Abundant AS events in barley core clock and clock-associated genes.

<p>Abundant AS events in barley core clock and clock-associated genes.</p

NMD sensitivity of alternatively spliced transcripts of <i>HvLHY</i>.

<p>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) <i>LHY</i> transcripts containing Exon 6a showing significantly increased abundance in CHX treatment at both temperature conditions. *** <i>P</i> < 0.001. (b) <i>LHY</i> transcripts containing I4R showing no increase in abundance in CHX-treated material at both temperature conditions.</p

<i>PPD-H1</i> AS during temperature changes.

<p>(a) WT plants assessed using HR RT-PCR primers spanning the N-terminus region. I1R –intron 1 retention. (b) <i>PPD-H1</i> 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).</p

Expression changes of clock genes during transfer from 20°C to 4°C.

<p>Total transcript levels of (a) <i>HvLHY</i>, (b) <i>HvPPD-H1</i> and (c) <i>HvPRR73</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168028#pone.0168028.s001" target="_blank">S1 Fig</a>). Results obtained with different HR RT-PCR primer pairs were considered as technical replicates. Error bars: SEM from three biological replicates. The <i>LHY</i> and <i>PPD-H1</i> 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.</p

Relative abundances of <i>LHY</i> FS and AS transcripts during temperature changes.

<p>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 (<i>P</i> < 0.01). FS—fully spliced; InR—intron retention of intron n.</p

Percentage of amino acid sequence identity between <i>Agl97</i>, <i>Agl1</i>, <i>Agl2</i>, <i>Agl3</i> and <i>Agl4</i>.

<p>Genbank accession numbers are shown.</p

Genomic structure and conserved AS events of Arabidopsis and barley <i>LHY</i> and <i>PRR37</i>/<i>PRR7</i>.

<p>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 <i>LHY</i> 5’UTR are explained in the text.</p

Conservation of AS events in barley core clock genes in Arabidopsis.

<p>Conservation of AS events in barley core clock genes in Arabidopsis.</p

Fractionation on a MonoQ anion exchange column of maltase activity from endosperm.

<p>All fractionations were reproduced at least twice, on independent batches of seedlings. Representative examples are shown. (A), (B). Extracts of endosperm of seedlings of cv NFC Tipple at 10 dpi were applied to a MonoQ column by FPLC. Proteins binding the column were eluted with a gradient of increasing NaCl concentration (dashed line). One-mL fractions were collected and assayed for α-glucosidase activity with pNPG (A), or maltase activity with maltose (B). I-III indicate the three peaks of activity. (C). Extracts were prepared, fractionated and assayed for maltase as in (B), except that seedlings were either transgenic lines in a cv Golden Promise background carrying an RNA interference (RNAi) silencing cassette for <i>Agl97</i> [lines 21 and 23 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151642#pone.0151642.ref031" target="_blank">31</a>] (squares)], or an out-segregant line (i.e. not carrying the silencing cassette) from the same transformation experiment (circles). Chromatographic conditions were the same as in (B) except that the column wash to remove unbound proteins was reduced from 20 to 10 mL. Note that peak II maltase activity elutes at the same NaCl concentration in the experiments shown in (B) and (C). (D), (E). Fractionation on a MonoS column of proteins not binding to a MonoQ column [peak I in (A) and (B)]. Proteins binding to the MonoS column were eluted with a gradient of increasing NaCl concentration (dashed line). One-mL fractions were collected and assayed for α-glucosidase activity with pNPG (D), or for maltase activity with maltose (E). Peaks of activity eluting from the column are designated Ia-Id.</p

Purification of maltase from endosperm extracts.

<p>Purification of maltase from endosperm extracts.</p