TOC1 functions as a molecular switch connecting the circadian clock with plant responses to drought

Abstract

Despite our increasing knowledge on the transcriptional networks connecting abscisic acid (ABA) signalling with the circadian clock, the molecular nodes in which both pathways converge to translate the environmental information into a physiological response are not known. Here, we provide evidence of a feedback mechanism linking the circadian clock with plant responses to drought. A key clock component (TOC1, timing of CAB expression 1) binds to the promoter of the ABA-related gene (ABAR/CHLH/GUN5) and controls its circadian expression. TOC1 is in turn acutely induced by ABA and this induction advances the phase of TOC1 binding and modulates ABAR circadian expression. Moreover, the gated induction of TOC1 by ABA is abolished in ABAR RNAi plants suggesting that the reciprocal regulation between ABAR and TOC1 expression is important for sensitized ABA activity. Genetic studies with TOC1 and ABAR over-expressing and RNAi plants showed defective responses to drought, which support the notion that clock-dependent gating of ABA function is important for cellular homeostasis under dry environments.

Figure 1

Genome-wide analysis of TOC1 transcriptional networks. (A) Percentages of cyclers and ABA-related genes at the whole-genome level, and in TOC1-ox and toc1-2 transcriptomic datasets (^**P-value<10⁻⁸; ^***P-value<10⁻¹¹; ^****P-value<10⁻¹⁵) (B) Venn diagrams showing the overlap between cyclers and ABA-related genes in TOC1-ox and toc1-2 transcriptomic datasets. (C) Hierarchical clustering of toc1-2 and TOC1-ox mis-regulated genes with transcripts involved in dehydration responses. Percentages of dehydration (D) at the whole-genome level, and in TOC1-ox and toc1-2 transcriptomic datasets, and among these, the ABA-related genes (E) (^*P-value<10⁻³; ^**P-value<10⁻⁶; ^***P-value<10⁻¹¹; ^****P-value<10⁻¹⁵). Details of datasets used for analysis are described in Supplementary data.

Figure 2

Altered responses to drought conditions of plants mis-expressing TOC1. (A) Plant survival to dehydration stress on agar plates. Data are means±s.e.m. of duplicate experiments with at least 25 plants per genotype. (B) Representative photographs of TOC1-ox (left), WT (middle) and TOC1 RNAi (right) plants of the dehydration experiments. (C) Stomatal aperture in rosette epidermis of TOC1-ox, WT and toc1-2 plants. Stomatal dimensions were measured after incubation for 2 h in a buffer containing 0, 1 or 5 μM ABA. Data are means±s.e.m. of duplicate experiments with at least 100 stomata per genotype and per treatment. (D) Representative images by light microscopy of the stomata guard cells. (E) Stomatal conductance of TOC1-ox, WT and toc1-2 mutant plants. Gas exchange measurements were performed with at least 10 rosettes for genotype. (F) Water-loss rates of detached rosettes from WT, TOC1-ox and toc1-2 plants. Data are means±s.e.m. of triplicate measurements with at least five rosettes for genotype at each time point. Compared with WT, the phenotypic differences were statistically significant with P-values below 0.01 in all cases.

Figure 3

Altered responses to drought conditions of plants mis-expressing ABAR. (A) Analysis by Q-PCR of ABAR mRNA expression in WT and several ABAR RNAi lines. (B) Stomatal aperture in rosette epidermis after incubation for 2 h in a buffer containing 0 or 5 μM ABA. Data are means±s.e.m. of duplicate experiments with at least 100 stomata per genotype and per treatment. (C) Representative images by light microscopy of the stomata guard cells. (D) Water-loss rates of detached rosettes from WT and ABAR RNAi plants. Data are means±s.e.m. of triplicate measurements with at least five rosettes for genotype at each time point. (E) Correlation between ABAR mRNA abundance and weight-loss changes of detached rosettes. Weight-loss changes were plotted relative to the WT value. (F) Plant survival to dehydration stress on agar plates. Data are means±s.e.m. of duplicate experiments with at least 25 plants per genotype. Compared with WT, the phenotypic differences were statistically significant with P-values below 0.005 in all cases.

Figure 4

TOC1 binds to the ABAR promoter and regulates ABAR circadian expression. (A, B) Northern blot analysis of TOC1 RNAi and TOC1-ox plants synchronized under LD cycles followed by 2 days under LL. The waveforms of ABAR expression after mRNA quantification are shown below the blots. CT, circadian time. (C) ChIP assays with TOC1-ox (right axis) and TMG plants (left axis) after TOC1 immuno-precipitation with an antibody to YFP followed by Q-PCR amplification of the ABAR promoter. (D) Comparison of the antiphasic waveforms of ABAR expression by northern blot and TOC1 binding to the ABAR promoter by ChIP assays. (E) Effects of ABA on the waveform of TOC1 binding to the ABAR promoter. (F) Effects of ABA on the ABAR mRNA expression. ChIP abundance was plotted relative to the maximum value.

Figure 5

TOC1 is acutely induced by ABA and this regulation is gated by the clock and requires a functional ABAR. (A, B) TOC1∷LUC expression in WT plants treated with 25 μM ABA at the indicated circadian times (CT) during the subjective day and night. (C) TOC1∷LUC expression in ABAR RNAi plants treated with 25 μM ABA at the indicated CT during the subjective day. Data are means±s.e.m. of luminescence from 6–12 plants. (D) Northern blot analysis of TOC1 mRNA expression in WT and ABAR RNAi plants. TOC1 expression was compared in the absence or in the presence of ABA.

Figure 6

ABAR and TOC1 interaction in plant responses to drought. (A, B) Water-loss percentages of detached rosettes from WT, ABAR RNAi, TOC1 RNAi, double ABAR/TOC1 RNAi, TOC1-ox and TOC1-ox/ABAR RNAi plants. Data are means±s.e.m. of triplicate measurements with at least 10 rosettes for genotype at each time point. (C) Survival percentages of WT, ABAR RNAi (A-R), TOC1 RNAi (T-R), double ABAR/TOC1 RNAi T/A-R, TOC1-ox (Tox) and TOC1-ox/ABAR RNAi (Tox/A-R) plants subjected to dehydration on agar plates. (D) Representative photographs of plants in the dehydration experiments. (E) Schematic representation depicting the reciprocal regulation between TOC1 and ABAR and the implication of ABA and the circadian clock in this regulation.