Table S1: dawn peak genes within each GO category (DONE)
—
There are multiple waves of gene expression after light
exposure at
dawn
While many regulatory genes have peak expression at the onset of light,
we do not know the precise timing of this burst. Additionally, we would
like to know how much variability there is in the timing of the gene
expression across all DNA binding genes in the early morning. To
resolve these questions, a high-resolution time series experiment was
performed at the onset of light, with 25 time points sampled at
22oC (Table S2 ).
To visualise this data, we clustered the gene expression values from the
22oC time series, focussing on genes with GO terms
associated with DNA binding– see Methods and Table
S3 . The dawn burst consists of multiple coordinated waves of gene
expression of genes associated with DNA binding. While there are genes
whose expression levels decrease (clusters 1-2) or increase (clusters
3-4) over the time course, there are clearly three distinct bursts of
expression at 16-24 minutes (clusters 7-8), 18-45 minutes (clusters
9-10), and 45-105 minutes (clusters 5-6), see Figure 2A .
These clusters contain many genes that are relevant to light signalling,
temperature response, and immunity. Clusters 7-8 include many genes
that are related to temperature, such as Heat SHOCK FACTOR A1A (HSFA1A)
and also includes a number of immunity genes such as the WRKY
transcription factor WRKY33 and BASIC LEUCINE ZIPPER 10 (bZIP10), which
is consistent with other research that suggests that plants may be less
sensitive to certain types of infections at dawn (Ingle et al. 2015).
Interestingly, clusters 5-6 include HEAT SHOCK FACTOR B2B (HSFB2B) that
suppresses the heat shock response and inhibits HSFA1A (Ikeda, Mitsuda,
and Ohme-Takagi 2011). It would make sense for such a gene to be
expressed later than HSFA1A. The clusters of genes that are expressed
at 45-105 minutes include genes that appear to be downstream of red
(RVE7, HY5, JMJ22, PNT1) and blue (MYC2, MYC4, CIB2, CRY3) light
signalling.
The dawn gene expression waves are sensitive to temperature
and
light
Since these clusters contained many genes associated with temperature
and light signalling pathways, we investigated the effect of temperature
and light perturbations on these expression waves. We repeated the high
resolution RNA-seq experiment at an elevated but ambient temperature of
27oC (Table S2 ), and observed that genes
continue to peak at the same time points, but the earlier expressed
genes (clusters 1-2 and 7-8) have elevated expression and the later
expressed genes (clusters 3-6) have lower expression than at
22oC, see Figure 2A . Please recall that the
clusters were determined using the 22oC time series
only.
Previously, (Rugnone et al. 2013) found sets of genes that were induced
or repressed by light, and moreover they identified genes whose
sensitivity to light was dependent on whether the light treatment
occurred at night or during the subjective day (after an extended
night). We compare their gene lists with ours in Figure 2B ,
also see Table S4 . There was significant enrichment for light
repressed genes among early expressing genes in clusters 1-2 (p-value
< 1e-14 for genes that were repressed by light either during
the day or in a time-neutral manner, based on Fisher exact test with
Bonferroni correction). In contrast, those genes that were expressed
late in the time series or that peaked at 45-105 minutes or 18-45
minutes tended to be light induced. Previously, we noticed that there
were a number of key light signalling genes in clusters 5-6 and we find
that over a third of genes within this group are light induced.
Intriguingly, the clusters of genes that includes HSFA1A (clusters 7-8)
are not enriched for light induced genes, except for a slight enrichment
for genes whose expression is induced by light at night (p-value
<0.037). However, there are only three genes from this
cluster that are light induced at night– ABA BINDING FACTOR 3 (ABF3),
G-BOX BINDING FACTOR 3 (GBF3) and HOMEOBOX-12 (HB-12). Interestingly,
all three of these light-sensitive-at-night transcription factors are
also ABA induced (Son et al. 2010; Song et al. 2016).
These results suggests that there are multiple coordinated bursts of
gene expression at dawn that are differentially regulated by temperature
and light, and moreover many of the dawn expressed genes are regulated
by light in a time-of-day dependent manner.