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.