Table 1.
Abundant AS events in barley core clock and clock-associated genes.
Table 2.
Conservation of AS events in barley core clock genes in Arabidopsis.
Fig 1.
Genomic structure and conserved AS events of Arabidopsis and barley LHY and PRR37/PRR7.
In the Arabidopsis genes, only those AS events which show conservation to barley are shown—for other Arabidopsis clock genes AS events see James et al. (2012a). Other abundant AS events in barley are also shown. Exons are numbered; 5’ and 3’ UTRs are dark boxes; coding sequences are open boxes. Alt, alternative; ss, splice site; I, intron; R, retention; E, exon; CrIn, cryptic intron; AUG, translation start site. Small black lines on the top right of each gene structure represent a scale of 200 bases. *, multiple intron retention events in the LHY 5’UTR are explained in the text.
Fig 2.
NMD sensitivity of alternatively spliced transcripts of HvLHY.
The relative abundance of different transcripts was measured in control (white) and CHX (grey) treatment conditions at 20°C and 4°C by high resolution RT-PCR; n = 3. RE, relative expression. (a) LHY transcripts containing Exon 6a showing significantly increased abundance in CHX treatment at both temperature conditions. *** P < 0.001. (b) LHY transcripts containing I4R showing no increase in abundance in CHX-treated material at both temperature conditions.
Fig 3.
Expression changes of clock genes during transfer from 20°C to 4°C.
Total transcript levels of (a) HvLHY, (b) HvPPD-H1 and (c) HvPRR73 in the morning (2.5 h after dawn) at 6 different time-points: 20°C, Day 1 at 4°C, Day 2 at 4°C, Day 4 at 4°C, Day 1 at 20°C and Day 2 at 20°C (see S1 Fig). Results obtained with different HR RT-PCR primer pairs were considered as technical replicates. Error bars: SEM from three biological replicates. The LHY and PPD-H1 levels on Day 1 at 20°C were low and probably reflected that this time point represents effectively one hour after switching to 20°C from 4°C, showing that recovery of transcript levels takes longer than one hour.
Fig 4.
Relative abundances of LHY FS and AS transcripts during temperature changes.
WT plants were assessed using HR RT-PCR primers spanning (a) the 5’ UTR region, (b) the MYB-encoding region and (c) the C-terminal coding region. Error bars are SEM of three biological replicates. In (a), the IRs fraction is made up of multiple transcripts containing intron retention: I2R & I3R, I3R & Alt3’ss I1, I3R, I1R & I2R, I1R, and I2R. The levels of total transcripts at the different time-points are shown by the height of the histogram bars relative to the 20°C value (100%). The relative amounts of AS variant transcripts are illustrated as a proportion of these totals. Relative expression values of abundant AS events are shown in the tables below the histograms with significantly different AS/FS ratio compared to 20°C data shown with an asterisk (P < 0.01). FS—fully spliced; InR—intron retention of intron n.
Fig 5.
PPD-H1 AS during temperature changes.
(a) WT plants assessed using HR RT-PCR primers spanning the N-terminus region. I1R –intron 1 retention. (b) PPD-H1 exon 6 undergoes AS to produce four different protein-coding isoforms: A, the fully spliced (FS) transcript; B, transcripts with only the Alt3’ss E6 (-6 nt); C, transcripts with only the Alt5’ss E6 (-45 nt); and D, transcripts with both Alt3’ss E6 (-6 nt) and Alt5’ss E6 (-45 nt). (c) WT plants assessed using HR RT-PCR primers spanning the region between exons 5 and 7. To simplify histograms, the PTC fraction is made up of multiple transcripts containing: I6R, I6R & Alt3’ss E6 (-6 nt), and Alt5’ss E6 (+5 nt). Error bars are SEM of three biological replicates. The levels of total transcripts at the different time-points are shown by the height of the histogram bars relative to the 20°C value (100%). The levels of AS variant transcripts are illustrated as a proportion of these totals. Relative expression values of abundant AS events are shown in tables below the histograms with significantly different AS/FS ratio compared to 20°C data shown with an asterisk (P < 0.01).