Gene Expression Analysis a Cold Responsive Gene from Poncirus trifoliata during Acclimation and Deacclimation

In Texas, the freezes of 1951 and 1962 together killed 125,000 acres of citrus trees and the freeze of 1983 killed 40,000 acres. The low temperature is one of the most important abiotic stresses to be understood and manipulated molecularly. Cold hardiness is found in the deciduous citrus relative, trifoliate orange, which can withstand temperatures as low as -26 °C when it is cold acclimated. Exposure of the cold hardy trifoliate orange plants to temperature from 28 °C to -5 °C enabled us to isolate and characterize one novel citrus low temperature gene (clt) with two transcripts, called clt-a and clt-b from leaves and twigs. Clt-a was produced when plants were subjected to low temperatures (starting at 10 °C), while cltb was constitutively expressed. Both clt-a and clt-b have the same open reading frame of 165 nucleotides and encodes a small protein of 54 amino acid. However, clt-a has an additional 98 bp nucleotides at the 3'-untranslated region (UTR), which is absent in clt-b. Expression analysis using relative quantitative RT-PCR demonstrated that clt-a is expressed exclusively at low temperatures, while clt-b is expressed constitutively (expression verified from 2 °C to -5 °C). In the process of deacclimation from -1 °C to 28 °C, the clt-a transcript degraded dramatically after 2 °C and was completely absent at 28 °C, while the clt-b transcript remain stable. When the acclimated plant was taken from -1 °C to room temperature, the clt-a gene degraded within 2 hours. Moreover, when acclimated plant was continuously exposed at -1 °C for 20 days, both transcripts clt-a and clt-b remained stable. Involvement of alternative splicing in transcript stability will be discussed.