Expression and functional characterisation of genes putatively involved in freezing tolerance in Arctic species of Vaccinium.

Abstract

Freezing temperatures are responsible for destroying vast areas of crops globally each year and are a major factor in explaining the size and distribution of plants. The ability of plants to survive freezing events, depends on predictable and gradual lowering of temperatures, allowing cold acclimation to occur before freezing temperatures arrive. Climate change is altering maximum and minimum temperatures globally and, making freezing events less predictable. A further understanding of the molecular mechanisms of acquired freezing tolerance would give insights into potential genetic targets for crop improvement. In Arabidopsis, CBF/DREB1 transcription factors are currently the most well characterised proteins associated with acquired freezing-tolerance.
Studies were performed on three species of Vaccinium collected from the Arctic, V. myrtillus, V. uliginosum and V. vitis-idaea. Genes encoding CBF/DREB1 transcription factors from each of these Arctic species were isolated, sequenced and expressed in Arabidopsis. Characterisation studies were performed on transgenic lines generated from each of the three Vaccinium species. Induced COR gene expression, freezing tolerance and altered phenotype were measured in these lines.
Results showed that V. myrtillus CBF/DREB1 can induce freezing tolerance in Arabidopsis. Western blot and reporter gene assay analysis of transiently-expressed CBF/DREB1 from Vaccinium, highlighted the possibility that the CBF/DREB1 from V. uliginosum was less stable than the CBF/DREB1 from the other two species. Site-directed mutagenesis of five regions of interest between the three Vaccinium species showed that the substitution of two amino acids improved COR gene binding/induction. This substitution could serve as a potential crop improvement site for commercial blueberry crops (V. corymbosum).
Other genes associated with the CBF/DREB1 pathway in Arabidopsis were cloned and sequenced from the three Vaccinium species. Cold-induced expression of these genes was tested and showed similarities in sequence and expression pattern between V. vitis-idaea and Arabidopsis.