In genome-scale datasets, Gene Ontology (GO) enrichment is a common analysis to highlight functions over-represented or under-represented in a subset of differentially expressed genes to elucidate the biological significance of the results. However, despite the diversity of existing tools to analyze GO enrichment, it is often difficult to integrate results in an article figure with sufficient clarity. This is partly due to the high number and to the redundancy of the enriched GO terms, especially when looking at large sets of differentially expressed genes. Here, we provide a simple method to plot representative enriched GO terms. The list of representative enriched GO terms is obtained using existing tools Panther and REVIGO and results are represented in different plots generated from a homemade R script and the ggplot2 R package. The generated plots are publication-quality figures. The diversity of represented parameters makes the plots highly informative (number of genes associated with the enriched GO terms, fold enrichment and level of statistical significance). Comparison of GO enrichment between different lists of genes in a single plot is possible. As proof of concept, we performed this analysis on an Arabidopsis heat responsive transcriptome dataset recently published. Bio-protocol is an online peer-reviewed protocol journal. Its mission is to make life science research more efficient and reproducible by curating and hosting high quality, free access protocols.

InArabidopsis, 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 genesCCA1,LHY,PRR7, andPRR9following 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 throughCCA1andLHY, appears to have a more profound role thanPRR7andPRR9in modulating heat stress responses during the day. Our results emphasize the importance of considering the dimension of time in studies on abiotic stress responses inArabidopsis.

The circadian clock helps organisms to anticipate and coordinate gene regulatory responses to changes in environmental stimuli. Under growth limiting temperatures, the time of the day modulates the accumulation of polyadenylated mRNAs. In response to heat stress, plants will conserve energy and selectively translate mRNAs. How the clock and/or the time of the day regulates polyadenylated mRNAs bound by ribosomes in response to heat stress is unknown. In-depth analysis of Arabidopsis thaliana translating mRNAs found that the time of the day gates the response of approximately one-third of the circadian-regulated heat-responsive translatome. Specifically, the time of the day and heat stress interact to prioritize the pool of mRNAs in cue to be translated. For a subset of mRNAs, we observed a stronger gated response during the day, and preferentially before the peak of expression. We propose previously overlooked transcription factors (TFs) as regulatory nodes and show that the clock plays a role in the temperature response for select TFs. When the stress was removed, the redefined priorities for translation recovered within 1 h, though slower recovery was observed for abiotic stress regulators. Through hierarchical network connections between clock genes and prioritized TFs, our work provides a framework to target key nodes underlying heat stress tolerance throughout the day.

Diel changes in the environment are perceived by the circadian clock which transmits temporal information throughout the plant cell to synchronize daily and seasonal environmental signals with internal biological processes. Dynamic modulations of diverse levels of clock gene regulation within the plant cell are impacted by stress. Recent insights into circadian control of cellular processes such as alternative splicing, polyadenylation, and noncoding RNAs are discussed. We highlight studies on the circadian regulation of reactive oxygen species, calcium signaling, and gating of temperature stress responses. Finally, we briefly summarize recent work on the translation-specific rhythmicity of cell cycle genes and the control of subcellular localization and relocalization of oscillator components. Together, this mini-review highlights these cellular events in the context of clock gene regulation and stress responses in Arabidopsis.

The circadian clock is found ubiquitously in nature, and helps organisms coordinate internal biological processes with environmental cues that inform the time of the day or year. Both temperature stress and the clock affect many important biological processes in plants. Specifically, clock-controlled gene regulation and growth are impacted by a compromised clock or heat stress. The interactions linking these two regulatory pathways include several rhythmic transcription factors that are important for coordinating the appropriate response to temperature stress. Here we review the current understanding of clock control of the regulators involved in heat stress responses in plants.

The circadian clock represents a critical regulatory network, which allows plants to anticipate environmental changes as inputs and promote plant survival by regulating various physiological outputs. Here, we examine the function of the clock-regulated transcription factor, CYCLING DOF FACTOR 6 (CDF6), during cold stress in Arabidopsis thaliana. We found that the clock gates CDF6 transcript accumulation in the vasculature during cold stress. CDF6 mis-expression results in an altered flowering phenotype during both ambient and cold stress. A genome-wide transcriptome analysis links CDF6 to genes associated with flowering and seed germination during cold and ambient temperatures, respectively. Analysis of key floral regulators indicates that CDF6 alters flowering during cold stress by repressing photoperiodic flowering components, FLOWERING LOCUS T (FT), CONSTANS (CO), and BROTHER OF FT (BFT). Gene ontology enrichment further suggests that CDF6 regulates circadian and developmental-associated genes. These results provide insights into how the clock-controlled CDF6 modulates plant development during moderate cold stress.