Genome-wide analysis of the AP2/ERF family in Eucalyptus grandis: an intriguing over-representation of stress-responsive DREB1/CBF genes.

BACKGROUND: The AP2/ERF family includes a large number of developmentally and physiologically important transcription factors sharing an AP2 DNA-binding domain. Among them DREB1/CBF and DREB2 factors are known as master regulators respectively of cold and heat/osmotic stress responses. EXPERIMENTAL APPROACHES: The manual annotation of AP2/ERF family from Eucalyptus grandis, Malus, Populus and Vitis genomes allowed a complete phylogenetic study for comparing the structure of this family in woody species and the model Arabidopsis thaliana. Expression profiles of the whole groups of EgrDREB1 and EgrDREB2 were investigated through RNAseq database survey and RT-qPCR analyses. RESULTS: The structure and the size of the AP2/ERF family show a global conservation for the plant species under comparison. In addition to an expansion of the ERF subfamily, the tree genomes mainly differ with respect to the group representation within the subfamilies. With regard to the E. grandis DREB subfamily, an obvious feature is the presence of 17 DREB1/CBF genes, the maximum reported to date for dicotyledons. In contrast, only six DREB2 have been identified, which is similar to the other plants species under study, except for Malus. All the DREB1/CBF and DREB2 genes from E. grandis are expressed in at least one condition and all are heat-responsive. Regulation by cold and drought depends on the genes but is not specific of one group; DREB1/CBF group is more cold-inducible than DREB2 which is mainly drought responsive. CONCLUSION: These features suggest that the dramatic expansion of the DREB1/CBF group might be related to the adaptation of this evergreen tree to climate changes when it expanded in Australia.

Complementary regulation of four Eucalyptus CBF genes under various cold conditions.

CBF transcription factors play central roles in the control of freezing tolerance in plants. The isolation of two additional CBF genes, EguCBF1c and EguCBF1d, from E. gunnii, one of the cold-hardiest Eucalyptus species, is described. While the EguCBF1D protein sequence is very similar to the previously characterized EguCBF1A and EguCBF1B sequences, EguCBF1C is more distinctive, in particular in the AP2-DBD (AP2-DNA binding domain). The expression analysis of the four genes by RT-qPCR reveals that none of them is specific to one stress but they are all preferentially induced by cold, except for the EguCBF1c gene which is more responsive to salt. The calculation of the transcript copy number enables the quantification of constitutive CBF gene expression. This basal level, significant for the four genes, greatly influences the final EguCBF1 transcript level in the cold. A cold shock at 4 degrees C, as well as a progressive freezing which mimics a natural frost episode, trigger a fast and strong response of the EguCBF1 genes, while growth at acclimating temperatures results in a lower but more durable induction. The differential expression of the four EguCBF1 genes under these cold regimes suggests that there is a complementary regulation. The high accumulation of the CBF transcript, observed in response to the different types of cold conditions, might be a key for the winter survival of this evergreen broad-leaved tree.

Enrichment in Specific Soluble Sugars of Two Eucalyptus Cell-Suspension Cultures by Various Treatments Enhances Their Frost Tolerance via a Noncolligative Mechanism.

A cell-suspension culture obtained from the hybrid Eucalyptus gunnii/Eucalyptus globulus was hardened by exposure to lower temperatures, whereas in the same conditions cells from a hybrid with a more frost-sensitive genotype, Eucalyptus cypellocarpa/Eucalyptus globulus, were not able to acclimate. During the cold exposure the resistant cells accumulated soluble sugars, in particular fructose and sucrose, with a limited increase in cell osmolality. In contrast, the cell suspension that was unable to acclimate did not accumulate soluble sugars in response to the same cold treatment. To an extent similar to that induced after a cold acclimation, frost-hardiness of the cells increased after a 14-h incubation with specific soluble sugars such as sucrose, raffinose, fructose, and mannitol. Such hardening was also observed for long-term cultures in mannitol-enriched medium. This cryoprotective effect of sugars without exposure to lower temperatures was observed in both the resistant and the sensitive genotypes. Mannitol was one of the most efficient carbohydrates for the cryoprotection of eucalyptus. The best hardiness (a 2.7-fold increase in relative freezing tolerance) was obtained for the resistant cells by the cumulative effect of cold-induced acclimation and mannitol treatment. This positive effect of certain sugars on eucalyptus freezing tolerance was not colligative, since it was independent of osmolality and total sugar content.

Introduction of specific carbohydrates into Eucalyptus gunnii cells increases their freezing tolerance.

The comparison of soluble sugar content in various cell lines of Eucalyptus gunnii exhibiting different freezing resistances revealed that the most resistant cell line contained the highest soluble sugar content. It was possible to increase the freezing resistance of the sensitive cell line by progressive exposure to low temperatures (acclimation). During the early stage of cold acclimation, an increase of soluble sugar concentration was observed in the cells confirming the correlation between freezing resistance and soluble carbohydrate content in this species. In addition, feeding experiments on the sensitive cell line were performed to introduce specific sugars into the cells. Both electropulsation and long-term incubation in the presence of fructose and raffinose led to an increase in the tolerance of the cells during a freezing programme. Using radioactive fructose, the uptake of the sugar into cells and protoplasts was checked. In the light of these results, hypotheses are presented concerning the possible role of intracellular sugars in cryoprotection.

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

DNA 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.

Lateral and Rotational Mobilities of Lipids in Specific Cellular Membranes of Eucalyptus gunnii Cultivars Exhibiting Different Freezing Tolerance.

Two cell lines of Eucalyptus gunnii have been shown to keep their differential frost tolerance at the cellular level after long-term culture. They have been used to investigate the fluidity of specific cell membranes in relation with frost tolerance. Protoplasts and isolated vacuoles were obtained from both cell lines. In addition, purified plasma membrane and tonoplast (the vacuolar membrane) were separated from a crude microsomal fraction through free-flow electrophoresis. The lateral and rotational mobilities of lipids in these different membranes were studied by two biophysical techniques: fluorescence recovery after photobleaching (FRAP) and fluorescence polarization. After labeling the vacuoles isolated from the frost-sensitive cells with 1-oleoyl-2-(7-nitro-2,1,3-benz-oxadiazol-4-yl)aminocaproyl phosphatidylcholine, a single mobile component was observed with a diffusion coefficient of 2.4 x 10(-9) cm(2) s(-1) and a mobile fraction close to 100% at a temperature of 23 degrees C. When using isolated vacuoles from the frost tolerant line, a higher lateral diffusion of tonoplast lipids was found with a diffusion coefficient of 3.2 x 10(-9) cm(2) s(-1), still with a mobile fraction close to 100%. No convincing data were obtained when performing fluorescence recovery after photobleaching experiments on protoplasts. Fluorescence polarization experiments confirmed the differential behavior of the two cell lines for tonoplast and also for plasma membrane. In addition, they showed that intrinsically tonoplast exhibited a higher fluidity than plasma membrane. Our results provide the first information on the fluidity of tonoplast and on the compared properties of two important plant membranes-tonoplast and plasma membrane-through the use of two complementary biophysical approaches. In addition, they suggest there is a correlation between membrane fluidity and cold tolerance. The potential interest of plant vacuole as a natural model system in membrane studies is emphasized.

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.

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.