Expression analysis of a cold responsive transcript from trifoliate orange by real-time PCR and RT-PCR.

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.).