Two AP2 domain containing genes isolated from the cold-hardy Citrus relative Poncirus trifoliata are induced in response to cold.

Poncirus trifoliata (L.) Raf. is a cold-hardy, interfertile Citrus relative able to tolerate temperatures as low as -26°C when cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus varieties. A cold-induced cDNA library was constructed by subtractive hybridisation of non-acclimated and 2-d cold-acclimated P. trifoliata seedlings and many genes induced in response to cold were identified. Two of these cDNAs, PI-B05 and PI-C10, were selected from this library for further characterisation. Full-length cDNA sequences of these genes were obtained by 5' and 3' rapid amplification of cDNA ends (RACE). Sequence analysis revealed that PI-B05 contained an apetala2 / ethylene response factor (AP2 / ERF) domain and showed homology with ERF proteins from other plants, some of which are involved in environmental stress-induced gene expression. PI-C10 contained both AP2 / ERF and B3 DNA binding domains and showed homology with other plant proteins in the RAV subfamily of the AP2 / ERF transcription factors, some of which are induced in response to cold and other environmental stresses. Expression patterns of these genes in cold-tolerant P. trifoliata and cold-sensitive pummelo [Citrus grandis (L.) Osb.] in response to cold and drought at different time points were analysed by northern blots. Expression analysis showed that both genes were induced in response to cold, but not under drought conditions in cold-hardy P. trifoliata. However, little or no expression of these genes was detected by northern analysis in cold-sensitive pummelo under cold or drought conditions. The sequence analysis and expression data indicated that these genes may play a role in cold-responsive gene expression in cold-hardy P. trifoliata and could possibly be used for improving cold tolerance in cold-sensitive citrus cultivars.