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.