Abstract

Three new CBF genes were isolated from E. globulus-denominated EglCBF1a, c, and d, coding for proteins of 220, 229, and 196 amino acids, respectively. The sequence analysis showed that the three predicted proteins contain an AP2 DNA-binding domain and two CBF signature sequences. Phylogenetic analysis demonstrated that these proteins were highly similar to those described in E. grandis and E. gunnii. Transcript abundance analysis in three different E. globulus genotypes exposed to a cold acclimation treatment showed that these CBF genes were highly related to the acclimation process and presented the highest relative expression at freezing temperatures. EglCBF1a showed the highest expression level (1311-fold change) in the cold-tolerant genotype (R1). EglCBF1a and d genes were induced by chilling and freezing temperatures, while EglCBF1c was constitutively expressed, increasing its transcript level when plants were exposed to freezing temperatures. The constitutive overexpression of each E. globulus CBF gene in Arabidopsis plants induces the endogenous CBF regulon gene expression of Arabidopsis and enhanced its tolerance to freezing, with additional phenotypic effects including growth inhibition and delayed flowering. These results indicate that the three EglCBF genes analyzed play important roles under cold acclimation processes in E. globulus and are involved in the signaling pathway of cold stress and the freezing tolerance phenotype observed on specific genotypes of this species and in transgenic Arabidopsis lines.