Introduction
Plant life is heavily influenced by the cycles of light, dark and temperature caused by the rotation of the planet on its axis, resulting in rhythmic outputs modulated by the circadian clock (Webb et al2019). Metabolism is an important output of the circadian system in plants, with the circadian oscillator regulating the expression and activity of the components of many metabolic pathways, including photosynthesis and starch metabolism (Dodd et al ., 2005; Grafet al ., 2010; Lu et al ., 2005), nutrient assimilation (Gutierez et al ., 2008), redox homeostasis (Lai et al ., 2012) and secondary metabolism (Kerwin et al ., 2011). However, metabolism has recently become recognized also to act as a crucial input regulating the circadian oscillators of both plants and animals. For example, in Arabidopsis, the period of the circadian oscillator is regulated by sugars (Haydon et al ., 2013; Frank et al ., 2018) and nicotinamide (Dodd et al ., 2007; Mombaerts et al ., 2019). Sugars fluctuate as a consequence of carbon homeostasis and photosynthesis (Webb et al ., 2019) whereas nicotinamide is a breakdown product of NAD acting as a substrate for post-translational modifications including poly(ADP-ribosyl)ation and protein deacetylation, and for the production of the Ca2+agonist cyclic ADP ribose (cADPR) (Hunt et al ., 2004). Nicotinamide lengthens circadian period in all organisms tested with proposed modes of action being through inhibition of poly (ADP-ribose) polymerases (PARPs), sirtuins (SRTs), ADPR cyclases, the reduction of H3K4me3 accumulation, TARGET OF RAPAMYCIN (TOR) and the action of BIG, a protein of unknown function (Dodd et al ., 2007; Asher et al ., 2008; Nakahata et al ., 2008; Asher et al ., 2010; Malapeira et al ., 2012; Hearn et al ., 2018; Mombaertset al ., 2019; Zhang et al . 2019).
In mammals, PARP1 participates in the phase entrainment of peripheral clocks (Asher et al ., 2010) and mice lacking PARP1 have a phase shift in the timing of complex formation between the circadian oscillator components CLOCK/BMAL1 and PER, and the entrainment of the peripheral circadian clock in the liver to inverted feeding cycles was significantly delayed (Kumar & Takahashi., 2010). PARPs also play roles in DNA repair, maintenance of genomic stability, transcription, chromatin structure, cell cycle and telomere length, energy metabolism and cell death (Krishnakumar & Kraus, 2010; Schreiber et al ., 2006). In plants, PARPs play a crucial role in the innate immune responses because parp1parp2 double mutant Arabidopsis have compromised immune gene activation and enhanced susceptibility to pathogen infections (Feng et al ., 2015). Decreased levels of PARP activity by chemical inhibition or genetic downregulation have been correlated with increased tolerance to abiotic stresses including oxidative, drought and heat stress (De Block et al., 2005; Vanderauwera et al., 2007) but parp loss-of-function mutants are not affected in abiotic stress responses (Rissel et al., 2017). Therefore, the role of PARPs in plant abiotic stress responses is still an open question and their effect on circadian function in plants has not been reported.
PARP enzymatic activity is counteracted by the enzyme poly(ADP-ribose) glycohydrolase (PARG), which hydrolyses PAR polymers and releases ADP-ribose subunits. Consistent with a role for PARPs in immune responses in plants, both PARG1 and PARG2 are required for stress responses to Botrytis cinerea in tomato and an increase in ADP-ribose polymer levels was observed in response to avirulentPst DC3000 (Adams-Phillips et al., 2010). Unlike the PARPs, PARG activity has been shown to regulate the circadian oscillator of Arabidopsis because the TEJ1 mutation in PARG1 has long free-running circadian period (Panda et al ., 2002).
Sirtuins, like the PARPs are NADases whose enzymatic activity is also inhibited by nicotinamide. Sirtuins are NAD+-dependent protein deacetylases homologous to the yeast Sir2 protein, which is responsible for heterochromatin formation in yeast (Imai et al ., 2000; Landry et al ., 2000; Smith et al ., 2000). In mammals, SIRTUIN1 (SIRT1) is closely associated with core circadian oscillator components. SIRT1 activity is rhythmic, and cultured mouse cells null for SIRT1 or transfected with SIRT1 siRNA had reduced amplitude of the BMAL1:LUC circadian oscillator reporter, suggesting SIRT1 is required to maintain the magnitude of oscillator gene expression (Asher et al ., 2008).
We have found evidence that nicotinamide affects the Arabidopsis circadian oscillator through its action on ADPRcyclase and the inhibition of Ca signalling (Dodd et al ., 2007; Awal et al2016; Marti et al 2018;Hearn et al ., 2018; Mombeartset al 2019). Here we have tested the counter hypothesis that members of the Arabidopsis PARP and SRT gene families participate in circadian regulation, and that this might explain the mode of action of nicotinamide in the circadian system. We performed this study because of the important role of the PARPs and SRTs in mammalian systems and the potential that this might be a conserved function between the circadian systems across Kingdoms. Using a combination of genetic and transcriptome analysis we find that the PARPs and SRTs are unlikely to be involved in circadian regulation in Arabidopsis and do not appear to explain the response of the Arabidopsis circadian oscillator to nicotinamide. These data define a major regulatory difference between the plant and mammalian circadian oscillators. We identify a function for PARPs in photoperiodic responses through the regulation of the flowering time regulator FLOWERING LOCUS C (FLC ). We demonstrate that SRT1 function might have been obscured in previous analysis of Arabidopsis because srt1knockouts are embryo lethal, which we conclude might be related to a profound effect of srt1 knockdown on gene expression during the day.