Figure 5.
Relative expression (with mean transcript values for the Italian ecotype set to 1) of (A) CBF1 (AT4G25490), (B) CBF2 (AT4G25480), and (C) CBF3 (AT4G25470) genes from leaves of Italian (Castelnuovo-12 [ABRC stock number: CS98761], sub-line 24) and Swedish (Rodasen-47 [ABRC stock number: CS98762], sub-line 29) ecotypes of A. thaliana grown under low light intensity (9-h photoperiod of 100 µmol photons m−2·s−1) at warm temperature (25 °C/20 °C [light/dark] air temperature). Mean values ± standard deviation (n = 4); significant differences (t-test) indicated by asterisks—* = p < 0.05, ** = p < 0.01, *** = p < 0.001. Leaf discs of 0.73 cm2 were homogenized in liquid nitrogen by bead beating and RNA was extracted and DNase-treated (Qiagen RNeasy Plant Mini Kit). cDNA synthesis was performed with 2 μg of DNase-treated RNA per sample (Qiagen Omniscript cDNA Synthesis Kit). Due to the sequence similarity of the three CBF genes, qPCR primers were designed using the NCBI Primer-BLAST Tool in order to minimize off-target amplification of paralogous genes. qPCR was performed with 40 ng of cDNA per sample (Applied Biosystem’s Fast Sybr Green Master Mix) and the housekeeping gene UBC21 (AT5G25760) was used as a control.