Identification of circadian transcripts that are co-regulated with [Ca2+]cyt

Our aim is to identify circadian transcripts that are co-regulated with [Ca2+]cyt, with the eventual goal of identifying genetic regulators and targets for circadian oscillations of [Ca2+]cyt. We have identified two conditions in which [Ca2+]cyt behaves differently to other circadian outputs. 1. Treatment of plants with nicotinamide, a metabolic inhibitor of ADPR cyclase, abolishes the circadian oscillations of [Ca 2+]cyt. However, leaf movement, CCA1, LHY, TOC1 and CAB transcript abundance and CAB promoter activity are all rhythmic albeit with a longer period (Dodd et al., 2007). 2. The toc1-1 mutant, which shortens the circadian period of all other rhythms tested, has no effect on the period of [Ca2+]cyt oscillations (Xu et al., 2007). We will measure the circadian regulation of transcript abundance in wild type (C24), toc1-1 and nicotinamide (C24)-treated plants. Method: Wild type (C24) and toc1-1 seeds were sown on 1/2MS 0.8% agar plates and imbibed at 4 C for 48 hours. Seedlings were grown in LD cycles of 12hL:12hD at 19 C for 11 days to entrain the oscillator. Following transfer to LL at dawn on the 12th day, 50% of the wild type seedlings were dosed with 50 mM nicotinamide every 2 hours over the entire course of the experiment. Wild type, toc1-1 and nicotinamide-treated seedlings (approx. 100 for each sample, excluding roots) were harvested at 4 hour intervals from 49 to 93 hours in LL (12 time points covering the entire third and fourth circadian cycles). Two independent replicates of the whole experiment will be hybridised. 72 samples were used in this experiment.