Discussion
Understanding of the mechanisms which regulate circadian timing has been
enhanced greatly in the past few decades, with the discovery of
post-transcriptional and post-translational modifications, chromatin
remodelling and cytosolic events. We have been investigating how
cellular metabolism, an important output of the circadian clock, might
reciprocally communicate with the circadian clock to modify circadian
clock behaviour. To this end, we screened a group of
NAD+-related mutants for circadian phenotypes. This
study was motivated by the extensive literature concerning the function
of the PARPs and SRT in regulating the circadian clock of mammals and
the effect of nicotinamide being common between the plant and mammalian
systems (Dodd et al ., 2007; Nakahata et al ., 2008;
Nakahata et al ., 2009; Asher et al ., 2010). Here, we found
no evidence that mutation of the PARPs or SRT affected
circadian period, the response to nicotinamide or affected specifically
the expression of circadian oscillator genes. Our finding that the
effects of nicotinamide on the circadian oscillator of plants are not
associated with inhibition of the PARP and SRT NADases means that the
mechanisms by which NAD regulates the circadian clock of Arabidopsis
might be different to that which occur in mammals (Dodd et al .,
2007; Nakahata et al ., 2008; Nakahata et al ., 2009; Asheret al ., 2010).. We cannot exclude the possibility that
unidentified members of the PARP and SRT gene families
could be targets for nicotinamide. Based on these findings and our
previous data we favour alternative explanations for the effects of
nicotinamide on the circadian oscillator. Nicotinamide is an inhibitor
also of ADPRcyclase which generates the Ca2+ agonist,
cADPR (Abdul-Awal et al 2016). and we have measured circadian
oscillations of cADPR in Arabidopsis (Dodd et al ., 2007)
Furthermore, we reported that Ca2+ affects circadian
period through regulation of TOC1 by CALMODULIN-LIKE 24 (CML24) (Martí
Ruiz et al ., 2018). Ca2+ is sensed by the
circadian oscillator by CML24 and the effects of mutations inCML24 on circadian period are not additive to the effects of
nicotinamide, which is consistent with the effect of nicotinamide being
to abolish the Ca2+ signal through inhibition of ADPR
cyclase activity (Martí Ruiz et al ., 2018). However nicotinamide
has a greater effect on circadian period than mutation of CML24alone, suggesting that either there are other Ca2+sensors in the circadian system or nicotinamide has other targets, such
as reduction of H3K4me3 accumulation (Malapeira et al ., 2012) and
the action of BIG (Hearn et al ., 2018). TOR seems to be required
for the response of the circadian oscillator to nicotinamide, and it has
been proposed this might be due to altered energy production by the
mitochondria (Zhang et al., 2019). The mitochondria are affected
by and regulate Ca2+ dynamics and therefore a dual
role for TOR and Ca2+ signalling in regulation of the
circadian oscillator due to mitochondrial responses to nicotinamide is
plausible (Bravo‐Sagua et al., 2017).
We also found a PARP inhibitor 3MB increases circadian period similarly
to nicotinamide (Figure 2). However, 3MB is a structural analogue of
nicotinamide, and therefore may also target enzymes other than PARP
which bind NAD+ or nicotinamide. In contrast, the use
of another chemical inhibitor of PARP activity, thymidine, did not
affect the period length or amplitude of circadian rhythms ofCCA1::LUC bioluminescence (Malapeira et al ., 2012).
Overall, based on these observations there is no strong evidence for the
role of PARPs in mediating the effect of nicotinamide on clock function
in Arabidopsis, and the specificity of the drugs used is questionable,
as different drugs targeting PARPs have conflicting effects on circadian
period. Whilst PARPs do not affect the circadian oscillator, we found
evidence that they affect seasonal timing through the regulation ofFLC expression and flowering time.
The lack of a role for the PARPs is at first sight surprising given the
reproducible role of PARG1 in setting circadian period (Figure
5). Panda et al ., (2002) predicted that parp mutants would
have reduced circadian period due to the period reducing effect of the
PARP inhibitor 3 AB, however our data indicate that the chemical
inhibition of PARP may have multiple effects and may act independently
of PARP. These results also lead us to speculate how PARGs could
specifically modulate circadian clock function without the apparent
reciprocal effect seen in parp mutants. It is possible that other
proteins encoded in the Arabidopsis genome which have ADP-ribosyl
transferase activity might be responsible for counteracting PARGs. For
example, the SRO (SIMILAR TO RCD-ONE) family of plant-specific proteins
have the conserved PARP catalytic domain, however, these have been found
not to possess ADP-ribosyl transferase activity (Jaspers et al .,
2010).
We also found no evidence for a role of sirtuins in modulating clock
behaviour in Arabidopsis. Neither srt single nor double mutants
had significantly different circadian periods to wild type in assays of
leaf movement or DF (Figure 3 and Supplemental Figure 3). This suggests
that unlike mammals, sirtuins are not regulators of the circadian
oscillator in Arabidopsis. Sirtuins are intimately associated with
central circadian clock components in mice (Asher et al ., 2008;
Chang & Guarente, 2013; Nakahata et al ., 2008; Nakahata et
al ., 2009). In mammals, there is significant evidence that aspects of
nicotinamide effects might be mediated through sirtuins, due to the long
period of rhythms in locomotor activity seen in brain-specificsirt1 mutants (Chang & Guarente, 2013). Furthermore,
resveratrol, an activator of SIRT1 (Lagouge et al ., 2006), causes
a shortened period of circadian rhythms in locomotor activity of grey
mouse lemur (Das et al ., 2010; Pifferi et al ., 2011) and
increased circadian clock gene expression in Rat-1 fibroblast cells
(Oike & Kobori, 2008). In Arabidopsis, acetylation has been established
as a regulatory mechanism controlling circadian clock gene expression
(Farinas & Mas, 2011; Malapeira et al ., 2012; Song & Noh,
2012), and flowering time by modifying chromatin acetylation at theFLC locus (Ausin et al ., 2004; He et al ., 2003; Kimet al ., 2004; Xiao et al ., 2013). As acetylation is
evidently involved within the circadian clock in Arabidopsis at a
transcriptional level (Farinas & Mas, 2011; Malapeira et al .,
2012; Perales & Mas, 2007), other non-NAD+-dependent
deacetylases must be responsible. A corepressor protein TOPLESS (TPL)
has been found to interact with PRR7 , 9 and 5 at
the promoter regions of CCA1 and LHY to repress
transcription (Wang et al ., 2013). This was found to require
histone deacetylase activity; treatment with Trichostatin A (TSA)
disrupts this repression, and histone deacetylase 6 (HDA6) forms a
complex with TPL and PRR9 in vitro (Wang et al ., 2013).
Further evidence that the effect of nicotinamide is independent of
sirtuin-like activity is provided by the finding that H3K56ac was
decreased in nicotinamide treated plants, opposite to the expectation if
deactylation is a nicotinamide-sensitive activity (Malapeira et
al ., 2012). C646, an acetylase inhibitor, phenocopies the effects of
nicotinamide also suggesting that sirtuin-like deactylase activity is
not a regulator of the Arabidopsis circadian oscillator (Malapeiraet al ., 2012). Interestingly, nicotinamide treatment increases
histone acetylation at the VIN3 locus, and induces FLCrepression and flowering (Bond et al ., 2009). However,VIN3 expression was not altered in sirtuin mutants, which
suggested the effect of nicotinamide on VIN3 expression also was
not mediated by sirtuins.
We found profound effects of SRT1 on transcript abundance only
during the day (Figure 9). Knockdown of srt1 affected the
abundance of nearly one third of the transcriptome in the day but not at
night. The transcripts affected were strongly associated with light and
sugar signalling pathways, and less so with those associated with heat
and salt stress (Figure 9). This was confirmed in microRNA lines and by
using qRT-PCR in independent experiments. Our finding that SRT1can have such profound effects on the transcriptome in the day and that
complete knock out of SRT1 is embryo lethal has not been reported
in previous studies, possibly because other investigations have focused
on lines with less strong effects on SRT1 expression. Liuet al ., (2017) found that the T-DNA insertion mutant linesrt1-2 (SALK_001493) has weak effects on SRT1 expression
and that RNAi lines had stronger effects on SRT1 expression and
metabolism. From RNAi and over expression studies Liu et al., (2017)
concluded that SRT1 interacts with Arabidopsis cMyc-Binding Protein 1
(AtMBP-1), which is a transcriptional repressor to regulate AtMBP-1
targets resulting in altered gene expression and metabolism. We also
found that srt1-2 has weak or no effects on SRT1expression, and that RNAi was more effective in reducing SRT1transcript abundance (Figure 4b; Supplemental Figure 8a). Zhang et
al ., (2018) reported that srt1-1 and srt1-2 completely
abolished SRT1 expression resulting in phenotypes associated with
ethylene responses. The qPCR primers used by Zhang et al ., (2018)
were down stream of the srt1-2 T-DNA insert representing the end
of the CDS, whereas we used primers upstream of the srt1-4insertion site in the deacetylase domain and Liu et al., (2017)
used primers in the same domain downstream of the location of thesrt1-4 insert (Figure 4A). Taking these together it is clear that
the srt1-1 and srt1-2 mutants make transcripts encoding an
intact deacetylase domain, which might explain why in our hands and
those of Liu et al., 2017 the srt1-1 and srt1-2mutants had weak or no phenotypes. Furthermore, we found that complete
knock out of SRT1 by insertion of a T-DNA in the acetylase domain
in the srt1-4 mutants was embryo lethal, which was not reported
by Zhang et al., (2018) for srt1-1 and srt1-2,which suggests the lines used in the study of Zhang et al.,(2018) were not abolished in SRT1 function. Based on our findings
that srt1-4 homozygous plants are embryo lethal and thatsrt1-4 heterozygous plants have lower expression of SRT1than RNAi lines and other T-DNA alleles, we conclude that the full
extent of the effects of SRT1 have been obscured previously by
investigation in lines which have little or no effect on the expression
of the SRT1 deacetylase domain (srt1-1, srt1-2 ), and
possibly weaker RNAi knock down lines. The profound regulation of gene
expression we find in srt1-4 heterozygous lines and the
associated embryo lethal effects of the srt1-4 homozygous lines
is associated with the loss of transcripts encoding the deacetylase
domain.
The strong correspondence between the transcripts mis-regulated in
plants with reduced SRT1 and those regulated by light and sugars
(Figure 9) and the opposite direction of the transcript regulation bysrt1-4 to the regulation by light signals (Figure 9) could
suggest that SRT1 participates in the regulation of transcripts
by pathways activated by light signalling and the light regulation of
photosynthesis. Our data suggest that SRT1 is required for the correct
regulation of gene expression during the day but independent of a
function in the circadian oscillator.