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Multiple feedback loops of the Arabidopsis circadian clock provide rhythmic robustness across environmental conditions.

ABSTRACT: Although circadian oscillators in diverse eukaryotes all depend on interlinked transcriptional feedback loops, specific components are not conserved across higher taxa. Moreover, the circadian network in the model plant Arabidopsis thaliana is notably more complex than those found in animals and fungi. Here, we combine mathematical modeling and experimental approaches to investigate the functions of two classes of Myb-like transcription factors that antagonistically regulate common target genes. Both CCA1/LHY- and RVE8-clade factors bind directly to the same cis-element, but the former proteins act primarily as repressors, while the latter act primarily as activators of gene expression. We find that simulation of either type of loss-of-function mutant recapitulates clock phenotypes previously reported in mutant plants, while simulated simultaneous loss of both type of factors largely rescues circadian phase at the expense of rhythmic amplitude. In accord with this prediction, we find that plants mutant for both activator- and repressor-type Mybs have near-normal circadian phase and period but reduced rhythmic amplitude. Although these mutants exhibit robust rhythms when grown at mild temperatures, they are largely arrhythmic at physiologically relevant but nonoptimal temperatures. LHY- and RVE8-type Mybs are found in separate clades across the land plant lineage and even in some unicellular green algae, suggesting that they both may have functioned in even the earliest arising plant circadian oscillators. Our data suggest that the complexity of the plant circadian network may have arisen to provide rhythmic robustness across the range of environmental extremes to which plants, as sessile organisms, are regularly subjected.

SUBMITTER: Shalit-Kaneh A

PROVIDER: S-EPMC6142266 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Multiple feedback loops of the Arabidopsis circadian clock provide rhythmic robustness across environmental conditions.

Shalit-Kaneh Akiva A Kumimoto Roderick W RW Filkov Vladimir V Harmer Stacey L SL

Proceedings of the National Academy of Sciences of the United States of America 20180618 27

Although circadian oscillators in diverse eukaryotes all depend on interlinked transcriptional feedback loops, specific components are not conserved across higher taxa. Moreover, the circadian network in the model plant <i>Arabidopsis thaliana</i> is notably more complex than those found in animals and fungi. Here, we combine mathematical modeling and experimental approaches to investigate the functions of two classes of Myb-like transcription factors that antagonistically regulate common target ...[more]

PMID: 29915068

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Project description:The circadian clock system is based on interlocked positive and negative transcriptional and translational feedback loops of core clock genes and their encoded proteins. The mammalian circadian clock system has been extensively investigated using mouse models, but has been poorly investigated in diurnal ruminants. In this study, goat embryonic fibroblasts (GEFs) were isolated and used as a cell model to elucidate the caprine circadian clock system. Real-time quantitative PCR analysis showed that several clock genes and clock-controlled genes were rhythmically expressed in GEFs over a 24 h period after dexamethasone stimulation. Immunofluorescence revealed that gBMAL1 and gNR1D1 proteins were expressed in GEFs, and western blotting analysis further verified that the proteins were expressed with circadian rhythmic changes. Diurnal changes in clock and clock-controlled gene expression at the mRNA and protein levels were also observed in goat liver and kidney tissues at two representative time points in vivo. Amino acid sequences and tertiary structures of goat BMAL1 and CLOCK proteins were found to be highly homologous to those in mice and humans. In addition, a set of goat representative clock gene orthologs and the promoter regions of two clock genes of goats and mice were cloned. Dual-luciferase reporter assays showed that gRORα could activate the promoter activity of the goat BMAL1, while gNR1D1 repressed it. The elevated pGL4.10-gNR1D1-Promoter-driven luciferase activity induced by mBMAL1/mCLOCK was much higher than that induced by gBMAL1/gCLOCK, and the addition of gCRY2 or mPER2 repressed it. Real-time bioluminescence assays revealed that the transcriptional activity of BMAL1 and NR1D1 in goats and mice exhibited rhythmic changes over a period of approximately 24 h in NIH3T3 cells or GEFs. Notably, the amplitudes of gBMAL1 and gNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells were higher than those in GEFs, while mBMAL1 and mNR1D1 promoter-driven luciferase oscillations in NIH3T3 cells had the highest amplitude. In sum, transcriptional and translational loops of the mammalian circadian clock system were found to be broadly conserved in goats and not as robust as those found in mice, at least in the current experimental models. Further studies are warranted to elucidate the specific molecular mechanisms involved.

Project description:BACKGROUND: The circadian clock enables living organisms to anticipate recurring daily and seasonal fluctuations in their growth habitats and synchronize their biology to the environmental cycle. The plant circadian clock consists of multiple transcription-translation feedback loops that are entrained by environmental signals, such as light and temperature. In recent years, alternative splicing emerges as an important molecular mechanism that modulates the clock function in plants. Several clock genes are known to undergo alternative splicing in response to changes in environmental conditions, suggesting that the clock function is intimately associated with environmental responses via the alternative splicing of the clock genes. However, the alternative splicing events of the clock genes have not been studied at the molecular level. RESULTS: We systematically examined whether major clock genes undergo alternative splicing under various environmental conditions in Arabidopsis. We also investigated the fates of the RNA splice variants of the clock genes. It was found that the clock genes, including EARLY FLOWERING 3 (ELF3) and ZEITLUPE (ZTL) that have not been studied in terms of alternative splicing, undergo extensive alternative splicing through diverse modes of splicing events, such as intron retention, exon skipping, and selection of alternative 5' splice site. Their alternative splicing patterns were differentially influenced by changes in photoperiod, temperature extremes, and salt stress. Notably, the RNA splice variants of TIMING OF CAB EXPRESSION 1 (TOC1) and ELF3 were degraded through the nonsense-mediated decay (NMD) pathway, whereas those of other clock genes were insensitive to NMD. CONCLUSION: Taken together, our observations demonstrate that the major clock genes examined undergo extensive alternative splicing under various environmental conditions, suggesting that alternative splicing is a molecular scheme that underlies the linkage between the clock and environmental stress adaptation in plants. It is also envisioned that alternative splicing of the clock genes plays more complex roles than previously expected.

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Multiple feedback loops of the Arabidopsis circadian clock provide rhythmic robustness across environmental conditions.

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Multiple feedback loops of the Arabidopsis circadian clock provide rhythmic robustness across environmental conditions.

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