Regulation of the expression levels of alternatively spliced CCA1 isoforms by environmental stress. (A) Semi-quantitative RT-PCR analysis of the I4R event in CCA1 transcripts under different abiotic stress treatments. The relative abundance of the PTC-harboring CCA1 I4R isoform changes compared to the full-length spliced variant (I4S) after cold stress treatment. Two-week-old seedlings were treated as previously described . Ctrl - untreated seedlings, Hi light - high intensity light, Heat - heat stress (42 °C), Cold - cold stress (4 °C), Salt - high salinity (0.5 M sodium chloride), Desiccation - dehydration (polyethylene glycol treatment). eF1α mRNA was used to demonstrate an equal PCR amplification of cDNAs. PCR was carried out for 16 cycles for all the reactions. PCR products were separated in 2 % agarose gels and stained by ethidium bromide. (B) qRT-PCR analysis of cold stress-induced changes in relative abundance of the CCA1 splice variants. The normalized expression of the CCA1 transcripts with spliced intron 4 (CCA1 I4S) increased after 12 and 168 hours of the cold treatment (4 °C). In contrast, the normalized fold expression of the I4R transcripts sharply decreased following the treatment at 4 °C. GOG was used as a reference housekeeping transcript. The sampling and conditions of the time course are described in detail in the Methods. The normalized fold change of expression of the target transcripts was calculated using a –ΔΔCt method and CFX Manager software. Vertical bars denote the standard error of the mean. Both RT-PCR and qRT-PCR were performed using splicing event-specific oligonucleotide primers (see Additional file 2). ZT - Zeitgeber time (hours).