The term cold hardiness or freezing tolerance is used to represent, in a general sense, the ability of plants to adapt to and withstand freezing temperatures. It is a complex, multigenic trait that is too often viewed as a single entity when in fact it is composed of many aspects, all of which can be to some extent viewed as genetically distinct. Advances in molecular biology and genomics have provided significant advances in understanding how plants respond to low temperature and acquire freezing tolerance. Among the most important discoveries has been the identification of the CBF/DREB transcription factor. This transcription factor, along with its regulators such as ICE transcription factors, play a major role in sensing low temperature, initiating the process of cold acclimation, and inducing the expression of a large set of cold-regulated genes. These latter genes are presumed to ameliorate injury to plant cells as a result of freeze-induced desiccation and the presence of extracellular ice. The present review provides a comprehensive overview of CBF and ICE genes in a number of woody plants whose genomes have been sequenced and provides information on the attempts to identify genetic markers for use in marker-assisted selection (MAS) or to improve cold hardiness using genetic transformation technologies. Functional studies of CBF genes in woody plants have indicated that their regulation and impact on abiotic stress resistance are more complex than in herbaceous plants. In particular, the possible relationship of CBF to dormancy is highlighted. Cold hardiness is a complex trait and the challenge in the future will be to use the molecular and genetic tools that are being developed, as well as new developments in bioinformatics, to integrate complex sets of data into a systems view of plant biology. This approach holds the best promise for developing the ability to significantly improve cold hardiness in economically important crops while still maintaining high levels of plant productivity and yield.

Plant organisms contain a large number of genes belonging to numerous multigenic families whose evolution size reflects some functional constraints. Sequences from eight multigenic families, involved in biotic and abiotic responses, have been analyzed in Eucalyptus grandis and compared with Arabidopsis thaliana. Two transcription factor families APETALA 2 (AP2)/ethylene responsive factor and GRAS, two auxin transporter families PIN-FORMED and AUX/LAX, two oxidoreductase families (ascorbate peroxidases [APx] and Class III peroxidases [CIII Prx]), and two families of protective molecules late embryogenesis abundant (LEA) and DNAj were annotated in expert and exhaustive manner. Many recent tandem duplications leading to the emergence of species-specific gene clusters and the explosion of the gene numbers have been observed for the AP2, GRAS, LEA, PIN, and CIII Prx in E. grandis, while the APx, the AUX/LAX and DNAj are conserved between species. Although no direct evidence has yet demonstrated the roles of these recent duplicated genes observed in E. grandis, this could indicate their putative implications in the morphological and physiological characteristics of E. grandis, and be the key factor for the survival of this nondormant species. Global analysis of key families would be a good criterion to evaluate the capabilities of some organisms to adapt to environmental variations.

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. 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. 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. 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.

Eucalypts are the most planted trees worldwide, but most of them are frost sensitive. Overexpressing transcription factors for CRT-repeat binding factors (CBFs) in transgenic Eucalyptus confer cold resistance both in leaves and stems. While wood plays crucial roles in trees and is affected by environmental cues, its potential role in adaptation to cold stress has been neglected. Here, we addressed this question by investigating the changes occurring in wood in response to the overexpression of two CBFs, taking advantage of available transgenic Eucalyptus lines. We performed histological, biochemical, and transcriptomic analyses on xylem samples. CBF ectopic expression led to a reduction of both primary and secondary growth, and triggered changes in xylem architecture with smaller and more frequent vessels and fibers exhibiting reduced lumens. In addition, lignin content and syringyl/guaiacyl (S/G) ratio increased. Consistently, many genes of the phenylpropanoid and lignin branch pathway were upregulated. Most of the features of xylem remodeling induced by CBF overexpression are reminiscent of those observed after long exposure of Eucalyptus trees to chilling temperatures. Altogether, these results suggest that CBF plays a central role in the cross-talk between response to cold and wood formation and that the remodeling of wood is part of the adaptive strategies to face cold stress.