Grape contains 4 ICE genes whose expression includes alternative polyadenylation, leading to transcripts encoding at least 7 different ICE proteins

- Vitis contains 4 constitutively expressed ICE genes producing spliced transcripts.
- Three alternative polyadenylated transcripts encode additional ICE-like proteins in leaves.
- Only one alternatively transcript is found in mature buds and could play a role in freezing tolerance.
- ICE from V. riparia and V. vinifera differs in only a few amino acids.

ICE (inducer of CBF expression) transcription factors play a role in cold acclimation, the low temperature-induced process leading to increased freezing tolerance, of plants from temperate regions, and stomatal development. Four ICE genes, each containing 4 exons, were sequenced from both freezing-tolerant Vitis riparia and freezing-sensitive Vitis vinifera. The predicted encoded ICE proteins all contain a bHLH domain with an ICE-specific sequence in their highly conserved C terminus, with only a few amino acid differences between orthologs. Putative phosphorylation sites were present in ICE1, 2 and 3, and sumoylation and ubiquitination motifs were present in ICE2 and ICE3 but not in ICE1 and ICE4. RT-PCR analysis followed by cloning of the predicted ORFs showed that conservatively spliced transcripts were common for all grape ICE genes at both ambient and cold conditions. In addition, these analyses identified alternative transcripts due to polyadenylation within intron 1 sequence from ICE1 and ICE4, or within intron 2 sequence from ICE2 in leaves but only ICE1 alternative transcript in mature buds. This is the first time that alternative polyadenylation for ICE genes is reported. These results imply that regulation of cold acclimation in grape is complicated by the possible synthesis of at least 5, and stomatal development by at least 7, different ICE-like proteins.