The role of CBF transcriptional activators in two Citrus species (Poncirus and Citrus) with contrasting levels of freezing tolerance

Citrus species are considered cold sensitive, yet Poncirus trifoliata, an interfertile Citrus relative, is extremely cold tolerant and can survive freezes at -20 degrees C when fully cold acclimated. To better understand the molecular basis of this difference in freezing tolerance, C-repeat-binding factors (CBFs) from P. trifoliata and Citrus paradisi (grapefruit) were isolated and characterized. Based on our results, P. trifoliata and C. paradisi do contain a CBF pathway for cold-regulated gene expression, and similar to other species, they both appear to contain small CBF families. The nucleotide and amino acid sequences of PtCBF1 and CoCBF1 are highly homologous. However, differences in cold-induced expression were observed. PtCBF1 accumulates both earlier and to higher levels than CpCBF1. Furthermore, CORc115, a cold-induced group II LEA gene, also accumulates earlier and to higher levels in P. trifoliata in response to cold temperatures. The regulatory region of CORc115 contains a putative C-repeat/dehydration-responsive element that is specifically recognized and bound by PtCBF, indicating that CORc115 is a target of PtCBF1. This study provides the first evidence of a correlation between CBF expression (timing and quantity) and the degree of cold tolerance in two closely related species with wide differences in cold tolerance. The importance of using Poncirus and Citrus, two non-model organisms, to address new questions about the CBF pathway is illustrated, as these results provide novel evidence that the CBF expression pattern may play an important role in the notable difference in cold tolerance between these two related species.