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 °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

<i>EgrCBF</i> and <i>EgrDREB2</i> gene induction rates for <i>E</i>. <i>grandis</i> detached leaves incubated for 8 hours under cold (4°C, blue), heat (45°C, red) or drought (yellow), in comparison to control conditions.

<p>Transcript abundance of <i>EgrCBF</i> (C1 to C17) and <i>EgrDREB2</i> (D2-1 to -6) was quantified through RT-qPCR on detached leaves from 10 individual <i>E</i>. <i>grandis</i> plants. The experimental values were normalized using two reference genes and the mean values issued from three independent biological replicates (n = 3) were analyzed for significance using the statistical T-test (p value < 0.01). The histograms represent the relative expression values as Log₁₀ of induction rates (stress/control). * means a significant 2-fold rate increase in gene expression (stress/control)</p

Genome-Wide Analysis of the AP2/ERF Family in <i>Eucalyptus grandis</i>: An Intriguing Over-Representation of Stress-Responsive <i>DREB1/CBF</i> Genes

<div><p>Background</p><p>The AP2/ERF family includes a large number of developmentally and physiologically important transcription factors sharing an AP2 DNA-binding domain. Among them <i>DREB1/CBF</i> and <i>DREB2</i> factors are known as master regulators respectively of cold and heat/osmotic stress responses.</p><p>Experimental Approaches</p><p>The manual annotation of AP2/ERF family from <i>Eucalyptus grandis</i>, <i>Malus</i>, <i>Populus</i> and <i>Vitis</i> genomes allowed a complete phylogenetic study for comparing the structure of this family in woody species and the model <i>Arabidopsis thaliana</i>. Expression profiles of the whole groups of <i>EgrDREB1</i> and <i>EgrDREB2</i> were investigated through RNAseq database survey and RT-qPCR analyses.</p><p>Results</p><p>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 <i>E</i>. <i>grandis DREB</i> subfamily, an obvious feature is the presence of 17 <i>DREB1/CBF</i> genes, the maximum reported to date for dicotyledons. In contrast, only six <i>DREB2</i> have been identified, which is similar to the other plants species under study, except for <i>Malus</i>. All the <i>DREB1/CBF</i> and <i>DREB2</i> genes from <i>E</i>. <i>grandis</i> 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.</p><p>Conclusion</p><p>These features suggest that the dramatic expansion of the <i>DREB1/CBF</i> group might be related to the adaptation of this evergreen tree to climate changes when it expanded in Australia.</p></div

Pairwise sequence comparison (% of identity) between EgrCBF proteins from <i>E</i>. <i>grandis</i>.

<p>Bolded numbers correspond to the most identical sequences (>70% identity).</p><p>Pairwise sequence comparison (% of identity) between EgrCBF proteins from <i>E</i>. <i>grandis</i>.</p

Schematic representation of the Egr<i>CBF</i> gene cluster on scaffold 1.

<p>The numbers correspond to <i>EgrCBF</i>1-14. The letters refer to <i>EguCBF1</i>A-, B-, C- or D-like, based on the percentage of identity between CBFs from <i>E</i>. <i>grandis</i> and <i>E</i>. <i>gunnii</i> (except for Egr<i>CBF</i>3, 4 and 5 unlabelled genes) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121041#pone.0121041.t003" target="_blank">Table 3</a>).</p

<i>EgrCBF</i> and <i>EgrDREB2</i> transcript abundance in leaves of <i>E</i>. <i>gunnii x E</i>. <i>dalrympleana</i> plants grown for 8h under 4°C cold (white) or control conditions (black).

<p>Gene expression levels of <i>EgrCBF</i> (C1 to C17) and <i>EgrDREB2</i> (D2-1 to -6) were quantified on leaves from cold-treated <i>Eucalyptus</i> plants. The experimental values from RT-qPCR (conducted on three biological replicates) were normalized using five reference genes and the mean values (n = 3) were analyzed for significance using the statistical T-test (p value < 0.05). The resulting histograms represent Log₁₀ of relative transcript abundance of the <i>CBF</i> and <i>DREB2</i> in control or cold conditions. * means a significant 2-fold rate increase in gene expression (cold/control)</p

Pairwise sequence comparison (% of identity) between EgrCBF and EguCBF proteins respectively from <i>E</i>. <i>grandis</i> and <i>E</i>. <i>gunnii</i>.

<p>Bolded numbers correspond to the most identical sequences (>90% identity).</p><p>Pairwise sequence comparison (% of identity) between EgrCBF and EguCBF proteins respectively from <i>E</i>. <i>grandis</i> and <i>E</i>. <i>gunnii</i>.</p

<i>CBF</i> and <i>DREB2</i> gene expression profiles from RNAseq data corresponding to various tissues from <i>E</i>. <i>grandis</i>.

<p>Raw expression data were obtained from the <i>Eucalyptus</i> genome integrative explorer (<a href="http://eucgenie.bi.up.ac.za/" target="_blank">http://eucgenie.bi.up.ac.za</a>). Expression analysis is presented for 14 <i>EgrCBF</i> and five <i>EgrDREB2</i> genes which were automatically annotated, but do not concerns the four additional CBF genes which were manually annotated in our hands. Gene name and accession numbers are mentioned respectively in Alias and ID columns. Heat map and hierarchical clustering of gene expression profiles (right panel) were performed by using expander software (ACGT, <a href="http://www.cs.tau.ac.il/" target="_blank">http://www.cs.tau.ac.il</a>). For each gene, the highest absolute expression value (Log₁₀ FKPM) is represented in the left panel with the indication of corresponding tissue. YL, young leaves; ML, mature leaves; F, flowers; ST, shoot tips; R, roots P, phloem; X, xylem.</p