PARP regulates flowering time in Arabidopsis by modulatingFLC expression
Since we found no evidence that PARPs and SRTs are implicated in circadian regulation, we performed RNAseq to investigate their wider role. We performed these measurements under 12 h L/12 h D light cycles, sampling in the middle of the day (ZT 6) and night (ZT18) to capture temporal regulation of gene expression. In parp mutants, only 11 genes were mis-regulated in all the parp mutants in the day (number of mis-regulated genes: parp1-2 day 27, night 57,parp2-1 day 43, night 39, parp3-1 day 79, night 44 andparp1-2x2-1x3-1 day227, night 41) (Figure 8 a, b and Supplemental Table 4). To attempt to confirm RNAseq data, we performed qRT-PCR to measure the transcript abundance of several genes that were found to have similar changes in all the parp lines. Genes were chosen representing no change (PRR7 ), a decrease (GP2, SUS4 ), a moderate increase (CCA1 ) or a large increase (AHA8 ) in abundance. The independent qRT-PCR experiments were consistent with the effects measured by RNAseq (Supplemental figure 6).
We were surprised that so few genes were differentially expressed in thePARP mutants and therefore we tested for any physiological consequences of the change in expression of the few genes we detected. We focused on FLC, because this was significantly upregulated in all PARP mutants in the day and night, except parp3-1 at night where the transcript counts were higher but the FDR did not reach statistical significance (FDR=0.14). It is PARP1 and 2that are expressed in vegetative tissue (Supplemental Figure 1) which might explain why parp3-1 had a less effect on FLCabundance than mutations in PARP1 and 2 . First, we confirmed that FLC is differentially expressed in PARPmutants by qRT-PCR of independent samples of the mutant plants (Figure 8 c, d). These data are strongly indicative that PARP activity can regulate FLC gene expression. We found that the effects onFLC might be meaningful because the elevation of the flowering suppressor was associated with a delay in the days to flowering and an increase of the number of leaves at flowering in the parpmutants, and this was specific to short days (Figure 8e,f). The flowering phenotype of the parp mutants is therefore consistent with the molecular phenotype as identified by the RNAseq, supporting the conclusion that PARP activity is not a major regulator of gene expression, at least in stress-free conditions, but can affectFLC expression and flowering time.