Functional Identification of a C-repeat Binding Factor Transcriptional Activator from Blueberry Associated with Cold Acclimation and Freezing Tolerance

Abstract

Highbush blueberry (Vaccinium corymbosum) is susceptible to winter freezing injury and frost damage in the spring. As part of an ongoing project to understand the process of cold acclimation, we isolated a C-repeat binding factor (CBF) transcriptional activator gene-coding region from the highbush blueberry cultivar Bluecrop. Expression of the highbush blueberry CBF gene was compared in floral buds of the cold-tolerant northern highbush cultivar Bluecrop and the more cold-sensitive southern rabbiteye (V. virgatum) blueberry cultivar Tifblue. Relative gene expression was higher in ‘Bluecrop’ than in ‘Tifblue’. Expression in both cultivars was highest at the earliest time point in the fall (coincident with the first stage of cold acclimation), declined during the later fall and winter, and, in ‘Bluecrop’, increased again as buds deacclimated, when temperatures tend to fluctuate. To confirm the putative identity of the gene as a member of the CBF gene family, and to determine if expression in a heterologous system could enhance freezing tolerance, the blueberry gene coding sequence was overexpressed in transgenic Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Transgenic plants expressing the putative blueberry CBF gene exhibited induced expression of the A. thaliana cold-regulated (COR) genes COR78 and COR6.6, under non-inducing conditions (i.e., 23 °C); however, expression of two other COR genes was unaffected. Transgenic plants also exhibited enhanced freezing tolerance under non-acclimated conditions, but not to the level of acclimated control plants. Thus, the expression pattern in floral buds and the ability of the isolated gene to turn on a subset of COR genes and increase freezing tolerance in a heterologous system suggest it is a functional member of the CBF gene family in blueberry.