Chantal Teulieres

DNA delivery into Eucalyptus globulus zygotic embryos through biolistics: optimization of the biological and physical parameters of bombardment for two different particle guns

SummaryDNA transfer into zygotic embryos of Eucalyptus globulus by microprojectile bombardment was studied with two devices: a gunpowder apparatus and a compressed-helium system. Using, as a test, the transient expression of a reporter gene, we optimized the physical and biological conditions of bombardment. Six-day-old cultured embryos were found to be the best target material, and osmotic treatment increased the expression rate. Conditions of bombardment (particle acceleration and quality of the particle: DNA mix) were studied. In optimal conditions, we were able to obtain up to 130 GUS expression events per embryo with a good distribution over the tissue.In our transient expression experiments, the gunpowder and helium devices exhibited similar efficiencies, reliabilities and reproducibilities.

Carbohydrate content of Eucalyptus gunnii leaves along an annual cycle in the field and during induced frost-hardening in controlled conditions

The annual changes in frost hardiness were studied for three Eucalyptus gunnii genotypes. Frost resistance evaluated on leaf discs by the electrolyte leakage method reached a maximum in the coldest period and a minimum in summer demonstrating winter frost hardening. Genotype 634 exhibited a higher intrinsic resistance than the other genotypes both in the hardened and in the non-hardened stages. Plants of this genotype were also frost acclimated in controlled conditions by a progressive decrease of culture temperature (25 to 0 °C) but the degree of hardening appeared to be lower in these conditions. The carbohydrate patterns in leaves varied with acclimation. In controlled conditions the leaves of genotype 634 exhibited a rise in sucrose, fructose and raffinose concentration up to a temperature of 10 to 7 °C which subsequently decreased. In natural conditions a comparison of the three genotypes allowed us to correlate the higher intrinsic resistance of genotype 634 to a higher soluble sugar content. During acclimation fructose and raffinose changes were also correlated to an increase in cold resistance even though the kinetics of these changes differed in controlled and natural conditions. The starch content was very low in the various genotypes in the different conditions but oligosaccharides such as stachyose and possibly verbascose were detected. The results point out the relationships occurring between increased frost resistance and changes in fructose and raffinose concentration in E. gunnii leaves.

Differential characteristics of cell suspension cultures initiated from Eucalyptus gunnii clones differing by their frost tolerance

Cell suspension cultures were initiated from two clones of Eucalyptus gunnii differing by their frost resistance.During cold treatments viability of the individual cell lines and of their protoplasts was correlated to the degree of frost resistance of the starting clones.Moreover, at moderate temperature (10°C) the growth rate was higher for the tolerant cells than for the sensitive ones.Free proline content was ten-fold higher in the resistant cell line than in the sensitive one whereas concentrations of other free amino-acids were equivalent.

Transient foreign gene expression in polyethylene/glycol treated or electropulsated Eucalyptus gunnii protoplasts

Conditions for optimal transient gene expression of reporter genes (chloramphenicol acetyl transferase and β-glucuronidase) by polyethylene glycol or electrical treatment of Eucalyptus gunnii protoplasts derived from callus or cell suspension cultures were investigated. The effciency of electropermeabilisation depended on several factors including electrical parameters, pH and the source of protoplasts. Polyethylene glycol mediated DNA uptake was highly stimulated by heat shock pretreatment and was also more efficient than the electrical treatment. For both treatments the nature of the promoter associated with the reporter gene was very important. A promoter corresponding to a protein synthesis elongation factor gene was more effective than the classical promoter of the 35 S transcript from cauliflower mosaic virus.

Reversible phosphorylation of tonoplast proteins involves tonoplast-bound calcium-calmodulin-dependent protein kinase(s) and protein phosphatase(s)

In highly purified tonoplast fractions from Acer pseudoplatanus cells, the in vitro reversible phosphorylation of proteins affected only a restricted set of polypeptides. The phosphorylation process has been shown to be dramatically stimulated by calcium via the mediation of calmodulin as the transducer. The protein kinase(s) was totally inhibited by micromolar concentrations of a calmodulin antagonist. Tonoplast appears to be potentially a good experimental system for the evaluation of the effects of protein phosphorylation on membrane properties in plants.

Isolation and Frost Resistance Screening of Protoplasts from Different Clones of Eucalyptus

Summary Protoplasts were enzymatically isolated from leaf mesophyll of in vitro or green-house grown Eucalyptus plants. Optimum yields were obtained using a new hydrolytic enzyme CAYLASE and different addenda in the digestion medium Ca ++ , PEG, DTT. The protoplasts were tested for frost resistance by estimating their viability after incubation at increasingly low temperatures. In particular experimental conditions this resistance is strictly correlated to the frost responses of the corresponding plants in nature.

ASPECTS OF THE CELLULAR AND MOLECULAR BASIS OF COLD TOLERANCE IN PLANTS

The diversity of the reactions of plants to low temperatures is illustrated by the classical distinction between chilling-sensitive and freezing-sensitive species. In an attempt to improve our understanding of the cellular and molecular basis underlying this diversity, we have investigated the fluidity of specific membranes in relation to cold resistance in Eucalyptus gunnii, a freezing-sensitive tree, and the changes in protein synthesis induced by cold acclimation in soybean (Glycine max), a chilling-sensitive species. We have been able to obtain isolated vacuoles and to purify, by free-flow electrophoresis, specific membranes (tonoplast and plasmalemma) from two cell lines of Eucalyptus gunnii which exhibit differential frost tolerance. The lateral and rotational mobilities of lipids in these different systems were studied by two biophysical techniques: fluorescence recovery after photobleaching (FRAP) and fluorescence polarisation. Our results show, for the first time, that intrinsically the tonoplast exhibits a higher fluidity than the plasma membrane. In addition, the membranes from the frost tolerant line or from acclimated lines were always more fluid than those from the sensitive line. These data strongly suggest a correlation between membrane fluidity and freezing tolerance in Eucalyptus. Through a progressive exposure to low temperatures, soybean plants were acclimated to a temperature of 8°C. As is the case with freezing-sensitive plants, acclimation improved the cold tolerance of chilling-sensitive plants. In order to assess changes in protein synthesis related to cold acclimation, proteins were labelled in vivo with 35S methionine, separated by two dimensional gel electrophoresis and the derived autoradiograms were subjected to computer analysis. The comparison of soluble proteins stimulated during acclimation and at low temperatures during the post-acclimation phase revealed that no new polypeptides were synthesised. The effects of acclimation on protein synthesis are essentially quantitative. Of the soluble proteins, the synthesis of a polypeptide homologous to the heat shock protein 70 family is stimulated. This polypeptide could protect soybean proteins from denaturation and aggregation at low, non-freezing temperatures.

Phosphorylation of Tonoplast Proteins in ACER PSEUDOPLATANUS Cell Suspension Cultures

Highly purified tonoplast preparations obtained from Acer cells allow us to study the autophosphorylation processes of intrinsic proteins in this membrane.