PARP mutants do not have altered circadian rhythms
The sensitivity of circadian rhythms to nicotinamide and 3MB might suggest a role of PARPs in the regulation of the plant circadian oscillator, which prompted us to investigate the regulation ofPARP transcripts and to isolate mutants in the three knownPARP encoding genes in Arabidopsis. None of the transcripts encoding PARPs in Arabidopsis oscillated with a circadian dynamic (Supplemental Figure 1). We were unable to detect PARP3 by qRT-PCR due to low abundance.
Homozygous T-DNA insertion alleles were identified for the parp1-2 (At4G02390; SAIL line 1250B03), parp2-1 (At2G31320; GABI line 380E06),and parp3-1 (At5G22470; SAIK line 108092 ) mutants (Supplemental Figure 2a and Supplemental Table 1) These were combined to generate aparp1-2x2-1x3-1 triple mutant (Supplemental Figure 2b) in which we could not detect transcripts of PARP1, 2 or 3 (Supplemental Figure 2c). We found no evidence that PARP activity contributes to circadian regulation as measured by leaf movement analysis (Figure 3a-d) or the measurement of circadian rhythms of delayed chlorophyll fluorescence (DF; Supplemental Figure 3) because circadian periods were indistinguishable from wild type background (leaf movement Col-0 24.4 ± 1.0 h, parp1 -2 25 ± 1.3 h, Col-0 25.0 ± 1.4 h, parp2 -1 25.3 ± 1.1 h, Col-0 24.1 ± 0.9 h, parp3 -1 24.3± 1.0 h P > 0.05 Figure 3; DF Col-0 23.8 ± 0.9 h, parp1-2 22.6 ± 2.7 h, Col-0 23.2 ± 0.7 h, parp2 -1 23.7 ± 1.0 h, Col-0 23.8 ± 0.9 h, parp3-1 23.9± 1.4 h P > 0.1). We can exclude genetic redundancy between the three known PARP-encoding genes because the triple mutant was also without effect on leaf movement (Col-0 23.8 ± 1.5 h, parp1-2x2-1x3-1 24.6 ± 1.9 h, P = 0.190; Figure 3d) and DF (Col-0 23.5 ± 0.6 h, parp1-2x2-1x3-1 24.0 ± 0.9 h, P = 0.029; Supplemental Figure 3d).