An abiotic stress-responsive WRKY gene is transiently induced in response to cold and drought stresses in Poncirus trifoliata

Abstract

A partial cDNA sequence sharing significant homology with WRKY transcription factors (TFs) was previously amplified from Poncirus trifoliata by reverse transcription polymerase chain reaction (RT-PCR) using degenerate primers. In this study, the full-length cDNA sequence of this cDNA, PtrWRKY3, was obtained by RACE method from P. trifoliata. The length of PtrWRKY3 cDNA was 1908 bp and contained open reading frame coding for a 512-amino-acid polypeptide. The ortholog of this gene, CpWRKY3, was also cloned and sequenced from Citrus paradisi (grapefruit). The gene consists of 1550 bp cDNA encoding a protein of 516 amino acids. Both genes have double WRKY domains with Cys2His2 signature motif and showed 98% amino acid sequence identity with each other. They were tightly clustered together and showed a close phylogenetic relation with putative WRKY TFs from different woody perennial plants. The expression of the WRKY3 gene was analyzed quantitatively by northern blot and real-time RT-PCR in response to cold and drought stresses in both cold-hardy Poncirus and one of the cold-sensitive Citrus species, C. paradisi. While the expression of the WRKY3 gene was initially induced transiently in response to cold in cold-hardy Poncirus, no significant changes in the expression of this gene were observed in cold-sensitive grapefruit plants. On the other hand, the expression of the WRKY3 gene was transiently induced by drought stress in both Poncirus and grapefruit plants. These results indicated that the WRKY3 gene from Poncirus and Citrus is conserved and its response to drought stress was similar in both species. However, its expression in response to cold was different in cold-hardy and cold-sensitive species indicating that it is transiently induced early in cold acclimation and may be involved in cold tolerance in Poncirus.