Isolation,culture and regeneration of protoplasts from birdsfoot trefoil (Lotus corniculatus)

A method was developed for rapid plant regeneration from protoplasts of birdsfoot trefoil (Lotus corniculatus L. cv. Leo). Green cotyledons from in vitro grown seedlings were preplasmolyzed in CPW salts containing 13% mannitol (CPW 13 M) for 1 h prior to the enzyme treatment. The enzyme formula consisted of 2% (w/v) Onozuka Cellulase R-10, 1% (w/v) Macerase and 0.1% (w/v) Pectolyase Y-23 in CPW 13 M. This method produced high yields of viable protoplasts after purification. The procedure is reproducible and takes approximately 2.5 months from protoplast isolation to plantlet establishment in a greenhouse. More than 100 plantlets were grown in soil. Two somaclonal variants, a chimeric plant for chlorophyll production and an albino cell line, have been obtained by this procedure.     

Genotype-specific response of a lycaenid herbivore to elevated carbon dioxide and phosphorus availability in calcareous grassland

Effects of elevated CO₂ and P availability on plant growth of the legume Lotus corniculatus and consequences for the butterfly larvae of Polyommatus icarus feeding on L. corniculatus were investigated in screen-aided CO₂ control chambers under natural conditions on a calcareous grassland in the Swiss Jura mountains. Elevated CO₂ conditions and P fertilisation increased the biomass production of L. corniculatus plants and affected the plant chemical composition. CO₂ enrichment increased the C/N ratio and sugar concentration and decreased the N and P concentrations. C- and N-based allelochemicals (cyanoglycosides, total polyphenols and condensed tannins) were only marginally affected by CO₂ enrichment. P fertilisation increased the specific leaf area and concentrations of water, N, sugar and P, while the C/N ratio and the concentration of total polyphenols decreased. Furthermore, P availability marginally enhanced the effect of elevated CO₂ on the total dry mass and sugar concentration while the opposite occurred for the total polyphenol concentration. The changes in food-plant chemistry as a result of P fertilisation positively affected larval mass gain and accelerated the development time of P. icarus. Only a marginal negative effect on larval mass gain was found for CO₂ enrichment. However, we found genotype-specific responses in the development time of P. icarus to elevated CO₂ conditions. Larvae originating from different mothers developed better either under elevated CO₂ or under ambient CO₂ but some did not react to CO₂ elevation. As far as we know this is the first finding of a genotype-specific response of an insect herbivore to elevated CO₂ which suggests genetic shifts in insect life history traits in response to elevated CO₂.     

Cheese whey: an alternative growth and protective medium for<Emphasis Type="Italic"> Rhizobium loti</Emphasis> cells

Cheese whey (CW)-based growth medium efficiently protects Rhizobium loti cells during freezing and desiccation and can maintain their growth in a manner similar to that of traditional mannitol-based medium (YEM). The cheese-whey-based medium (CW) improved viability when used to re-suspend cell pellets kept at –20 °C and –80 °C and resulted in the survival of over 90% of the cells. Moreover, bacterial pellets obtained from cells grown in CW withstand desiccation better than cells grown in YEM. Survival was over 60% after 30 days at 4 °C. No differences were observed in nodulation efficiency between YEM-grown and CW-grown cells. Fast protein liquid chromatography (FPLC) protocols are presented for total protein profile analyses of sweet and acid cheese whey.In memoriam of Sylvio Cortina Vicepresident of Fundación COREPRO     

Epidermal cells of a symbiosis-defective mutant of Lotus japonicus show altered cytoskeleton organisation in the presence of a mycorrhizal fungus

The influence of the mycorrhizal fungus Gigaspora margarita on cytoskeleton organisation in epidermal cells of Lotus japonicus roots was compared between plants of the wild type Gifu and the mutant Ljsym4-2, in which the fungus is confined to the epidermal cells. Immunofluorescence labelling of plant microtubules and microfilaments showed only limited alterations in the peripheral cytoskeleton of epidermal cells during early stages of fungal interaction with the wild type. Later, microtubules and microfilaments enveloped the growing hypha, while the host cell nucleus moved close to the fungus. In contrast, epidermal cells of the mutant responded with disorganisation and disassembly of microtubules and microfilaments before and during fungal penetration attempts. The fungus penetrated only as far as to epidermal cells, whose cytoplasm became devoid of tubulin and actin, suggesting cell death. The close relationship between host cytoskeleton organisation and compatibility with the fungus suggests that a functional Ljsym4 gene is necessary for correct reorganisation of the epidermal cell cytoskeleton in the presence of the fungus and for avoiding hypersensitivity-like reactions.     

Large Scale Structural Analysis of cDNAs in the Model Legume, Lotus japonicus

Lotus japonicus possesses certain characteristics suited to molecular genetic and genomic analyses and has been adopted as a model species in the study of legume plants. To make a catalogue of genes expressed in L. japonicus and understand biological processes specific to legume plants, large scale EST analyses have been performed. To date, more than 26,000 EST sequences of L. japonicus have been deposited in the public databases. These sequences were developed by five laboratories using different organs. In this review, information obtained from two EST projects carried out in Japan is presented. Some 7137 non-redundant EST groups from young plants and 718 groups from flower buds were generated. A similarity search revealed that homologues of nodulin genes in other legume plants, as well as genes related to secondary metabolism, seed development and the reproductive process, were included in the EST collection, indicating the usefulness of the EST clones in the study of biological phenomena distinctive to legume plant species. Received 23 August 2000/ Accepted in revised form 22 September 2000     

Molecular characterization of two glutathione peroxidase genes of Panax ginseng and their expression analysis against environmental stresses

Glutathione peroxidases (GPXs) are a group of enzymes that protect cells against oxidative damage generated by reactive oxygen species (ROS). GPX catalyzes the reduction of hydrogen peroxide (H₂O₂) or organic hydroperoxides to water or alcohols by reduced glutathione. The presence of GPXs in plants has been reported by several groups, but the roles of individual members of this family in a single plant species have not been studied. Two GPX cDNAs were isolated and characterized from the embryogenic callus of Panax ginseng. The two cDNAs had an open reading frame (ORF) of 723 and 681 bp with a deduced amino acid sequence of 240 and 226 residues, respectively. The calculated molecular mass of the matured proteins are approximately 26.4 kDa or 25.7 kDa with a predicated isoelectric point of 9.16 or 6.11, respectively. The two PgGPXs were elevated strongly by salt stress and chilling stress in a ginseng seedling. In addition, the two PgGPXs showed different responses against biotic stress. The positive responses of PgGPX to the environmental stimuli suggested that ginseng GPX may help to protect against environmental stresses.     

FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca²⁺ dynamics

Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca(2+) -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca(2+) fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca(2+) imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca(2+) dynamics in Lotus japonicus revealed distinct Nod factor induced Ca(2+) spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca(2+) dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures.     

Influence of temperature and salinity on the germination of Lotus creticus (L.) from the arid land of Tunisia

Effects of salinity, temperature and their interactions on the rate and final percentage of germination were evaluated for two populations (Msarref, Oued dkouk) of the invasive glycophyte Lotus creticus Linné, grown under arid environmental conditions of the Tunisia. Seeds that were not treated with NaCl germinated well in a wide range of temperatures. For both populations, maximum germination occurred in distilled water at 25°C and lowest germination for all salinities was at 35°C. Germination was substantially delayed and significantly reduced with an increase in NaCl to levels above 300 mm . Compared to the Oued dkouk population, final germination and germination rate of the Msarref population was completely inhibited at 300 mm NaCl. The interactive effect of temperature and NaCl concentration on final germination and germination rate was significant (P < 0.01), indicating that the germination response to salinity depended on temperature. The inhibition of Oued dkouk population seed germination at high salt concentration was mostly due to osmotic effects while ionic effects were noted at Msarref population. The germination behaviour of the Oued dkouk population would therefore imply adaptive mechanisms to saline environments, while in the Msarref population such mechanisms seem to be absent. Since seed germination is more sensitive to salinity stress than the growth of established plants, the greater tolerance to salinity of Oued dkouk population would be an adaptive feature of this population to saline environment.