The interplay between the circadian clock and abiotic stress responses mediated by ABF3 and CCA1/LHY

Significance Climate change poses a global threat to plants and humans. Understanding how plants respond to abiotic stresses is crucial for addressing this challenge. Here, we reveal the important role of the circadian clock in regulating abiotic stress responses through the reciprocal regulation between CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) and ABSCISIC ACID RESPONSIVE ELEMENTS-BINDING FACTOR3 (ABF3). We demonstrate how the circadian clock influences ABF3 expression, which in turn delivers stress signals to core clock genes and adjusts the circadian period in response to stress. These findings offer valuable insights for developing genetic and molecular approaches to enhance plant resilience in the face of climate change.


Figures S1 to S3
Primer List Other supporting materials for this manuscript include the following:

SI Materials and Methods
ChIP assay.For ChIP against ABF3, Arabidopsis seedlings for pABF3::ABF3-YPET or Col-0 wild type control were grown on ½ MS plates for 12 d under 8-h:16-h short-day cycles at 22 °C and treated with or without 1 µM ABA for 2 hours before harvesting samples.For ChIP against LHY, Col-0 or cca1-1 lhy-20 mutant control were grown on ½ MS plates for 12 d under 12-h:12-h longday cycles at 22 °C before harvesting samples.Two grams of seedlings were harvested and crosslinked with 1% (v/v) formaldehyde (Sigma-Aldrich) for 15 min under a vacuum, and then Glycin was added to quench the crosslink.The seedlings were ground with Mixer mills (Retsch MM400) and the powder was homogenized in nuclear extraction buffer 1 and precipitated by centrifugation at 2000g for 20 min, washed with nuclear extraction buffer 2, and lysed in nuclei lysis buffer.The chromatin was sheared by sonication (Diagenode Bioruptor 300).The chromatin solution was diluted 10-fold with ChIP dilution buffer and incubated with anti-GFP antibody (Thermo Fisher Scientific, cat.#A11122) or anti-LHY antibody (gift from Isabelle A. Carre,) prebound to Dynabeads Protein G (Thermo Fisher Scientific, cat.#10003D) at 4°C.After washing the immunocomplexes, the bound chromatin fragments were eluted with elution buffer and were reversed the crosslink with 200 mM NaCl at 65°C overnight.The genomic DNA was purified with phenol after digesting the proteins with Proteinase-K.Glycogen and NaAc were added to help recover DNA.RT-qPCR was conducted to analyze the immunoprecipitated DNA and input DNA.

RT-qPCR.
For RT-qPCR, total RNA of seedling plants was isolated using GeneJET Plant RNA Purification Kit (Thermo Scientific, cat.#K0801).cDNA was synthesized from 500 ng of total RNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara, cat.#RR047A).SYBR Greeb Master Mix (applieddbiosystems, cat.#A25742) was used for RT-qPCR on the Real-Time PCR System (Bio-Rad CFX Opus 384).The level of PP2A mRNA accumulation (AT1G69960) was used as an internal control.RT-qPCR data for each sample were normalized to the respective PP2A expression level.The cDNA was amplified following denaturation via 40 cycles of PCR (95°C, 5 s; 60°C, 20 s per cycle).Three biological replicates and two technical replicates were performed per experiment.Primers are listed in Supplemental Primer List.
Seed Germination Assays.Seeds collected at the same time and same growing conditions were used for germination assays.Harvested seeds were air-dried in a silica gel environment at room temperature for 3 weeks before the germination assays.Seeds were sterilized with 70% ethanol and sown on plates containing ½ Murashige and Skoog (MS) medium + 0.7 % agar (wt/vol) + 1% Sugar with or without NaCl treatment.Plates were placed at 4°C in the dark for three days before transferring to a growth chamber with 16-h:8-h long-day (22°C, approximately 80 μmol•m−2•s−1).
Radicle protrusion was regarded as seed germination completion.

Fig
Fig.S1.(A) ABF1 and ABF3 oscillate in the diel condition.RT-qPCR analysis of gene expression of ABF1 and ABF3 in wild type (Col-0) in the diel condition.Col-0 was grown in ½ MS under 12-h:12-h long-day cycles for 10 d and collected samples every 3 hours as indicated in the diagram.The PP2A gene was analyzed as an internal control.Error bars, SDs of three biological replicates.(B) The expression of ABF1 is regulated by LHY and/or CCA1.RT-qPCR analysis of ABF1 in Col-0, LHY-OX, and cca1 lhy in the diel condition.The seeds were grown in ½ MS under 12-h:12h long-day cycles for 10 d and collected samples every 3 hours as indicated in the diagram.The PP2A gene was analyzed as an internal control.Error bars, SDs of three biological replicates.*P < 0.05, compared to corresponding Col-0 of same time point, by t-test using Excel.(C) The experiment schema for investigating stress-induction at different time points.Col-0 wild type was entrained in ½ MS under 12-h:12-h long-day cycles for 8 d before releasing to long light conditions.The plants were transferred to ½ MS liquid medium containing 50 µM ABA or 120 mM NaCl separately at ZT1 and ZT10, and samples were collected for a time course.(D) RT-qPCR analysis of gene expression in response to ABA.ABA treatment (50 µM) was applied as indicated in Fig. S1C.The expression of ABF3 was quantified using PP2A gene as an internal control.Error bars, SDs of three biological replicates.(E) The quantified gene induction rate in response to ABA.The gene induction rate in response to ABA was calculated by dividing ABA with its respective MS medium control at each time point.**P < 0.01 by t-test using Excel.(F) RT-qPCR analysis of gene expression in response to NaCl.NaCl treatment (120 mM) was applied as indicated in Fig. S1C.The expression of ABF3 was quantified using PP2A gene as an internal control.Error bars, SDs of three biological replicates.(G) The quantified gene induction rate in response to NaCl.The gene induction rate in response to NaCl was calculated by dividing NaCl with its respective MS medium control at each time point.*P < 0.05, **P < 0.01 by t-test using Excel.

Fig
Fig. S2.(A) RT-qPCR verifies the overexpression of ABF3 in ABF3-OX/ pLHY::LUC.The expression of ABF3 was quantified using PP2A gene as an internal control and then normalized to pLHY::LUC.Error bars, SDs of three biological replicates.*P < 0.05, by t-test using Excel.(B) Bioluminescence analysis of pLHY::LUC and ABF3-OX/ pLHY::LUC.The indicated genotypes were grown in ½ MS under 12-h:12-h long-day cycles for 8d, then released to free-running LL conditions and detected by a bioluminescence reader.Each sample's bioluminescence value of all time points was normalized to its average level.Error bars, mean ± SEM, n = 12.(C) The circadian period of pLHY::LUC, treated with MS medium control, or 120 mM KCl or 120 mM NaCl, as indicated in Fig. 3C.Values are shown as means ± SEM; n = 12.**P < 0.01, by t-test using Excel.

Fig
Fig. S3.(A-B) ABF3 regulates the expression of core clock genes in Arabidopsis seedlings.RT-qPCR analysis of gene expression TOC1 (A), and PRR9 (B) in Col-0, ABF3-OX and abf1234.The indicated genotypes were grown in ½ MS under 12-h:12-h long-day cycles for 10 d, then released to free-running LL conditions.The seedlings were harvested every 3 hours, as indicated in the diagram.The PP2A gene was analyzed as an internal control.Error bars, SDs of three biological replicates.