Nicotinamide acts through a pathway independent of PARP1, 2 and 3, PARG2, and SIRTUIN 1 and 2
We have previously proposed that nicotinamide regulates circadian period in Arabidopsis through inhibition of ADPRcyclase activity (Dodd et al., 2007). Here, we tested a counter hypothesis that nicotinamide regulates circadian clocks through the inhibition of PARP or SRT activity. We reasoned that in knock downs of PARP or SRTthat nicotinamide would be less effective if its mode of action in the circadian system was associated with the activity of the encoded proteins. We found no evidence that PARPs or sirtuins are the target for nicotinamide to affect circadian rhythms. The period of circadian rhythms of leaf movement in the parp1-2 , parp2-1 andparp3-1 single mutants was longer in the presence of nicotinamide in comparison to untreated controls (Figure 6 a,b,c; circadian period estimates for each genotype minus and plus 50 mM nicotinamide follow:parp1-2 24.8 ± 2.1, 25.3 ± 2.4 h, P = 0.002; parp2-1 25.0 ± 0.8 h, 26.1 ± 1.1 h, P = 0.007; parp3-1 24.5 ± 2.1 h, 25.7 ± 0.8 h, P = 0.001). Circadian rhythms of leaf movement in theparp1-2x2-1x3-1 mutant also had a long period with the addition of nicotinamide (Figure 6d; parp1-2x2-1x3-1 25.5 ± 1.0 h,parp1-2x2-1x3-1 50 mM nicotinamide 26.8 ± 1.2 h, two-way ANOVA, P(genotype) = 0.146, P(treatment)< 0.001). We also confirmed the lack of interaction between PARPs and nicotinamide by examining the circadian regulation of transcript abundance (Figure 7). Nicotinamide treatment delayed the peak time of CCA1, PRR7 and TOC1 expression by 4 hours in both Col- 0 and parp1-2x2-1x3-1 with no difference between the genotypes (P > 0.2).
Sirtuins also are not required for the response to nicotinamide.srt1-4 hete and srt2-1 mutants had a significantly longer period in the presence of nicotinamide (Figure 6e,f,g circadian period estimates for each genotype minus and plus 50 mM nicotinamide follow:srt1-4 hete 24.7 ± 0.9 h, srt1-4 hete 25.7 ± 0.8 h, two-way ANOVA, P(genotype) = 0.521, P(treatment) = 0.05; srt2-1 24.8 ± 1.3 h, 25.5 ± 1.7 h, two-way ANOVA, P(genotype) = 0.717, P(treatment) = 0.002; srt1-4 hetex2-1 24.2 ± 0.7 h, 25.0 ± 0.5 h, two-way ANOVA P(genotype) = 0.9, P(treatment) = 0.013).
We also examined the effects of nicotinamide in the PARG mutants, whilst these are not expected to be targets, there is a possibility of an interaction because PARG reverses the effects of PARP. Nicotinamide treatment lengthens the period of circadian rhythms of leaf movement inparg1-1 mutants (Figure 6h, parg1-1 25.37 ± 1.5 h,parg1-1 + nicotinamide 26.59 ± 1.2 h, P < 0.001) andparg2-1 (figure 6i, parg2-1 25.00 ± 1.7 h, parg2-1+ nicotinamide 26.1 ± 1.1 h, P < 0.001). Taken together we found no evidence that nicotinamide regulates the circadian period of Arabidopsis through the action of PARPs, PARGs or sirtuins.