Citrus is a cold-sensitive genus and most commercially important varieties of citrus are susceptible to freezes. On the other hand, Poncirus trifoliata (L.) Raf. is an interfertile Citrus relative that can tolerate temperatures as low as -26 degrees C when fully cold acclimated. Therefore, it has been used for improving cold tolerance in cold-sensitive commercial citrus rootstock varieties and in attempts to improve scion varieties. In this study, cDNA libraries were constructed from both 2-day cold-acclimated and from non-acclimated Poncirus seedlings using a subtractive hybridization method with the objective of identifying cold-regulated genes. A total of 192 randomly picked clones, 136 from the cold-induced library and 56 from the cold-repressed library, were sequenced. The majority of these clones showed sequence homology to previously identified cold-induced and/or environmental stress-regulated genes in Arabidopsis. In addition, some of them shared homology with cold and/or environmental stress-induced genes previously identified in other herbaceous and woody perennial plants and some showed no homology with sequences in GenBank. When these 192 cDNAs were analyzed by reverse northern blot with cold-acclimated and non-acclimated probes, 92 of the cDNAs displayed significantly increased expression, ranging from 2 to 49-fold, during cold acclimation; all 92 were from the cold-induced library. Surprisingly no clones displayed significantly repressed expression in response to cold. Analysis of a number of selected genes individually in northern blots of mRNA from cold-acclimated and non-acclimated plants largely confirmed the reverse northern analysis, verifying induction of expression of selected cDNAs in response to cold. The results showed that subtractive hybridization is an efficient method for identification of cold-induced genes in plants with limited sequence information available. This study also revealed that genes induced during cold acclimation of the cold-hardy citrus relative P. trifoliata are similar to those in Arabidopsis, indicating that similar pathways may be present and activated during cold acclimation in woody perennial plants.
Many genes in different organisms encode proteins with really interesting gene (RING) finger domain(s). The RING zinc finger domain is involved in a wide variety of functions in diverse organisms. A cDNA clone showing homology with RING zinc finger genes and nine-fold induction in response to cold was previously identified during a gene expression study in the interfertile Citrus relative Poncirus trifoliata (L.) Raf. In this study, the fulllength cDNA of this clone was isolated from 2-day cold-acclimated P. trifoliata by a rapid amplification of cDNA ends method using genespecific primers. The full-length cDNA was 956 bp containing a complete open reading frame of 474 bp encoding a polypeptide of 158 amino acids. The full-length cDNA showed a high level of homology with genes encoding putative RING zinc finger proteins in plants. The deduced amino acid sequence of this gene contained a signature sequence motif for a RING zinc finger close to the C terminus of the protein. The RING zinc finger domain was significantly similar to previously characterized RING zinc finger proteins from different organisms. Additionally, it had a histidine residue at the fifth co-ordination site, indicating that this gene encodes a RING-H2 finger protein. Northern blot hybridization showed that the expression of the RING finger gene was induced in response to cold in cold-hardy P. trifoliata but not to the same extent in cold-sensitive Citrus grandis L. Osb. (pummelo). However, the gene was induced by drought stress similarly in both the species. To our knowledge, this study presents the first isolation of the full-length sequence of a RING zinc finger gene induced in response to abiotic stress in plants and the initial characterization of this gene in Citrus.
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