Circadian Clock Experiments: rhythmic transcriptome of dark-grown, temperature-entrained seedlings

NASCARRAYS-2;

Plants have endogenous clocks which are entrained by the day-night cycle. For a seedling germinating under the soil temperature may be the most important entraining signal. Two groups have published data showing that the circadian clock regulates 2-6% of the genome in Arabidopsis (Harmer et al2000; Schaffer et al.2001). Many of the genes identified function in photosynthesis and associated metabolism because the RNA was extracted from light-grown tissues. Other clock-controlled genes included functions in cell expansion, photo-protection and regulatory genes. The circadian clock can therefore affect many studies of gene regulation including those that are not overtly related to timing. The clock is already functioning in dark-grown seedlings in which the highly abundant photosynthesis-related RNAs are not expressed (Zhong et al. 1998). The clock in dark-grown seedlings can be entrained by temperature; it rhythmically regulates CCR2 expression and the light response of CAB (our unpublished results). Aim This experiment aims to identify clock-regulated genes in dark-grown seedlings for direct comparison with the published results from light-grown tissue. We will use the same microarray the same ecotype and analysis criteria that match Harmer et al. The experiment will identify genes that are: 1. Specifically regulated by the clock only in dark-grown or in light-grown seedlings. 2. Genes that are clock-regulated in both conditionsor in neither. Our results will directly address several questions: * What functions might circadian rhythms serve in dark-grown seedlings as revealed by the clock-regulated physiological or biochemical pathways ? * Are candidate regulators that control rhythmic light responses in the light also rhythmic in darkness ? * Are all the genes that are current candidates for "central oscillator components" rhythmically regulated in both conditions ? Our results will permit future studies (with Nagy Schaefer): * characterising the transcriptome in clock mutants * identifying of a minimal set of target genes for functional studies of the rhythmic proteome. N.B. our protocol avoids the most abundant proteins of light-grown seedlings potentially revealing more low-abundance regulatory proteins. Experimental Plan: Col-0 Arabidopsis seed will be sown on MS plates _plus 3% sucrose and imbibed at 4 C in the dark for 48h. Germination will be synchronised with a 3h white light pulse. The seed will be entrained for 4 days in darkness with temperature cycles then transferred to constant 22 C. Tissue will be harvested after 24h 30h 36h 42h and 48h at 22 C to avoid direct responses to temperature.