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. 2019 Feb 28;10:235. doi: 10.3389/fpls.2019.00235

Figure 4.

Figure 4

Cold-induced alternative splicing of Trans-acting siRNA 1a. (A) Transcript structures of TAS1a isoforms showing the intron and the position in the intron of the region containing the miR173 binding site and phased cleaved siRNAs. The up arrow shows the cleavage site of the TAS1a RNA by miR173 and the down arrows the cleavage sites which release the various siRNAs (Allen et al., 2005). (B) Expression profile of TAS1a transcript isoforms and gene; TAS1a is DAS-only (no significant change in gene level expression). (C) 5-week-old Arabidopsis rosettes harvested at dawn (arrow) after 12 h of variable reductions in temperature. Reductions (Δ) of 2, 4, 8, 12, and 16°C in temperature applied at dusk provides evidence of temperature-sensitive, long-term changes in AS. (D) Unspliced (intron retention – IR) of TAS1a (AT2G27400_ID1) is sensitive to reductions in temperature of 8°C. Student’s t-tests were performed to compare each temperature reduction results against 20°C control. (E) 5-week-old Arabidopsis rosettes harvested rapidly after transfer to cold. The temperature was gradually reduced from 20°C at 0 h to 11°C at 40 min and eventually 4°C at 120 min into the cold treatment; rosettes were harvested across the first 3 h of cold at the times shown allowing the measurement of the speed of transcriptional and AS changes due to temperature reduction. (F) The unspliced (IR) transcript of TAS1a (AT2G27400_ID1) responded rapidly to cold within 90 min, when the temperature reaches 5°C. RT-qPCR was used to measure relative expression levels for data presented in D and F, see Section “Materials and Methods.” Student’s t-tests were performed to compare each temperature reduction results against 20°C control. Significant differences are labeled with asterisks (∗∗p < 0.01; ∗∗∗p < 0.001).