Grape (Vitis spp) has four ICE genes encoding proteins with possible functions as transcription factors in an ICE-CBF cold acclimation pathway. Using an Agrobacterium-mediated transactivation system, it was determined that Vitis riparia ICE activates the VrCBF4 promoter after a 4-day incubation period at 22 C when effector and reporter bacteria were at an OD600 of at least 0.25 and 0.5, respectively. All four grape ICE proteins activated VrCBF4 and VrCBF6 promoters. These promoters were used as reporter because both these CBF genes are abundantly expressed in overwintering mature grape buds and therefore thought to be important for freezing tolerance in grape. VrCBF4 was activated most by VrICE2 and VrICE3 whereas VrCBF6 was activated most by VrICE1 and VrICE4. An additional 2 hours of cold treatment increased activation by only the ICE proteins, VrICE2 and VrICE3 that contain a sumoylation site. This supports the notion that low temperature stabilizes VrICE2 and VrICE3 protein by sumoylation. Using a 5’ deletion series of the VrCBF4 promoter it was found that the -281 to -342 bp region contains sequence sufficient for activation by the VrICE proteins or stomatal protein VrFAMA(L). Analyses with a wild type or mutated MYC2g element plus min35S promoter showed that VrICE2, VrICE3 and FAMA(L) activated via this element. The higher activation by combinations of VrICE and VrFAMA(L) suggests that they interact with each other to direct VrCBF4 activation which showed possible overlap between stomatal development and cold acclimation pathways.
