NADase inhibitors lengthen the period of circadian rhythms
We confirmed previous reports that nicotinamide increases circadian period in Arabidopsis (Dodd et al 2007; Mombearts et al ., 2019). The period of the circadian rhythms of leaf position of untreated Col-0 was 24.5 ± 1.5 h which increased to 26.00 ± 0.5h, P < 0.001 in the presence of 50 mM nicotinamide (Figure 1a,b). Similarly, using CLOCK ASSOCIATED 1 (CCA1 ) fused to the luciferase reporter gene (CCA1:luc)we measured an increase of circadian period from 23.7 ± 0.3 h, to 26.0 ± 0.5 h following treatment with 50 mM nicotinamide (Figure 1c).
To investigate if PARPs might be mediating the effect of nicotinamide on the circadian oscillator, we measured the response to 3-Methoxybenzamide (3MB), an inhibitor that has been reported to be more specific than nicotinamide for PARP activity (Chen et al ., 1994). 3MB increased the period of circadian oscillations of CCA1::LUC luminescence (Figure 2a, untreated 25.2 ± 0.6 h, DMSO solvent control 25.4 ±0.7 h, 1 mM 3MB 26.0 ± 0.6 h, 2 mM 3MB 27.1 ± 0.7 h P < 0.001) and leaf movement (Figure 2b; untreated 23.2 ± 0.7 h; 3MB 23.9 ± 0.8 h, P = 0.012).