Contribution of time of day and the circadian clock to the heat stress responsive transcriptome in Arabidopsis

In Arabidopsis, a large subset of heat responsive genes exhibits diurnal or circadian oscillations. However, to what extent the dimension of time and/or the circadian clock contribute to heat stress responses remains largely unknown. To determine the direct contribution of time of day and/or the clock to differential heat stress responses, we probed wild-type and mutants of the circadian clock genes CCA1, LHY, PRR7, and PRR9 following exposure to heat (37°C) and moderate cold (10°C) in the early morning (ZT1) and afternoon (ZT6). Thousands of genes were differentially expressed in response to temperature, time of day, and/or the clock mutation. Approximately 30% more genes were differentially expressed in the afternoon compared to the morning, and heat stress significantly perturbed the transcriptome. Of the DEGs (~3000) specifically responsive to heat stress, ~70% showed time of day (ZT1 or ZT6) occurrence of the transcriptional response. For the DEGs (~1400) that are shared between ZT1 and ZT6, we observed changes to the magnitude of the transcriptional response. In addition, ~2% of all DEGs showed differential responses to temperature stress in the clock mutants. The findings in this study highlight a significant role for time of day in the heat stress responsive transcriptome, and the clock through CCA1 and LHY, appears to have a more profound role than PRR7 and PRR9 in modulating heat stress responses during the day. Our results emphasize the importance of considering the dimension of time in studies on abiotic stress responses in Arabidopsis. We used RNA-sequencing to measure transcriptomic changes in Columbia-0 (WT), cca1-1/lhy-20 (cca1lhy) and prr7-3/prr9-1 (prr7prr9) under 1 h of moderate cold (10°C) and 1 h of heat stress (37°C). Samples were collected at ZT1 and ZT6. Seedlings were grown on plates containing Murashige and Skoog (MS) medium supplemented with 1.5% sucrose (wt/vol) in 12 h light and 12 h dark (LD) cycles for 12 days at constant 22°C and 90 µm light intensity. Single-end 75 base pair sequences were generated for each mRNA library using the NextSeq 500 (Illumina).