Exposure of cold-hardy Rubidoux trifoliate orange [Poncirus trifoliata (L) Raf.] plants to temperatures from 28 degrees C to -5 degrees C enabled us to isolate and characterize a novel citrus low-temperature gene (CLT) with two transcripts, called CLTa and CLTb, from leaves and stems. CLTa was produced when plants were subjected to low temperatures (starting at 10 degrees C), while CLTb was constitutively expressed. Both CLTa and CLTb have the same open reading frame (ORF) of 165 nucleotides and encode a small (54 deduced amino acid) protein. However, CLTa has an additional 98 nucleotides in the 3'-untranslated region (UTR) that are absent in CLTb. Expression analysis using relative quantitative RT-PCR demonstrated that CLTa is expressed exclusively at low temperatures, while CLTb is expressed constitutively (expression verified from 33 degrees C to -5 degrees C). A GenBank database search identified 61 nucleotides inside of the ORF that are highly similar to low-temperature-responsive genes from Arabidopsis thaliana and Solanum tuberosum. The deduced amino acid sequence revealed similarity with low-temperature-responsive proteins from A. thaliana, Oryza sativa, and S. tuberosum of 77%, 81%, and 73.9%, respectively. A genomic clone was isolated, and the genome organization revealed the presence of three exons and two introns, the second of which is in the 3' UTR and participates in alternative 3' splice site selection. One of the 3' splice sites of the second intron was located immediately before the additional 98-bp non-coding fragment of CLTa, and the second at the very end of the 98-bp fragment. Additionally, the presence of the tetranucleotides TCTT and TTCT, which are involved in the regulation of transcript processing in animals and possibly also active in peach, was found in this intron. Competition for splicing sites on the pre-mRNA in the spliceosome, which is induced by low temperature, may be involved in the production of the two transcripts of the CLT gene.

CLTa and CLTb are two transcripts produced by the citrus CLT gene. CLTb is constitutively expressed, while CLTa is a low temperature responsive transcript expressed in temperatures below 10 degrees C during the acclimation process of some species of citrus. Real-time PCR was used to study CLTa expression pattern in trifoliate orange during acclimation, gradual deacclimation and abrupt deacclimation. A standard curve of nine dilution series from 10 x 10(6) to 10 x 10(-3) fg was constructed, using part of the CLTa transcript, which enabled us to determine the concentration of the transcript at different temperatures and conditions. It was found that during acclimation, CLTa expression is first detected at 10 degrees C, reaching a maximum after 24 h of exposure at -1 degrees C. During gradual deacclimation, the transcript accumulated at 5 degrees C and after this point it degrades, reaching a low level at 10 degrees C. When the plants were abruptly transferred to room temperature after being acclimated to -1 degrees C, the transcript reduced from 11,303 fg to levels below the threshold 3 h later, indicating a rapid degradation and how the expression of CLTa is dependent on low temperature. Exposure of trifoliate plants to abscisic acid (ABA) and salts indicated that CLTa is not induced by these treatments at the concentrations used. CLTa expression was also verified in other citrus species and was not detected in the acclimated cold-sensitive species "Mexican lime" (Citrus aurantifolia Swing.) and in the cold hardy "Satsuma mandarin (C. unchiu Marc.).

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