (plasmid pGA-GUSGFrolC) or left uncoated. Meristems bombarded with uncoated particles were co-cultivated with Agrobacterium tumefaciens strain EHA 101 harboring the binary vector pGA-GUSGFrolC. Whole-plant transformants were produced from 4 of 40 hyperhydric meristems bombarded with uncoated particles followed by co-cultivation with A. tumefaciens. One transgenic plant was obtained from 40 normal, non-hyperhydric meristems treated. Transformation was verified by growth on kanamycin-containing medium, GUS assays, PCR, and Southern analysis. The plants tested through Southern analysis appeared to have 2 or more copies of the transgene insert. Seeds obtained from self-pollination of these transgenic plants segregated 3:1 or 15:1 (kanamycin resistant:sensitive) when germinated on medium containing 100 mg/l kanamycin, indicating transfer of foreign genes through the sexual cycle. Whole-plant transformants were not produced from 50 normal tobacco meristems bombarded with plasmid-coated gold particles and not exposed to engineered A. tumefaciens, but 1 plant of 60 bombarded hyperhydric meristems produced transgenic roots, the result of a chimera. We suggest that hyperhydric meristems are more readily transformed.     

Variable karyotype with B-chromosomes in Bellevalia saviczii (Liliaceae)

Bellevalia saviczii (Liliaceae), a hexaploid (2n = 6x = 24), was collected from mountain slopes in the Shiraz valley, southern Iran. The basic karyotype consists of one long metacentric chromosome, a slightly shorter acrocentric, and two shorter submetacentrics. In 32% of the plants, one and sometimes two of the acrocentrics (IIa) appeared with a pericentric inversion that changed it into a metacentric (IIm). This metacentric IIm was present in a Hardy--Weinberg ratio throughout the collection range of more than 300 km. A number of aneuploids were found, pentasomics [2n – 1 = 23 (4.9%)], heptasomics [2n + 1 = 25 (2.5%)], and one octasomic [2n + 2 = 26 (0.3%)]. Re-assorted karyotypes with 2n = 24, but with odd numbers of two of the chromosomes, were also present (6.6%). B-chromosomes were found in root meristems and in pedicels in a number (19.1%) of the plants that grew around a 30 km marsh. Their numbers ranged from 1B to 8Bs per plant peaking in the 2B mode. The B-chromosomes (two polymorphs with terminal and near-terminal sticking points) were telocentric with a few metacentric iso-Bs. No B-carrying plants were found in the drier regions away from the marsh. The B-frequency distribution for aneuploids and pericentric inversions was much different than for the standard euploid. Dr Mark Gettner died on November 2nd, 2002. He is fondly remembered by his family, his many students and friends for his kindness and generosity, and for his dedication to his scientific work. Dr Broun is the son-in-law of Dr Gettner and he will be the corresponding author for this article (pb22@york.ac.uk).     

Transfer of a marine DNA virus fromEctocarpus toFeldmannia (Ectocarpales,Phaeophyceae): aberrant symptoms and restitution of the host

Summary The marine brown algaEctocarpus siliculosus is invaded by a polyhedric virus, whose genome consists of circular, double-stranded DNA. In laboratory experiments this virus can infect a different host species,Feldmannia simplex. InfectedFeldmannia plants show severe somatic malformations. However, no functional virus particles are formed. SuchFeldmannia plants recover to resume a normal, symptom-free appearance. This result raises the possibility of intergeneric gene transfer in the natural habitat.     

Synthesis and properties of glutathione reductase in stressed peas

We have subjected peas (Pisum sativum L.) to four different oxidative stresses: cold conditions (4 °C) in conjunction with light, treatment with paraquat, fumigation with ozone, and illumination of etiolated seedlings (greening). In crude extracts of leaves from stressed plants, an increase (up to twofold) in activity of glutathione reductase (GR) was observed which was consistent with previous reports from several laboratories. In all cases, except for ozone fumigation, the increase in activity was not due to an elevation in the steady-state levels of GR protein. None of the applied stresses had any effect on steady-state levels of GR mRNA. In contrast to the small increase in GR activity, the K _m of GR for glutathione disulphide showed a marked decrease when determined for extracts of stressed leaves, compared with that from unstressed plants. This indicates that GR from stressed plants has an increased affinity for glutathione disulphide. The profile of GR activity bands fractionated on non-denaturing acrylamide gels varied for extracts from differently stressed leaves and when compared with GR from unstressed plants. The changes in GR-band profiles and the alteration in the kinetic properties are best explained as changes in the isoform population of pea GR in response to stress.Abbreviations GR glutathione reductase - GSSG glutathione disulphide - Rubisco Ribulose-1,5-bisphosphate carboxylase-oxygenase - RNase A/T1 ribonucleases A and T1 We are grateful to Prof. Alan Wellburn and Dr. Phil Beckett (Division of Biological Sciences, University of Lancaster, UK) for providing ozone-fumigated material and Dr. Jeremy Harbinson for providing material grown at 4° C. This work was supported by a grant-in-aid to the John Innes Institute from the Agricultural and Food Research Council. E.A.E. and C.E. gratefully acknowledge the support of a John Innes Foundation studentship and a European Molecular Biology Organisation Fellowship respectively.     

Effect of osmotic stress on thylakoid fine structure inPorphyra umbilicalis

Summary The fine structural organization of thylakoid membranes in intact cells ofPorphyra umbilicalis, an intertidal red alga, was studied using the freeze-fracture method with special emphasis on changes induced by hypo- and hyperosmotic stresses. In osmotically adapted plants the density of intramembraneous particles on the PF-face increases considerably in the osmotic range from 5-fold diluted to 6-fold concentrated artifical seawater medium ASP₁₂, while that on the EF-face remains constant. The size of the particles on both fracture faces decreases strongly from extreme hypoosmotic to extreme hyperosmotic stress. These findings are discussed with relation to their biological significance.The author is member of the Arbeitsgemeinschaft für Elektronenmikroskopie an der Tierärztlichen Hochschule Hannover.     

Constitutive expression of an inducible lipoxygenase in transgenic tobacco decreases susceptibility to Phytophthora parasitica var. nicotianae

Plant lipoxygenases (LOXs) are key enzymes involved in the generation of fatty acid derivatives, called oxylipins. In tobacco, LOX gene expression and activity are very low in healthy tissues and are highly enhanced in response to infection by Phytophthora parasitica nicotianae and to elicitor treatment. We previously showed, using antisense-LOX1 plants, that expression of the tobacco LOX1 gene is required for the race-cultivar specific resistance of tobacco to Phytophthora parasitica nicotianae. In order to investigate the effect of over-expressing a LOX gene on plant resistance, we transformed tobacco plants with the LOX1 coding sequence fused to the CaMV 35S promoter. Four transgenic lines with enhanced levels of LOX protein and specific activity over control plants were selected for further analysis. These plants were macroscopically indistinguishable from WT plants. Upon stem inoculation, the sense-LOX1 plants displayed a significantly decreased susceptibility to virulent races of Phytophthora parasitica nicotianae, stem lesions being 2- to 3-fold shorter in the transgenic lines than in WT plants. Using a root inoculation assay, the survival rate of sense-LOX1 seedlings was increased about 4-fold compared to their WT counterparts, with 60 to 80% of transgenic plants vs 15 to 20% of WT controls remaining healthy following inoculation with Phytophthora parasitica nicotianae. This is the first demonstration that the over-expression of a LOX gene is sufficient to reduce the susceptibility of a host plant to an oomycete pathogen.