MYB96 shapes the circadian gating of ABA signaling in Arabidopsis

Abstract

Circadian clocks regulate the rhythms of biological activities with a period of approximately 24-hours and synchronize plant metabolism and physiology with the environmental cycles. The clock also gates responses to environmental stresses to maximize fitness advantages. Here we report that the MYB96 transcription factor is connected with the clock oscillator to shape the circadian gating of abscisic acid (ABA) responses. MYB96 directly binds to the TIMING OF CAB EXPRESSION 1 (TOC1) promoter to positively regulate its expression. The use of myb96 mutant plants shows that this regulation is essential for the gated induction of TOC1 by ABA. In turn, MYB96 induction by ABA is also altered in toc1-3 mutant plants. The increased tolerance to drought of MYB96 over-expressing plants is decreased in the toc1-3 mutant background, suggesting that MYB96 and TOC1 intersect the circadian clock and ABA signaling. The MYB96-TOC1 function might be also regulated by the clock component CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), which binds to the MYB96 promoter and alters its circadian expression. Thus, a complex circuitry of CCA1-MYB96-TOC1 regulatory interactions provides the mechanistic basis underlying the connection between circadian and stress signaling to optimize plant fitness to ambient stresses.

Figure 1. Circadian clock regulation of MYB96 expression.

(a) Circadian expression of MYB96. Seedlings grown under neutral day conditions (ND) for 10 days were transferred to continuous light conditions (LL) at Zeitgeber Time 0 (ZT0). Transcript levels were determined by quantitative real-time RT-PCR (RT-qPCR). Gene expression values were normalized to the EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (eIF4A) expression and represented as n-fold compared to the value of the sample at ZT24. Biological triplicates were averaged. Bars represent the standard error of the mean. (b,c) Circadian gating of ABA induction of MYB96 (b) and RD22 (c). Ten-day-old seedlings grown under ND were transferred to MS-liquid medium supplemented with or without 20 μM ABA for 2 h under LL and harvested at the indicated ZT points. Gene expression values were normalized to the eIF4A expression and represented as n-fold compared to the value of the mock-treated wild-type sample at ZT2. Three biological replicates were averaged and statistically significant differences between mock and ABA values are indicated by asterisks (Student’s t-test, **P < 0.01; *P < 0.05). Bars indicate standard error of the mean. The numbers above the bars indicate the ratio of expression in mock and ABA-treated samples (ABA/mock). The white and pale grey boxes indicate the subjective day and night, respectively.

Figure 2. MYB96 regulation of TOC1 expression and binding to the TOC1 promoter.

(a) ChIP assays showing the enrichment of putative R2R3-MYB binding regions in promoters of core clock genes analyzed by ChIP-PCR. Seedlings grown under ND were transferred to LL at ZT0. The regions for PCR amplification were shown in Supplementary Figure S2. Biological triplicates were averaged and statistical significance of the measurements was determined by a Student’s t-test (*P < 0.05). Bars indicate the standard error of the mean. (b) Expression of TOC1 in myb96-1. (c) Expression of CCA1 in myb96-1. In (b,c), seedlings grown under ND were transferred to LL at ZT0. Whole seedlings were harvested from ZT24 to ZT68 to analyze transcript accumulation. Gene expression values were normalized to the eIF4A expression and represented as n-fold compared to the value of the wild-type sample at ZT24. Biological triplicates were averaged (Student’s t-test, **P < 0.01; *P < 0.05; difference between wild-type and myb96-1 plants). Bars indicate the standard error of the mean. The white and pale grey boxes indicate the subjective day and night, respectively.

Figure 3. Regulation of TOC1 circadian gating by MYB96.

(a) Induction of TOC1 by ABA. Ten-day-old seedlings grown under ND conditions were transferred to MS-liquid medium supplemented with or without 20 μM ABA at ZT8 and incubated for 2 h under LL. Transcript accumulation was analyzed by RT-qPCR. Gene expression values were normalized to the eIF4A expression and represented as n-fold compared to the value of the mock-treated wild-type sample. Biological triplicates were averaged (Student’s t-test, *P < 0.05). Bars indicate the standard error of the mean. (b) Time course analysis of TOC1circadian gating in myb96-1. Gene expression values were normalized to the eIF4A expression and represented as n-fold compared to the value of the mock-treated wild-type sample at ZT2. Three biological replicates were averaged. Different letters represent a significant difference at P < 0.05 (one-way anova with Fisher’s post hoc test). Bars indicate standard error of the mean. The numbers above the bars indicate the ratio of expression in mock and ABA-treated samples (ABA/mock).

Figure 4. Role of MYB96 regulating the changes on ABA-mediated rhythms.

(a) Effects of ABA on CCA1 expression in wild-type plants. (b) Analysis of pCCA1:LUC circadian activity. Ten-day-old pCCA1:LUC transgenic plants grown under ND were transferred to LL. Luciferase activities were examined in the presence or absence of 20 μM ABA that was added at ZT0. Waveforms represent the average of 42 plants for each condition. (c) Period estimates of pCCA1:LUC activity in the presence or absence of 20 μM ABA. Bars indicate the standard error of the mean (*P < 0.05; Student’s t-test). (d) Phase plot of pCCA1:LUC activity in the presence or absence of 20 μM ABA. Phases were plotted against the strength of the rhythm expressed as relative amplitude error. The rhythm strength is graphed from 0 (center of the plot) to 0.12 (periphery of the circle). (e) Effects of ABA on CCA1 expression in myb96-1. In (a,e), ten-day-old seedlings grown under ND were transferred to LL in the absence or presence of 20 μM ABA. Transcript accumulation was analyzed by RT-qPCR. Gene expression values were normalized to the eIF4A expression and represented as n-fold compared to the value of the mock-treated wild-type sample at ZT0 (a). Biological triplicates were averaged (Student’s t-test, **P < 0.01; *P < 0.05; difference between ABA-treated and mock-treated plants). Bars indicate the standard error of the mean. The white and pale grey boxes indicate the subjective day and night, respectively.

Figure 5. Regulation of MYB96 by TOC1.

(a) Time course analysis of MYB96 expression in toc1-3. Student’s t-test, **P < 0.01; *P < 0.05; difference between wild-type and toc1-3 plants. (b) Disruption of circadian gating of MYB96 (upper panel) and RD22 (lower panel) in toc1-3. Seedlings grown under ND conditions for 10 days were transferred to LL. Gene expression values were normalized to the eIF4A expression and represented as n-fold compared to the value of the mock-treated wild-type sample at ZT2. Three biological replicates were averaged. Different letters represent a significant difference at P < 0.05 (one-way anova with Fisher’s post hoc test). Bars indicate standard error of the mean. The white and pale grey boxes indicate the subjective day and night, respectively. (c) Analysis of plant survival to drought conditions. Two-week-old plants were subjected to drought conditions by withholding water for two weeks. Plant survival rate was determined 3 d after rewatering. Biological triplicates were averaged. Different letters represent a significant difference at P < 0.05 (one-way anova with Fisher’s post hoc test). Bars indicate standard error of the mean.