Redox rhythm reinforces the circadian clock to gate immune response

Abstract

Recent studies have shown that in addition to the transcriptional circadian clock, many organisms, including Arabidopsis, have a circadian redox rhythm driven by the organism’s metabolic activities1,2,3. It has been hypothesized that the redox rhythm is linked to the circadian clock, but the mechanism and the biological significance of this link have only begun to be investigated4,5,6,7. Here we report that the master immune regulator NPR1 (non-expressor of pathogenesis-related gene 1) of Arabidopsis is a sensor of the plant’s redox state and regulates transcription of core circadian clock genes even in the absence of pathogen challenge. Surprisingly, acute perturbation in the redox status triggered by the immune signal salicylic acid does not compromise the circadian clock but rather leads to its reinforcement. Mathematical modelling and subsequent experiments show that NPR1 reinforces the circadian clock without changing the period by regulating both the morning and the evening clock genes. This balanced network architecture helps plants gate their immune responses towards the morning and minimize costs on growth at night. Our study demonstrates how a sensitive redox rhythm interacts with a robust circadian clock to ensure proper responsiveness to environmental stimuli without compromising fitness of the organism.

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Figure 1: SA disrupts redox rhythm but boosts TOC1 expression without changing its period.
Figure 2: SA-regulation of TOC1 depends on nuclear NPR1.
Figure 3: NPR1 regulates transcription of multiple clock genes.
Figure 4: SA reinforces the circadian clock to gate immune response.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

The microarray data have been deposited in Gene Expression Omnibus under accession number GSE61059. The computer code is available upon request.

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Acknowledgements

We thank R. McClung for sharing the TOC1p:LUC, LHYp:LUC and CAB2p:LUC transgenic lines; S. H. Wu for providing the toc1-101 mutant; A. Millar for discussion on the project and advice on the modelling; S. Spoel for suggestions on an experiment; and P. Benfey for critiquing the manuscript. This work was supported by grants from the National Institutes of Health (NIH) (1R01-GM099839-01, 2R01-GM069594-09) and by the Howard Hughes Medical Institute and the Gordon and Betty Moore Foundation (through grant GBMF3032) to X.D., and a Defense Advanced Research Projects Agency (DARPA) Biochronicity Grant (DARPA-BAA-11-66), NIH Director’s New Innovator Award (DP2 OD008654-01), and Burroughs Wellcome Fund CASI Award (BWF 1005769.01) to N.E.B.

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Authors

Contributions

M.Z., W.W., M.M., and J.M. performed the experiments and statistical analysis. S.K. and N.E.B. identified additional links of NPR1 to the circadian clock through mathematical modelling. X.D. supervised the project. M.Z., W.W., S.K., N.E.B., and X.D. wrote paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Nicolas E. Buchler or Xinnian Dong.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Circadian oscillation of the NAPDH/NADP+ ratio.

NADPH/NADP+ ratios in 3-week-old soil-grown plants derived from Fig. 1a, b. Water (CK) or 1 mM SA was applied at 0 h. Data are mean ± s.e.m. (n = 3). White bars represent subjective days and grey bars represent subjective nights. Harmonic regression analysis suggests significant circadian oscillation of water-treated NADPH/NADP+ ratio (P < 0.0001).

Extended Data Figure 2 The effects of exogenous and endogenous SA on TOC1 expression.

a, Luciferase activity measurements using the TOC1p:LUC plant extracts. Relative luciferase activity of the fifth and sixth leaves from 3-week-old soil-grown TOC1p:LUC plants. Water (CK) or 1 mM SA was applied at ZT24. LL, constant light. a.u., arbitrary unit. Data are mean ± s.e.m. (n = 6 biological replicates; t-test; ***P < 0.001). b, TOC1p:LUC activity rhythms in 3-week-old soil-grown WT and sid2 plants treated with water (CK) or 1 mM SA at subjective dusk (black arrow) (mean ± s.e.m., n = 8 plants). White bars represent subjective days and grey bars represent subjective nights. The bar graphs represent the estimates of amplitude and average expression of TOC1p:LUC, respectively (mean ± s.e.m.). The letters above the bars indicate statistically significant differences between groups at P < 0.05 (Tukey’s multiple comparisons test). NS, non-significant (two-way ANOVA, non-significant interaction between genotype and treatment). This experiment was repeated three times with similar results.

Extended Data Figure 3 NPR1 regulates the amplitude and average expression of TOC1p:LUC.

a, TOC1p:LUC activity rhythms in 3-week-old soil-grown WT and npr1-3 plants treated with water (CK) or 1 mM SA at subjective dusk (black arrow) (mean ± s.e.m.; n = 6 plants). LL, constant light. a.u., arbitrary unit. White bars represent subjective days and grey bars represent subjective nights. The bar graphs show the estimates of amplitude and average expression level (mean ± s.e.m.; two-way ANOVA; *P < 0.05; ****P < 0.0001). bd, Estimates of amplitude (b), average expression (c), and period (d) of TOC1p:LUC in WT and npr1-3. Data are mean ± s.e.m. (t-test; ****P < 0.0001). These experiments were repeated three times with similar results.

Extended Data Figure 4 The abundance of NPR1 monomer under constant light conditions.

NPR1 monomer (M) abundance in 3-week-old soil-grown plants without treatment (a; uncropped version of Fig. 2b) and after 1 mM SA treatment at 0 h (b) under constant light (LL) conditions. NPR1 protein were detected using western blot after non-reducing SDS–PAGE (a, b). NPR1 monomer protein was quantified using the non-specific band (*) as a loading control (b; mean ± s.e.m.; n = 3 biological replicates). O, NPR1 oligomer. White bars represent subjective days and grey bars represent subjective nights.

Extended Data Figure 5 Redox perturbations affect the amplitude and average expression of TOC1p:LUC in an NPR1-dependent manner.

a, TOC1p:LUC activity rhythms in 3-week-old soil-grown WT and trx-h3 trx-h5 (trx-h3 h5) (mean ± s.e.m., n = 6 plants). LL, constant light. White bars represent subjective days and grey bars represent subjective nights. The bar graphs show the estimates of amplitude and average expression (mean ± s.e.m.; t-test; ****P < 0.0001). b, TOC1p:LUC activity rhythms in 3-week-old soil-grown WT and npr1 plants treated with water (CK) or 3 mM GSHmee at subjective dusk (black arrow) (mean ± s.e.m., n = 8 plants). The bar graphs represent the estimates of amplitude and average expression of TOC1p:LUC, respectively (mean ± s.e.m.). The letters above the bars indicate statistically significant differences between groups at P < 0.01 (Tukey’s multiple comparisons test). **P < 0.01; ****P < 0.0001 (two-way ANOVA). These experiments were repeated three times with similar results.

Extended Data Figure 6 Model prediction and validation.

a, Comparison of best-fit solutions for the TOC1-only and the TOC1-and-PRR7 coupling in npr1. LL, constant light. White bars represent subjective days and grey bars represent subjective nights. b, Addition of PRR7 coupling improves the fitness and mostly rescues the short period phenotype of the TOC1-only model (mean ± s.e.m.; n = 715, n is degree of freedom derived from nonlinear regression). ce, The transcript levels of CCA1 (c), LHY (d), and ELF3 (e) in WT plants after water (CK) or 1 mM SA treatment. fh, The transcript levels of CCA1 (f), LHY (g), and ELF3 (h) in WT and npr1 plants. The expression was normalized to UBQ5 (ch). The bar graphs show the estimates of amplitude and average expression level, respectively (ch; mean ± s.e.m.; n = 3 biological replicates; t-test; *P < 0.05; ***P < 0.001; ****P < 0.0001). i, j, Comparison of best-fit solutions for NPR1 activation of TOC1-only (i) and NPR1 activation of TOC1 and LHY/CCA1 (j) after SA treatment.

Extended Data Figure 7 Validation and analysis of microarray data.

a, b, The transcript levels of CML40 (a) and AT4G33960 (b) in 3-week-old soil-grown plants 0 or 3 h after application of 1 mM SA either in the subjective morning (ZT24) or in the subjective evening (ZT36) normalized to UBQ5 under constant light conditions. Data are mean ± s.e.m. (n = 3 biological replicates; two-way ANOVA; ***P < 0.001; ****P < 0.0001). c, d, Enrichment of cis-elements affecting time-of-day-specific sensitivity to induction. Promoter analysis of genes that were more induced by SA when treated at ZT24 (c) or more repressed by SA when treated at ZT36 (d). The heat maps show the average expression levels based on the microarray. Circadian correlation coefficients were extracted from Diurnal (http://diurnal.mocklerlab.org/diurnal_data_finders/new). Yellow represents a high value or a target of CCA1/LHY or TOC1. Blue represents a low value or not a target of CCA1/LHY or TOC1. X represents a gene that was more induced by SA when treated at ZT24 (c) or more repressed by SA when treated at ZT36 (d). Arrows represent activation. Blocked arrows represent repression. P values were determined on the basis of hypergeometric distribution.

Extended Data Figure 8 NPR1 senses and transduces redox signals to trigger transcriptional reprogramming.

SA-triggered redox changes induce the oligomer-to-monomer switch of NPR1. The monomer then enters the nucleus and upregulates both defence genes and clock genes through interaction with TGA transcription factors.

Extended Data Figure 9 Technical details for model fitting.

a, Normalized NPR1 monomer abundance in mock-treated samples. The blue line presents the mean values from Fig. 2b, where the value at 48 h (marked with an open star) was inferred to be the same as that at 0 h. The red line represents the smoothened values used for modelling by averaging over 2 days to create a 1-day trace, which was then repeated over 2 days. The smoothened data were normalized, such that the time average of NPR1 was equal to 1. LL, constant light. White bars represent subjective days and grey bars represent subjective nights. b, SA-treated NPR1 monomer abundance. NPR1 monomer abundance after SA treatment from Extended Data Fig. 4b was normalized so that 0 h has the same value as the corresponding mock-treated NPR1 monomer level. On the basis of the assumption that the SA induction lasted for 2 days, the value of the last time point was inferred to be equal to the basal level (marked with an open star). c, Coefficient of variation (CV) of least-squares residual Σ for 15 different, random initial parameters for the model fitting of npr1 data. d, Coefficient of variation of nb* for 15 different, random initial parameters for the model fitting of npr1 data. e, Optimal na*,Kd* exhibit a linear relationship. log(Σ) was plotted as a function of na and Kd for mock-treated TOC1-only coupling (no query pairs). A ‘low’, linear Σ region is evident and is described by a simple analytical linear relationship, nb* = 0.5689 h−1. f, Coefficient of variation of Σ for 15 different, random initial parameters for the model fitting of SA-treated data. g, Coefficient of variation of Kd* for 15 different, random initial parameters for the model fitting of SA-treated data.

Extended Data Table 1 Primer sequences

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Zhou, M., Wang, W., Karapetyan, S. et al. Redox rhythm reinforces the circadian clock to gate immune response. Nature 523, 472–476 (2015). https://doi.org/10.1038/nature14449

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